KR101677279B1 - Globo h and related anti-cancer vaccines with novel glycolipid adjuvants - Google Patents

Abstract

Methods of treating immunogenic compositions, cancer vaccines and cancers are provided. (a) glycans such as Globo H or an immunogenic fragment thereof conjugated to a carrier protein by a linker such as para-nitrophenyl; And (b) an adjuvant, such as an? -Galactosyl-ceramide derivative, comprising a glycolipid capable of binding to CD1d on a dendritic cell, wherein the immunogenic composition is compared to an IgM isotype antibody Leading to an immune response that leads to relatively higher levels of IgG isotype antibodies. An immunogenic composition comprising a carrier protein diphtheria toxin cross-reacting substance 197 (DT-CRM 197) and an adjuvant C34 is provided. The antibodies produced by the immunogenic compositions disclosed herein also neutralize at least one of the antigen Globo H, step-specific embryonic antigen-3 (SSEA-3) and step-specific embryonic antigen-4 (SSEA-4) . Therapeutic agents for breast cancer stem cells comprising an immunogenic composition comprising Globo H, SSEA-3 or SSEA-4 conjugated with DT-CRM 197 are provided.

Description

[0001] GLOBO H AND RELATED ANTI-CANCER VACCINES WITH NOVEL GLYCOLIPID ADJUVANTS [0002]

Cross reference of related application

This patent application is a continuation-in-part of co-pending U.S. Patent Application No. 12 / 485,546, filed on June 16, 2009, which claims priority to U.S. Provisional Patent Application No. 61 / 061,968 (filed June 16, 2008) , The title of the invention: "Compositions for inducing immune responses specific to Globo H and SSEA-3 and uses thereof in cancer treatment". The contents of these patent applications are incorporated herein by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of cancer vaccines. In particular, the present application relates to a carbohydrate-based vaccine containing Globo H, a B cell epitope conjugated to an immunogenic carrier DT-CRM 197. More particularly, the present invention relates to an anticancer Globo H-DT vaccine administered with a novel glycolipid adjuvant, for example C34.

In order to design a treatment for cancer, it is desirable to obtain a molecular target of cancer or cancer stem cells not in normal cells. Aberrant glycosylation is often associated with tumor progression and was first described by Meezan et al . In 1969 with an explanation that amglycan is different from healthy cells (Meezan E, et al . (1969) Biochemistry 8: 2518- 2524). Abnormalization includes loss or overexpression of a particular structure, persistence of the truncated structure, and generation of a new structure. Structural differences were subsequently demonstrated by many histological evidence using lectin-staining to compare healthy and malignant tissues (Turner GA (1992) Clin Chim Acta 208: 149-171; Gabius HJ (2000) Naturwissenschaften 87: 108-121).

More recently, tumor-associated carbohydrate antigens have been identified by monoclonal antibodies and mass spectrometry (Shriver Z, et al . (2004) Nat Rev Drug Disc 3: 863-873; Pacino G, et al . (1991) Br J Cancer 63: 390-398). To date, a number of tumor-associated antigens expressed on cancer cells in the form of glycolipids or glycoproteins have been characterized and associated with specific cancer types (Bertozzi CR, Dube DH (2005) Nat Rev Drug Discovery 4: 477-488). Although relatively little is known about the role of surface carbohydrates in malignant cells, antibodies to these antigens, either passively administered or vaccine-induced, were associated with an improvement in prognosis.

Among the reported tumor-associated glycans, the glycolytic antigen Globo H (Fucα1 → 2 Galβ1 → 3 GalNAcβ1 → 3 Galα1 → 4 Galβ1 → 4 Glc) was first isolated and identified from breast cancer MCF-7 cells by Hakomori et al. In 1984 EG, meat al . (1984) J Biol Chem 259: 14773-14777). Further studies using anti-Globo H monoclonal antibodies have shown that Globo H is present in many other cancers, including prostate cancer, stomach cancer, pancreatic cancer, lung cancer, ovarian cancer and colon cancer, (Ragupathi G, et al., 1987). However, there is little expression on the luminal surface of the secretory tissue (Ragupathi G, meat al . (1997) Angew Chem Int Ed. 36: 125-128). In addition, sera from breast cancer patients contain high levels of anti-Globo H antibodies (Gilewski T. el al . (2001) Proc Natl Acad Sci 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), patients with Globo H-positive tumors were found to have shorter survival times than those with Globo H-negative tumors (Chang, YJ, et al. Proc Natl Acad Sci USA 104 (25): 10299-10304). These findings make Globo H, a six-saccharide epitope, an attractive tumor marker for cancer vaccine development and a suitable target.

Globo H is a cancer antigen that is overexpressed in various epithelial cancers. It has been suggested that this antigen can be a target in cancer immunotherapy. A vaccine was developed to elicit an antibody response to Globo H, but due to the low antigenicity of Globo H, its anti-cancer efficacy is unsatisfactory. A new vaccine is needed to bring Globo H-targeted immune responses to a higher level.

Stem cells are defined as a self-renewing ability and a multitude of cells that have the ability to differentiate into different types of cells and tissues (Reya T et al., (2001) Nature 414: 105-111). Because both malignant and normal tissues contain heterogeneous cell populations, cancer stem cells can play an important role in maintaining tumor growth and tumor heterogeneity. Cancer stem cells have been identified from a variety of solid tumors such as brain, breast, colon and prostate cancers. Breast cancer stem cells (BCSCs) were isolated from the CD24 ^- CD44 ⁺ subpopulation of breast cancers by Al-Hajj et al., Based on their ability to produce phenotypic tumors in xenotransplantation with NOD / SCID mice (Al-Hajj M, et al., (2003) Proc Natl Acad Sci USA 100: 3983-3988). Most early disseminated cancer cells in the bone marrow of breast cancer patients showed a phenotype of CD24 ^- CD44 ⁺ (Balic M et al., (2006) Clin Cancer Res 12: 5615-5621), suggesting that BCSC can be metastasized. BCSCs are a major target for the treatment of breast cancer based on their growth, differentiation and metastatic potential and their resistance to radiation (Tang C. et al., (2007) FASEB J. 21: 1-9).

Globo H expression was observed in more than 60% of ductal, lobular, and tubular carcinomas in breast cancer, but not in non-epithelial breast tumors (Mariani-Constantini R et al., (1984) Am . J. Pathol 115:. 47-56) . Globo H is not expressed in normal tissues except for weak expression in the apical epithelial cell at the luminal edge, where it appears to be inaccessible to the immune system ( Id . ; Zhang S. et al., 1997) Int . J. Cancer 73: 42-49).

Globo H is also expressed in breast cancer stem cells (BCSC). As a flow cytometer, Globo H was found to be expressed in 25 of 41 breast cancer specimens (61.0%). Non-BCSCs from 25 out of 25 and BCSCs from 8 out of 40 (20%) express Globo H. Step-specific embryonic antigen 3 (SSEA-3), a five-saccharide precursor of Globo H, is expressed in 31 (77.5%) of 40 tumors. (62.5%) from 25 of the 40 and non-BCSCs from 29 of 31 express SSEA-3 (Chang WW. Et al., (2008) Proc Natl Acad Sci USA 105 : 11667-11672).

Danishefsky and Livingston previously reported that Globo H-KLH vaccine for various cancers (Gilewski T el al . (2001) Proc Natl Acad Sci 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 7-valent vaccine (containing GM2, Globo H, Lewis Y, Tn, STn, TF and Tn-MUC1 individually conjugated to KLH; Sabbatini PJ et get (2007) Clin Cancer Res 13: 4170-4177). However, in patients immunized with the 7-valent vaccine, antibody responses to only 5 of the 7 antigens were induced, except for the GM2 and Lewis Y antibodies. Globo H, which is expressed only at a very low level in normal secretory tissues and specifically expressed in tumor cells, is a desirable target for vaccine development, rather than antigens expressed everywhere like GM2. In these studies, ozonolysis of the Globo H aglycone followed by reductive amination using the KLH carrier protein resulted in about 150 carbohydrate units per protein (Ragupathi G, et al . (1997) Angew Chem lnt Ed 36: 125-128). Further improvements have increased the carbohydrate conjugation ratio to about 720: 1 by using the MMCCH linker (Wang SK, et al . (2008) Proc Natl Acad Sci USA 105: 3690-3695). However, it is difficult to characterize the glycoconjugate precisely. In addition, the synthetic vaccine in combination with the immunological Adjuvant QS-21 appears to induce IgM mainly in both prostate and metastatic breast cancer patients and induce a smaller amount of IgG antibody. In addition, the vaccine showed minimal toxicity with transient focal skin reactions at the vaccination site in Phase I trials (Gilewski T. el al . (2001) Proc Natl Acad Sci USA 98: 3270-3275; Ragupathi G, et al . (1997) Angew Chem lnt Ed. 36: 125-128; Slovin SF et al (1997) Proc Natl Acad Sci USA 96: 5710-5715). Weak flu-like symptoms observed in some patients will probably be associated with side effects of QS-21. Five vaccines containing five prostate and breast cancer-associated carbohydrate antigens Globo-H, GM2, STn, TF and Tn - conjugated to the maleimide-modified carrier protein KLH had a higher potency than the IgM in the ELISA assay Gt; IgG < / RTI > serum (Zhu J. et < RTI ID = al . (2009) J. Am . Chem . Soc . 131 (26): 9298-9303).

Accordingly, it is desirable to identify alternative carriers and adjuvants for increasing antibody response to Globo H, especially with high titers of IgG, and to improve vaccine efficacy with minimal side effects.

The present invention relates to a carbohydrate-based vaccine containing a Globo H (B cell epitope) chemically conjugated to an immunogenic carrier diphtheria toxin cross-reacting substance 197 (DT-CRM 197) (Th epitope) via a p-nitrophenyl linker . Synthetic vaccines in combination with galangal adjuvant induce IgG, IgG1 and IgM antibodies in breast cancer models and provide exceptional immunogenicity indicating delayed tumor formation in xenotransplantation studies. Glycan array analysis of the antigens induced by Globo H-DT and glycolipid C34 showed that the antibodies were not only Globo H, but also SSEA-3 (Gb5) and SSEA-4 Sialyl Gb5) glycans.

(A) a glycan consisting essentially of Globo H or an immunogenic fragment thereof and conjugated to a carrier protein via a linker; And (b) an adjuvant comprising a glycolipid capable of binding to a CD1d molecule on a dendritic cell, wherein the immunogenic composition is relative to an IgM isotype antibody Lt; RTI ID = 0.0 > IgG isotype < / RTI > antibody.

In some aspects, the carrier protein is diphtheria toxin cross-reacting substance 197 (DT-CRM 197). In some aspects, the linker is a p-nitrophenyl linker.

In some aspects, azuban is a synthetic analogue of alpha -galactosyl-ceramide (alpha -GalCer). In some embodiments, the adjuvant is C34 comprising the structure:

In some aspects, 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 is capable of inducing humoral and cellular immune responses.

In some aspects, the antibody produced by the immune response neutralizes the antigen expressed on cancer cells or cancer stem cells. In some embodiments, the antibody produced by the immune response neutralizes at least one of antigen Gb4, step-specific embryonic antigen-3 (SSEA-3) and step-specific embryonic antigen-4 (SSEA-4) . In some embodiments, the antibody that neutralizes at least one of antigen Gb4, step-specific embryonic antigen-3 (SSEA-3) and step-specific embryonic antigen-4 (SSEA-4) And relatively higher levels of IgG isotype antibodies.

The present invention relates to a cancer vaccine comprising an immunogenic composition capable of inducing an anti-cancer immune response in a subject. In some aspects, the cancer vaccine is selected from the group consisting of breast cancer, lung cancer, liver cancer, buccal cancer, stomach cancer, colon cancer, nasopharyngeal cancer, dermal cancer, kidney cancer, brain tumor, prostate cancer, ovarian cancer, cervical cancer, ) ≪ / RTI > and bladder cancer.

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

In some embodiments, the cancer vaccine comprises an antibody that neutralizes at least one of the antigen Globo H, Gb4, phase-specific embryonic antigen-3 (SSEA-3) and step- specific embryonic antigen-4 (SSEA-4) . In some aspects, the antigen is expressed on breast cancer stem cells.

The present invention relates to an immunogenic composition comprising (a) an immunogenic composition comprising a globin consisting essentially of Globo H or an immunogenic fragment thereof and a glycans conjugated with a carrier protein via a linker, and an adjuvant comprising a glycolipid capable of binding CD1d molecules on dendritic cells Administering the pharmaceutical composition to a subject in need thereof; And (b) inducing an immune response that induces a greater amount of an IgG isotype antibody relative to the IgM isotype antibody.

In some embodiments of the method, the linker is p - nitrophenol, the carrier protein is diphtheria toxin cross-reacting substance 197 (DT-CRM 197), the adjuvant is alpha -galactosyl- Lt; / RTI > In one embodiment, the adjuvant is C34.

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

In some embodiments of the method, the immune response is preferably at least one of antigen Globo H, Gb4, step-specific embryonic antigen-3 (SSEA-3) and step- specific embryonic antigen- Lt; RTI ID = 0.0 > IgG < / RTI > isotype antibody. In some aspects, at least one of the antigen Globo H, step-specific embryonic antigen-3 (SSEA-3) and step-specific embryonic antigen-4 (SSEA-4) is expressed on breast cancer stem cells. In some aspects, the Globo H antigen is expressed on epithelial cells of breast tumors.

(A) an adjuvant comprising glycans consisting predominantly of Globo H or an immunogenic fragment thereof and conjugated with a carrier protein through a linker, and a glue capable of binding CD1d molecules on dendritic cells, An immunogenic composition that elicits an immune response that induces a higher level of IgG isotype antibody relative to an IgM isotype antibody; And (b) a pharmaceutically acceptable excipient.

In some aspects, the cancer vaccine is a vaccine in which the linker is p-nitrophenol, the carrier protein is diphtheria toxin crossreactant 197 (DT-CRM 197), and the adjuvant is a synthesis of alpha -galactosyl-ceramide (alpha-GalCer) ≪ / RTI > In one embodiment, the adjuvant is C34.

In some aspects, the cancer vaccine is used to treat cancer, wherein more than one treatment with an effective amount of a cancer vaccine inhibits tumor growth. In some embodiments, administration of a cancer vaccine reduces the size of the tumor. In some embodiments, the cancer is selected from the group consisting of breast cancer, lung cancer, liver cancer, ball cancer, stomach cancer, colon cancer, nasopharyngeal cancer, skin cancer, kidney cancer, brain tumor, prostate cancer, ovarian cancer, cervical cancer,

(A) glycans consisting predominantly of Globo H-related glycans or immunogenic fragments thereof and conjugated to a carrier protein through a linker; And (b) an adjuvant comprising a glycolipid capable of binding to a CD1d molecule on a dendritic cell, wherein 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 leading to a relatively higher level of IgG isotype antibody relative to the IgM isotype antibody.

In some aspects of the immunogenic composition, the carrier protein is a diphtheria toxin cross-reacting substance 197 (DT-CRM 197), an adjuvant is a synthetic analog of? -Galactosyl-ceramide (? -GalCer), the linker is p- Phenyl linker. In one embodiment, the adjuvant is C34.

The present invention is based on the finding that Globo H conjugated to diphtheria toxin cross-reacting substance 197 (DT-CRM 197) carrier protein via a p-nitrophenyl linker; And an adjuvant comprising a glycolipid capable of binding to CD1d molecules on dendritic cells. In some embodiments of the therapeutic agent, the adjuvant is C34.

The present invention is directed to a method for the treatment and / or prophylaxis of SSEA-3 conjugated with diphtheria toxin cross-reacting substance 197 (DT-CRM 197) carrier protein via a p-nitrophenyl linker; And an adjuvant comprising a glycolipid C34 capable of binding to CD1d molecules on dendritic cells.

The present invention relates to a therapeutic agent for breast cancer stem cells comprising SSEA-4 conjugated with a diphtheria toxin cross-reacting substance 197 (DT-CRM 197) carrier protein via a p-nitrophenyl linker. In some embodiments, the therapeutic agent further comprises an adjuvant comprising a glycolipid capable of binding to a CD1d molecule on a dendritic cell.

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

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the detailed description of the present invention and are included to further demonstrate certain aspects of the present disclosure and, together with the detailed description of specific embodiments presented herein, Can be further understood with reference to FIG. The patent or application file contains at least one drawing made in color. A copy of this patent or patent application publication with colored drawing (s) will be provided by the relevant patent office upon request and payment of the required fee.

Figure 1 shows the structure of Globo H and truncated derivatives;
Figures 2A-2C show the binding specificity of the monoclonal antibodies VK9 and Mbr1 (for Globo H) and anti-SSEA-3, respectively;
Figures 3A and 3B show the serological responses of various Globo H conjugates and vaccinated mice with [alpha] -GalCer, with 3 C57BL / 6 mouse groups subcutaneously, with 2 [mu] g of glycolipid or Without this, 1 μg of synthetic glyco conjugate was vaccinated. Mouse serum was diluted to 1: 60 and 1: 240 for IgM (Figure 3A) and IgG (Figure 3B) antibody analysis, respectively. Cy3-anti-mouse IgG or IgM secondary antibody was used for fluorescence detection under 532 nm, PMT 500. Data were expressed as the mean fluorescence intensity +/- SEM of 3 mice;
4 is a view showing the structure of? -GalCer and an analogue thereof;
Figure 5 shows the IgM levels of mice vaccinated with the Globo H conjugate and the alpha -GalCer derivative. Mouse sera were collected and analyzed after the second and third vaccinations, as shown in the figure. Cy3 secondary anti-mouse IgM was used for detection under 532 nm, PMT 400. Results were expressed as the mean fluorescence intensity +/- SEM of 3 mice;
Figure 6 shows the sophisticated specificity of mouse polyclonal antibodies (anti-Globo H, anti-Gb5, anti-SSEA-4 and anti-Gb4) after vaccination, in which mouse serum was incubated with 2 ug of adjuvant (Female, Balb / c, intramuscularly) after three weeks of the third vaccination with 1.6 [mu] g of GH-DT. The IgG titers were analyzed by a glycan microarray and defined as the highest dilution that yielded an MFI of greater than 1000 (10-fold over background) under PMT 400. Each point represents an individual mouse titer;
Figure 7 shows IgM to IgG antibody titers of Globo H-DT with different adjuvants;
Figure 8 shows an evaluation of the adjuvant activity with the GH-KLH vaccine; Female Balb / c mice were injected intramuscularly with 1.6 ug of GH-KLH and 2 ug of the designated adjuvant and blood was collected every 2 weeks after vaccination. Serum was diluted and introduced into microarray analysis;
Figure 9 shows the antibody isotype profile after immunization, in which the mice were vaccinated as described. Serum (1: 60 dilution) was introduced into the microarray for antibody subclassification analysis (532 nm, PMT 300). Data were expressed as the mean fluorescence ± SEM of 3 mice;
Figure 10 shows antibody titers of IgM to IgG induced by SSEA-3-DT or SSEA-4-DT with different classes of glycolipid adjuvants;
Figure 11 shows the structure of 24 glycans on the cell surface;
Figures 12a-12c illustrate a cross-reactivity study of IgGs induced by different vaccines, with Figure 12a: anti-Globo H IgG induced by Globo H-DT with Cl adjuvant; Figure 12b: anti-Gb5 IgG induced by Gb5-DT with C1 < RTI ID = 0.0 > 12C: anti-SSEA-4 IgG induced by SSEA-4-DT with C1 adjuvant;
Figure 13 were injected subcutaneously to a mouse model transplanted with two kinds of the diagram, 2 × 10 ⁵ 4T1 mouse metastatic breast tumor cells prepared in a PBS sterilized and vaccination of Balb / c mice. Mouse tumor size was measured with a Vernier caliper and defined as (length × height × width) / 2 (㎣);
Figure 14 is a diagrammatic representation of the synthesis of a Globo H half ester and a glyco conjugate;
FIG. 15 is a flow cytometric analysis of expression of SSEA-4 in primary breast cancer stem cells. Expression of SSEA-4 on the surface of BCSC and non-BCSC was evaluated by 4-color immunofluorescence staining followed by flow cytometry. BCSCs are defined as CD45 ^- / CD24 ^- / CD44 ⁺ cells, as shown in the left panel, and non ^- BCSCs are defined as different populations of CD45 ^- cells. Expression of the antigen of interest on BCSC and non-BCSC was shown in the middle and right panel, respectively. The dotted line represents the isotype control group, the number represents the percentage of benign cells;
Figure 16 shows the limited expression of SSEA-4 in normal tissues, in which immunohistochemical staining of normal tissue arrays was used to test the expression of SSEA-4 in breast, small intestine and rectum. Positive staining for SSEA-4 was restricted to the apical surface of epithelial cells.

The present invention relates to the surprising discovery that DT-CRM 197 is a promising carrier protein for Globo H and SSEA-4 due to its high immunogenicity as well as the reason that DT-CRM 197 has been widely used for human vaccination against diphtheria for decades. Above all, it was approved by the FDA for various glycon conjugate vaccines. Diphtheria toxin cross-reacting substance 197 (DT-CRM 197) shares the immunological properties of natural molecules and is often associated with heparin-binding epithelial growth factor (HB-EGF), a specific cell- membrane receptor for DT over- (G52E), which shares the binding capacity of the T-cell to the T-cell receptor (Buzzi S. et al ., Cancer Immunology, Immunotherapy (2004), 53 (11): 1041-1048).

By using C34 as an adjuvant, both GH-DT and SSEA-4-DT showed the most effective immune response to further induce IgG against the tumor antigen than the IgM antibody. GH-DT in combination with C34 induced antibodies that neutralize not only Globo H, but also SSEA-3 (Gb5) and SSEA-4 (both specific for breast cancer cells and cancer stem cells).

In addition, the disclosed glycan microarrays provide a strong foothold for antibody specificity testing and are useful for identifying patients for vaccine testing and monitoring their immune response after immunization.

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

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications and patents specifically mentioned herein are incorporated herein by reference for all purposes, including describing and disclosing the chemicals, cell lines, vectors, animals, instruments, statistical analyzes and methods reported in the published literature, which may be used in connection with the present invention For example. All references listed in this specification should be taken to suggest a level of skill in the industry. In this specification, the present invention should not be construed as an admission that such disclosure is not allowed to precede the preceding invention.

Before describing the materials and methods of the present invention, it is to be understood that the invention is not limited to the particular methods, protocols, materials and reagents described, but that they may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, but is only limited by the scope of the appended claims.

Justice

The singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. In addition, the terms " a "or" an ", "one or more" It is also to be understood that the terms " comprising ", "including ", and" having "

The practice of the present invention will employ the techniques of conventional molecular biology, microbiology, recombinant DNA and immunology within the skill of the art, unless otherwise indicated. These techniques are fully described in the literature. For example, the following references [Molecular Cloning A Laboratory Manual, 2nd Ed., Ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Culture of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. Eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, by 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 lipophilic (lipophilic) molecule that participates in the cellular signaling pathway.

The term "glycolipid " as used herein refers to a carbohydrate-adherent lipid that serves as a marker for cell recognition.

The terms "alpha-galactosylceramide" and "alpha-GalCer ", as used herein, refer to glycolipids that stimulate natural killer T cells to produce both T helper (TH) 1 and TH2 cytokines. The glycolipid derivative C34 used herein has the following structure:

The? -GalCer analogs of the present disclosure can be prepared by reacting? -GalCer analogs of bacterial origin (Group I: C2, C3 and C14), sulfonated? -GalCer analogs (Group II: C4, C5 and C9) C12 and C8-6) and phytosphingosine-cleaved alpha -GalCer analogs (Group IV: C12, C13, C18- And C 17). The structures of C34 and other alpha-galactosyl ceramide analogs and their use as adjuvants are described in detail in PCT Patent Application No. PCT / US2008 / 060275 filed on April 14, 2008.

A synthetic α-GalCer analog, including C34, can form complexes with CD1d molecules. Synthetic a-GalCer analogs can be recognized by NKT T-cell receptors. Synthetic a-GalCer analogs can elicit T _H 1-, T _H 2-, or THl-and TH2-type reactions. α-GalCer analogs can activate NKT in vitro. α-GalCer analogs can activate NKT in vivo.

The term "glycan" as used herein refers to polysaccharides or oligosaccharides. Glycans are also used herein to refer to carbohydrate moieties of glycoconjugates such as glycoproteins, glycolipids, glycopeptides, glycoproteomes, peptidoglycans, lipid polysaccharides, or proteoglycans. Glycans usually consist solely of O-glycosidic bonds between monosaccharides. For example, cellulose is a glycan (or more specifically, glucan) composed of? -1,4-linked D-glucose and chitin is a glycine composed of? -1,4-linked N-acetyl-D-glucosamine It is Khan. Glycans may be homo- or heteropolymers of monosaccharide residues and may be linear or branched. Glycans attached to proteins such as in glycoproteins and proteoglycans can be observed. These are generally observed on the outer surface of the cells. O- and N-linked glycans are very common in eukaryotes, but are not common, they can also be observed in prokaryotes. N-linked glycans attached to the R-group nitrogen (N) of asparagine are observed in the sequon. Sequon is an Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid except proline.

As used herein, the term "glycoprotein" refers to a protein that is covalently modified to glycan (s). There are four types of glycoproteins: 1) N-linked glycoproteins, 2) O-linked glycoproteins (mucin), 3) glucosaminoglycan (GAG, also referred to as proteoglycans), 4 GPI-anchored. Most glycoproteins have structural micro-heterogeneity (multiple different glycan structures attached within the same glycosylation site) and structural macro-heterogeneity (multiple glycan attachment sites and types) .

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

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

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

The term "immunotherapy ", as used herein, refers to a number of therapeutic strategies based on the concept of modulating the immune system to achieve a preventive and / or therapeutic goal.

The term "CD1d" as used herein refers to a member of the CD1 (differentiation cluster 1) family of glycoproteins expressed on the surface of various human antigen-presenting cells. CD1d presenting lipid antigens activates natural killer T cells. CD1d has a deep antigen-binding groove to which the glycolytic antigen binds. CD1d molecules expressed on dendritic cells can bind and suggest glycolipids, including alpha-GalCer analogs, such as C34.

The term "adaptive immune system " as used herein refers to highly specialized systemic cells and processes that eliminate pathogenic challenge. Cells in the adaptive immune system are a type of white blood cell called lymphocytes. B cells and T cells are the major types of lymphocytes.

As used herein, the terms "T cell" and "Ts " refer to a leukocyte group known as a lymphocyte that plays a pivotal role in cell-mediated immunity. T cells can be distinguished from other lymphocyte types such as B cells and NK by the presence of special receptors on their cell surface, called T cell receptors (TCRs). A subset of several different T cells, each with a unique function, has been described. Helper T (T _H ) cells are "mediators" of the adaptive immune system. Once activated, they are rapidly cleaved and secrete small proteins called cytokines that regulate or "help" the immune response. Dependent on the accepted cytokine signal, these cells differentiate into one of T _H 1, T _H 2, T _H 17, or another subset, which secrete different cytokines.

The term "antigen-presenting cell" (APC), as used herein, refers to a cell that expresses a foreign antigen that complexes with its major histocompatibility complex (MHC) on its surface. T-cells can recognize this complex using their TCR. APC is divided into two categories: professional and non-professional. Dendritic cells (DCs) fall into the professional category and can present antigens to T cells in the context of CD1. In an exemplary implementation, the DC used in the methods of the present disclosure may be any of a subset of DCs that in one embodiment is differentiated from a lymphocyte, or in another embodiment, from a myeloid bone marrow progenitor cell.

The term "naive cell ", as used herein, refers to undifferentiated immune system cells such as CD4 T-cells that have not yet been specialized to recognize a particular pathogen.

As used herein, the terms "natural killer cell" and "NK" refer to the classification of lymphocytes activated by interferon that is responsible for innate host defense against viruses and other intracellular pathogens.

The term " natural killer T cell "(NKT) as used herein refers to a subset of T cells that share a characteristic / receptor with both conventional T and NK. Many of these cells recognize non-polymorphic CD1d molecules that are antigen presenting molecules that bind to self-and exogenous lipids and glycolipids. The TCR of NKT is capable of recognizing (chaperoned) glycolytic antigens presented by CD1d molecules. The primary response of NKT is the rapid secretion of cytokines, including IL-4, IFN-y and IL-10, after stimulation, thus affecting a variety of immune responses and pathogenesis. NKT can be homogeneous population or heterogeneous recruitment single. In one exemplary embodiment, the population includes, for example, CD1d-reactive non-invariant T cells expressing various TCRs and also capable of producing large amounts of IL-4 and IFN- Quot; non-invariant NKT ", which may include liver T cell populations. The best known subset of CD1d-dependent NKTs express the constant TCR-alpha (TCR-alpha) chain. These are referred to as Form I or Invariant NKT (iNKT). These cells are conserved between human (Vα24i NKT) and mouse (Vα14i NKT) and are involved in many immunological processes.

The term "cytokine ", as used herein, refers to a protein that modulates the intensity and duration of an immune response by affecting the process of immune cell differentiation, including changes in gene expression that typically lead to precursor cells becoming distinct specialized cell types Lt; RTI ID = 0.0 > secreted < / RTI > Cytokines have been variously named as lymphokines, interleukins and chemokines, based on their putative function, secretory cells or activity targets. For example, some common interleukins include IL-12, IL-18, IL-2, IFN-y, TNF, IL-4, IL-10, IL-13, IL-21 and TGF- But is not limited thereto.

The term "chemokine" as used herein refers to any of a variety of small chemotactic cytokines released into the site of infection, providing a means for activation and mobilization of lymphocytes. Chemokines attract white blood cells to the site of infection. Chemokines have conserved cysteine residues that cause them to be assigned to four groups. The group, along with the typical chemokines, is a member of the CC chemokine family (RANTES, MCP-1, MIP-1α and MIP-1β), CXC chemokine (IL-8), C chemokine (Lymphotactin) and CXXXC chemokine (Fractalkine ))to be.

As used herein, the term " T _H 2-type reaction "refers to a cytokine expression pattern that allows certain types of cytokines, interferons, and chemokines to be produced. Typical T _H 2 cytokines include, but are not limited to IL-4, IL-5, IL-6 and IL-10.

As used herein, the term " T _H 1-type reaction "refers to a cytokine expression pattern that allows certain types of cytokines, interferons, and chemokines to be produced. Typical T _H 1 cytokines include, but are not limited to IL-2, IFN-y, GMCSF and TNF-β.

As used herein, "T _H 1 ubiquity (biased)" refers to an immune response is the production of T _H 1 cytokines and / or chemokines increased to a greater extent than the T _H 2 cytokines, and / or creation of a chemokine .

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

The term "vaccine" as used herein includes allergy-induced organisms (structurally or weakly) as below or components of these organisms, such as proteins, peptides or polysaccharides, Refers to an agent used to impart immunity. Vaccine preparations can be natural, synthetic, or derived from recombinant DNA technology.

The term " immunological adjuvant " as used herein refers to a substance that is conjugated with an immunogen and that enhances or alters the immune response to the immunogen. The? -GalCer analogs of the present disclosure are used as immunological adjuvants that alter or increase the effect of the vaccine by stimulating the immune system of the patient to which the vaccine is administered to react more vigorously to the vaccine. In an exemplary implementation, analog C34 is used as an adjuvant.

The term "alum adjuvant " as used herein refers to an aluminum salt having immunosuppressant activity. This preparation absorbs and precipitates protein antigens in solution; The resulting precipitate enhances vaccine immunogenicity by promoting slow release of the antigen from the vaccine depot formed at the inoculation site.

The term " anti-tumor immunotherapeutic " as used herein refers to an antibody produced by a vaccine of this disclosure that inhibits, reduces and / or eliminates tumors.

The term " antigen specific ", as used herein, refers to the property of a cell population that causes the supply of a particular antigen, or fragment of an antigen, to cause a particular cell proliferation.

The term "flow cytometry" or "FACS ", as used herein, refers to techniques for testing the physical and chemical properties of suspended particles or cells in a fluid stream through optical and electronic detection devices.

The amino acid residues in the peptide will be abbreviated as follows: phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic acid is Asp or D; Glutamic acid is Glu or E; Cysteine is Cys or C; Tryptophan may be Trp or W; Arginine is Arg or R; Glycine refers to Gly or G. amino acids for further explanation, see Proteins: Structure and Molecular Properties by Creighton, T. E., W. H. Freeman & Co., New York 1983.

The compositions disclosed herein may be included in a pharmaceutical or functional food composition in conjunction with additional active agents, carriers (or carriers), vehicles, excipients, or adjuvants that can be ascertained by one skilled in the art upon reading this disclosure .

The pharmaceutical or functional food composition preferably comprises at least one pharmaceutically acceptable carrier. In such pharmaceutical compositions, the compositions disclosed herein form "active compounds" also referred to as "active agents ". The term " pharmaceutically acceptable carrier "as used herein includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like which are compatible with pharmaceutical administration. Supplementary active compounds may also be incorporated into the compositions. The pharmaceutical composition is formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal and rectal administration. Solutions or suspensions used in parenteral, intradermal, or subcutaneous applications may include the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other Synthetic solvent; Antimicrobial agents such as benzyl alcohol or methyl paraben; Antioxidants such as ascorbic acid or sodium bisulfite; Chelating agents such as ethylenediaminetetraacetic acid; Buffers, such as acetates, citrates, or phosphates, and agents for controlling isotonicity, such as sodium chloride or dextrose. The pH can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable needles, or in multivalent vials made of glass or plastic.

As used herein, the term " animal " includes humans and non-human primates such as gorillas, macaques, and marmots, livestock such as sheep, cows, horses, donkeys and pigs, companion animals such as dogs and cats, Such as a mouse, a rabbit, a rat, a guinea pig, a hamster, caught wild animals (such as fox, deer) and any other organism that may benefit from the preparations of the present disclosure. There is no limit to the type of animal that may benefit from the formulations disclosed herein. A subject may be referred to as a patient, an individual, an animal, a host, or a recipient, whether human or non-human organism is recognized.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (from BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to be readily present in the syringe. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, a polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be desirable to include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable composition may be caused by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound in an appropriate solvent with one or a combination of the above listed ingredients, if necessary, followed by sterile filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other ingredients required from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the process comprises vacuum drying and freeze-drying, whereby a powder of the active ingredient and any desired additional ingredients are obtained from the previously sterile-filtered solution.

Oral compositions generally include an inert diluent or an edible carrier. For oral therapeutic administration purposes, the active compounds may be incorporated with excipients and used in the form of tablets, troches, or capsules, such as gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as an oral cleanser. A pharmaceutically compatible binder, or an adjuvant material may be included as part of the composition. Tablets, pills, capsules, ointments, etc. may contain the following components or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth gum or gelatin; Excipients such as starch or lactose; Disintegrants such as alginic acid, Primogel, or corn starch; Lubricants such as magnesium stearate or sterotes; Polishes such as colloidal silicon dioxide; Sweeteners such as sucrose or saccharin; Or a flavoring agent such as peppermint, methyl salacillate or orange flavor.

To be administered by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer.

Systemic administration may also be transmucosal or transdermal. For transmucosal or transdermal administration, a suitable penetrator is used in the formulation for the barrier to be permeated. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of a propellant spray or suppository. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams as is generally known in the art. The compounds may also be prepared in the form of suppositories (for example using conventional suppository bases such as cocoa butter and other glycerides) or rectal delivery for rectal delivery.

According to the practice, the active compound is prepared with a carrier that will protect the compound from rapid removal from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods for making such formulations will be known to those skilled in the art. Materials may be obtained from Alza Corporation and Nova Pharmaceuticals, Inc. In addition, liposomal suspensions (including liposomes targeting infected cells with monoclonal antibodies to cell-specific antigens) can be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, according to the method described in U.S. Patent No. 4,522,811, which is incorporated herein by reference.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of administration. Dosage unit form as used herein refers to physically discrete units suitable as a uniform dosage for the subject to be treated; Each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier.

Toxicity and therapeutic efficacy of these compounds can be assessed by standard pharmaceuticals in cell cultures or laboratory animals to determine, for example, the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅₀ (the therapeutically effective dose in 50% of the population) Can be determined by the process. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the LD ₅₀ / ED ₅₀ ratio. Compounds that exhibit a high therapeutic index are preferred. Compounds that exhibit toxic side effects can be used, but care must be taken to devise a delivery system that targets such compounds at the site of infection to minimize side effects by minimizing possible damage to non-infected cells.

Data from cell culture assays and animal studies can be used to determine the dosage range for use in humans. The dose of such compounds is preferably within a circulating concentration range that includes the ED ₅₀ with little or no toxicity. The dose may vary within this range depending on the dosage form employed and the route of administration utilized. For compounds used in the methods of the invention, therapeutically effective doses can be estimated initially from cell culture assays. The dose can be formulated in an animal model that achieves a circulating plasma concentration range that includes an IC ₅₀ (i. E., The concentration of the test compound that achieves half the maximum inhibition of symptoms) as measured in cell culture. This information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount (i.e., an effective dosage) of the active compound is in the range of from about 0.001 to 100 g / kg per kilogram of body weight, or in another range that is apparent and understandable to those skilled in the art without undue experimentation . Those skilled in the art will recognize that certain factors, including the severity of the disease or symptom, the prior treatment, the general health or age of the subject, and other conditions present, may influence the dosage and timing necessary to effectively treat the subject will be.

According to another aspect, a kit of one or more parts may be conceived by a person of ordinary skill in the art, and may comprise a kit of parts that implement at least one of the methods described herein, a kit of parts comprising two or more compositions, ≪ RTI ID = 0.0 > and / or < / RTI > the effective amount of the composition disclosed herein alone or in combination.

The kits may also include active agents, identifiers of biological events, or other compounds that can be ascertainable by one of ordinary skill in the art upon reading this disclosure. The kit may also comprise at least one composition or cell line comprising an effective amount of a composition as disclosed herein. The compositions and cell lines of the kit of parts are used to carry out at least one of the methods disclosed herein according to procedures that are identifiable by those skilled in the art.

The term "polypeptide" as used herein refers to any multimer or polymer of amino acid residues. A polypeptide may consist of two or more polypeptide chains. Polypeptides include proteins, peptides, and oligopeptides. The polypeptide may be linear or branched. The polypeptide may comprise a modified amino acid residue, an amino acid analog or a non-naturally occurring amino acid residue and may be intervened by a non-amino acid residue. Whether natural or by intervention, such as formation of disulfide bonds, glycosylation, lipidation, methylation, acetylation, phosphorylation, or by manipulation, such as conjugation with a labeling component , Modified amino acid polymers are included within the definition.

As used herein, the term "specifically binding" refers to the interaction between a binding pair (e.g., an antibody and an antigen). In various examples, specific binding may be achieved by affinity constants of about 10 ^-6 mol / liter, about 10 ^-7 mol / liter, or about 10 ^-8 mol / liter or less.

The cancer vaccine of the present invention

One embodiment of the present invention is directed to a method of screening for an effective amount of an immunogenic composition comprising Globo H or a fragment thereof (e.g., one of step-specific embryonic antigen-3 (SSEA-3, also known as Gb5), or SSEA- To a subject in need of treatment for cancer. Examples of target cancer types include breast cancer (including 1-4), lung cancer (such as small cell lung cancer), liver cancer (such as hepatocellular carcinoma), oral cancer, gastric cancer (including T1-T4), colon cancer, , But are not limited to, renal cancer, brain tumors (such as astrocytomas, polymorphic glioblastomas and meningiomas), prostate cancer, ovarian cancer, cervical cancer, bladder cancer and endometrium, rhabdomyosarcoma, osteosarcoma, leiomyosarcoma and gastrointestinal stromal tumors .

Cancers classified as areas include the oral and pharyngeal cancers (lips, tongue, salivary glands, mouth bottoms, gums and other mouths, nasopharynx, one way, the following structures, hypopharynx, other oral / pharynx); Cancer of the digestive system (esophagus, stomach, small intestine, colon and rectum, rectum, anal and anorectal rectum, liver, bile duct, gallbladder, other bile duct, pancreas, retroperitoneum, peritoneum, mesentery and mesentery; Cancer of the respiratory system (nasal cavity, middle ear, and sinus cavity; larynx; lung and bronchial; pleura; airway, mediastinum and other respiratory systems); Cancer of mesothelioma; Bones and joints; Soft tissues including heart; Skin cancer, including melanoma and other non-epithelial skin cancer; Kaposi ' s sarcoma and breast cancer; Cancer of the female reproductive system (uterine cervix; other unspecified uterus; ovary; vagina; vulva; and other female reproductive system); Cancer of the male reproductive system (prostate; testes; penis; and other male reproductive organs); Urinary cancer (bladder; kidney and renal; ureter; and other urinary); Cancer of the eye and orbit; Cancer of the brain and nervous system (brain; and other nervous systems); Cancers of the endocrine system (other endocrine including thyroid and thymus); Lymphoma (Hodgkin's disease and non-Hodgkin's lymphoma), multiple myeloma and leukemia (lymphoid leukemia, myeloid leukemia, monocytic leukemia, and other leukemia).

Other cancers classified by histological type, which may be suitable targets for the cancer vaccine according to the invention, include malignant neoplasms; Other unspecified carcinomas; Other undifferentiated carcinomas not specified; Giant and spindle cell carcinoma; Other small cell carcinomas not specified; Other papillary carcinomas not specified; Other squamous cell carcinomas not specified; Lymphoid carcinoma; Other basal cell carcinomas not specified; Hair cell carcinoma; Other non-specific transitional cell carcinomas; Nipple transitional cell carcinoma; Other unexplained adenocarcinomas; Malignant gastrin; Cholangiocarcinoma; Other unspecified hepatocellular carcinoma; Complex hepatocellular carcinoma and cholangiocarcinoma; Adolescent adenocarcinoma; Adenocarcinoma; Adenocarcinoma in adenomatous polyposis; Adenocarcinoma, familial polyposis; Other solid carcinomas not specified; Malignant carcinoid tumors; Bronchoalveolar carcinoma; Other papillary adenocarcinomas not specified; Cyanotic carcinoma; Eosinophilic carcinoma; Oxyphilic adenocarcinoma; Basophil carcinoma; Other clear cell adenocarcinomas not specified; Granulocytic carcinoma; Other unspecified sporangial carcinomas; Papillary and follicular adenocarcinomas; Nonencapsulating sclerosing carcinoma; Adrenocortical carcinoma; Endometrial carcinoma (endometrial carcinoma); Skin adnexal carcinoma; Apocrine adenocarcinoma; Adenocarcinoma; Earwax adenocarcinoma; Mucoepidermoid carcinoma; Other unspecified adenocarcinoma; Other papillary adenocarcinomas not specified; Papillary serous adenocarcinoma; Other unspecified mucocellular adenocarcinomas; Mucinous adenocarcinoma; Ring cell carcinoma; Invasive ductal carcinoma; Other non-specific carcinomas; Lobular carcinoma; Inflammatory carcinoma; Weeping Paddle Jets; Squamous cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Malignant thymoma; Malignant ovarian stromal tumor; Malignant ovarian carcinoma; Malignant granulosa cell tumor; Malignant male blastoma; Sertoli cell carcinoma; Malignant lady cell tumor; Malignant lipo - cell tumor; Malignant cranial necrosis; Malignant extramural extramedullary nodule; Chromium-rich cell tumor; Dwarf vascular sarcoma (Glomangiosarcoma); Other malignant melanoma not specified; Colorless melanoma; Shallow diffuse melanoma; Malignant melanoma in giant pigmentary nevus; Epithelial cell melanoma; Malignant blue birth; Other breeding not specified; Other non-specific fibrosarcoma; Malignant fibrous histiocytoma; Mucosal sarcoma; Other non-specific liposarcoma; Other leiomyosarcoma not specified; Other rhabdomyosarcoma not specified; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Other non-specific epileptic sarcoma; Other malignant mixed tumors not specified; Müllerian mixed tumors; Nephroblastoma; Hepatoblastoma; Other non-specific carcinosarcoma; Malignant mesenchymal species; Malignant Brenner tumor; Malignant follicular tumor; Other non-specific lubricating sarcoma; Malignant mesothelioma; Undifferentiated cell tumor; Other embryo carcinomas not specified; Other malignant teratomas not specified; Malignant ovarian thyroid gland; Chorionic villus carcinoma; Malignant mesothelioma; Angiosarcoma; Malignant vascular endothelial; Kaposi's sarcoma; Malignant pericytes; Lymphatic sarcoma; Other unspecified osteosarcoma; Juxtacortical osteosarcoma; Other unspecified chondrosarcoma; Malignant chondroblastoma; Mesenchymal osteosarcoma; Giant cell tumor of bone; Yingong breeding; Malignant giant tumor; Ameloblastic odontosarcoma; Malignant enameloblastoma; Encephaloblast fibrosarcoma; Malignant pineal species; Chordoma; Malignant glioma; Other unspecified ventricular cell tumors; Other astrocytomas not specified; Protoplasmic astrocytoma; Fibrillar astrocytoma; Aortoblastoma; Other unspecified glioblastomas; Other rarely prominent glial cell types not specified; Rare dendritic glioblastoma; Unwanted neuroectodermal tumors; Other cerebellar sarcoma not specified; Glioblastoma; Other non-specific neuroblastoma; Other non-specific retinoblastoma; Posterior neurogenic tumor; Malignant meningioma; Nerve fiber sarcoma; Malignant neuroendocrine; Malignant granular cell tumor; Other malignant lymphoma not specified; Other non-specific Hodgkin's disease; Hodgkin's disease; Other adnexal granulomas not specified; Malignant lymphoma lymphoma; Large cell malignant lymphoma; Other malignant vesicular lymphoma not specified; Bacterial sarcoma; Other specified non-Hodgkin's lymphoma; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferative intestinal diseases; Other unspecified leukemia; Other non-specific lymphoid leukemia; Plasma cell leukemia; Leukemia; Lymphoma sarcoma cell leukemia; Other non-specific myeloid leukemia; Basophil leukemia; Eosinophil leukemia; Other unspecified monocytic leukemia; Mast cell leukemia; Leukemia; Myeloid sarcoma; ≪ / RTI > and hairy cell leukemia.

As used herein, the term "treating" is intended to refer to the treatment of cancer, signs of cancer, or signs of cancer, to a person suffering from cancer, signs of cancer or cancer, Quot; refers to the application or administration of a composition comprising one or more active agents, for the purpose of improving, ameliorating, or affecting the composition. As used herein, "effective amount" refers to the amount of each active agent required to impart a therapeutic effect to a subject, alone or in combination with one or more other active agents. Effective amounts will vary with the route of administration, the use of excipients and co-use with other active agents, as will be appreciated by those skilled in the art.

The immunogenic composition used in the methods described above may contain Globo H or a fragment thereof, glycan (i.e., a molecule containing a sugar moiety) and an adjuvant. Globo H is a glycans containing a six saccharide epitope (Fucα1 → 2 Galβ1 → 3 GalNAcβ1 → 3 Galα1 → 4 Galβ1 → 4 Glc) and optionally a non-sugar moiety. Its fragments are glycan containing fragments of the six saccharide epitopes and, if applicable, non-sugar moieties. These oligosaccharides may be prepared by conventional methods (as described [Huang et al, Proc Natl Acad Scl USA 103:..... 15-20 (2006)] reference). If desired, they can be attached to the non-sugar moiety.

U.S. Patent Application No. 12 / 485,546, filed by the present applicant, (1) SSEA-3, an immediate precursor of Globo H, is expressed at high levels in breast cancer stem cells and may be a suitable target for breast cancer treatment , And (2) an unexpected discovery that? -Galactosyl-ceramide (? -GalCer) is an effective adjuvant that promotes the production of anti-Globo H and anti-SSEA-3 antibodies.

U.S. Patent Application No. 12 / 485,546 features an immunogenic composition comprising Globo H or a fragment thereof (e.g., SSEA-3) and an adjuvant (e.g.,? -GalCer). Globo H or a fragment thereof can be conjugated to keyhole-lipopememycin (KLH). When administered to a subject (e. G., A human), the immunogenic composition can elicit an immune response (e. G., Antibody production) targeting Globo H or a fragment thereof to treat cancer (e. G., Breast cancer, prostate cancer, Ovarian cancer and lung cancer).

U.S. Patent Application No. 12 / 485,546 discloses the administration of the immunological composition described above to a non-human mammal (e.g., mouse, rabbit, goat, sheep or horse) and isolation from a mammalian antibody that binds Globo H or a fragment thereof To a method for producing antibodies specific for Globo H or fragments thereof.

Globo H or other glycans described in this disclosure is conjugated to a protein carrier, such as DT-CRM 197. They are then mixed, via conventional means, with an adjuvant such as C34 and optionally with a pharmaceutically acceptable carrier (e.g., phosphate buffered saline or bicarbonate solution) to form an immunogenic composition (e.g., a vaccine) can do. See, for example, U.S. Patent Nos. 4,601,903; 4,599,231; 4,599,230; And 4,596,792. The composition may be prepared as an injectable material, a liquid, or an emulsion, and the carrier is selected according to the standard pharmaceutical practice as well as the mode and route of administration. Suitable pharmaceutical carriers and diluents, and pharmaceutical needing substances for their use, are described in Remington ' s Pharmaceutical Sciences. The immunogenic composition preferably contains [alpha] -GalCer as an adjuvant. Other examples of adjuvants include cholera toxin, Escherichia coli heat-labile enterotoxin (LT), liposomes, immunostimulatory complex (ISCOM) or immunostimulatory sequences (immunostimulatory sequences oligodeoxynucleotide (ISS-ODN). The composition may also contain a polymer that facilitates in vivo delivery. Audran R. et al. Vaccine 21: 1250-5, 2003]; And Denis-Mize et al. Cell Immunol., 225: 12-20, 2003). Optionally, a small amount of auxiliary material, such as a wetting or emulsifying agent, or a pH buffering agent, is added to enhance the ability of the composition to elicit an immune response to the sugar moiety of Globo H or a fragment thereof ≪ / RTI > The immunogenic compositions described herein may be administered parenterally (e.g., by intravenous, subcutaneous, or intramuscular injection). Alternatively, other modes of administration, including suppositories and oral formulations, may be desirable. For suppositories, the binder and carrier may comprise, for example, polyalkylene glycols or triglycerides. Oral formulations may include, for example, commonly used incipients such as pharmaceutical grades of saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of the immunogenic compositions described herein.

The immunogenic compositions described herein may be administered parenterally (e.g., by intravenous, subcutaneous, or intramuscular injection). Alternatively, other modes of administration, including suppositories and oral formulations, may be desirable. For suppositories, the binder and carrier may comprise, for example, polyalkylene glycols or triglycerides. Oral preparations may include, for example, commonly used inhaledients such as pharmaceutical grades of saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of the immunogenic compositions described herein.

The immunogenic composition is administered in a therapeutically effective, protective, immunogenic amount in a manner compatible with the dosage formulation. The amount administered will depend on the subject being treated, including, for example, the ability of the individual immune system to synthesize antibodies and, if necessary, the ability to produce a cell-mediated immune response. The exact amount of active ingredient required for administration will depend on the judgment of the practitioner. However, suitable dosage ranges can be readily determined by those skilled in the art. Suitable treatments for initial administration and booster doses are also variable, but may include initial administration followed by subsequent administration. The dose of the vaccine may also vary depending on the route of administration and will vary with the size of the host.

The immunogenic compositions of the present invention may also be used to produce antibodies in animals for the production of antibodies that can be used in both cancer therapy and diagnosis. Methods for producing monoclonal and polyclonal antibodies and fragments thereof in animals (e. G., Mouse, rabbit, goat, sheep or horse) are well known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The term "antibody" refers not only to the original immunoglobulin molecule but also to fragments thereof such as Fab, F (ab ') ₂ , Fv, scFv (single chain antibody) and dAb (domain antibody; Ward, (1989) Nature, 341, 544).

Globo  H- DT -CRM 197 and related vaccines

Globo H (1) and its fragments (2-10) were synthesized by the methods described herein. For protein conjugation, the purified Globo H half ester 12 was incubated with the individual carrier proteins as shown in Fig.

The Globo H-protein conjugate was characterized by MALDI-TOF analysis to determine the number of Globo H molecules on each carrier protein. The Globo H average incorporation numbers are listed in Table 1.

Globo  H-incorporated MALDI - TOF  analysis Reference Protein molecular weight After saccharification ^a Average incorporation (n) Carbohydrate percent GH-BSA 66431 66449 76029 8 14.4% GH-DT 58472 58326 62138 2 to 4 6.8% GH-TT 150682 155609 162902 6 4.5% GH-KLH ^* 8.6 × 10 ⁶ ~ 700 14.7% GH-Bamboo 25 kD x 1600
   N.D. ^a Peak m / z at MALDI-TOF; ND: not determined; ^* GH-KLH was obtained from Optimer Inc.

The GH-KLH conjugate showed the highest Globo H incorporation count, mostly due to the larger size of KLH and more Lys residues. In addition, the same coupling procedure using a p-nitrophenyl linker was applied to a bamboo mosaic virus containing over 100,000 lysine residues on a virus coat. However, the instability of the virus during the reaction in sodium phosphate buffer (pH = 7.2) at 4 ° C is a major concern for further development. In addition, GH-BaMV (16) is limited in its detection by its MALDI-TOF analysis because of its large size.

Synthetic Globo H and cleaved fragments (Figure 1) are attached to the pentylamine linker at the reducing end and are covalently immobilized on NHS-coated glass slides. Nine of the 11 oligosaccharides were selected for printing on the microarray. 50 μM of each of 9 Globo H analogues (SSEA-4, GH, Gb5, Gb4, Gb3, Gb2, BB4, BB3 and BB2) was spotted on each microarray slide in 12 batches.

To demonstrate the carbohydrate on the microarray, a mouse monoclonal antibody (VK9 and Mbr1 for Globo H, and anti-SSEA-3) was used and each secondary antibody (goat anti-mouse IgG and IgM) The binding specificity was tested and the results are shown in Figures 2A-2C. The data suggest that MBrl recognizes BB3 slightly, but both VK9 and Mbr1 recognize Globo H and the external tetracycline BB4. In addition, the anti-SSEA-3 antibody specifically recognized the SSEA-3 antigen (Gb5) without any cross reactivity. The results indicate that the Globo H microarray can be used to profiling the specificity and efficacy of polyclonal antibodies from immunized mice.

As described above, the immunization of a fully synthetic Globo H vaccine and QS-21 co-administered mice results in the generation of antibodies to human breast cancer cells; Even after several booster vaccinations, the mouse antibody was mainly IgM (Ragupathi G, et al . (1997) Angew Chem Int Ed. 36: 125-128).

One group of mice was immunized subcutaneously with 1 [mu] g of synthetic Globo H-conjugate with or without agonist alpha-GalCer (C1). As summarized in Figure 3A, GH-KLH, GH-DT and GH-BV are the most effective immunogens for IgM induction, followed by GH-TT and GH-BSA, It has been observed that it can stimulate an immune response to induce antibodies. A similar trend was also observed with mouse IgG antibody (Figure 3B) and the relative level of IgG was higher than the IgM level. In summary, despite the lower carbohydrate density of synthetic glycoconjugates, GH-DT possesses similar immunogenicity as GH-KLH, and the adjuvant alpha-GalCer appears to enhance the immune response.

Since α-GalCer appears to be an effective adjuvant for GH-DT, other glycolipids with better adjuvant activity than C1 were tested as shown in FIG. Groups of mice were immunized with GH-DT and GH-BV with or without glycolipids. Serum was obtained and introduced into the glycan microarray assay. In general, mouse anti-Globo H IgG reverse transcripts increased as immunization progressed, but IgM levels were hardly affected by the number of vaccinations (FIG. 5). There was no significant difference in IgM levels between the glycolipid-vaccine and the single vaccine in the GH-BV vaccine group. The results suggest that GH-BV in combination with glycolipid is not an effective immunization regimen, but low immunogenicity can be attributed to the unstable nature of BaMV. Nevertheless, the α-GalCer analog, particularly 7DW8-5, cooperated well with GH-DT to elicit a mouse immune response.

Interestingly, mouse polyclonal IgG antibodies produced by GH-DT and various glycolipid adjuvants not only neutralize Globo H, but also cross-react with Gb5, SSEA-4 and Gb4, and C34 is the most effective glycolipid adjuvant (Fig. 6). In order to find a novel vaccine composition capable of inducing IgGs with a much higher potency than IgM, the Globo H-DT conjugate and the glycogen C1 or C34 or the commercially available adjuvant AlPO ₄ (aluminum phosphate) or MF59 were tested Respectively.

Surprisingly, Globo H-DT induced IgG antibodies almost exclusively after the third vaccination with glycolipid C34 (Figure 7). In summary, the novel glycolipid Adjuvant 7DW8-5 in combination with the GH-DT conjugate was able to enhance both anti-Globo H IgG and IgM antibodies, and the glycolipid adjuvant C34, in combination with GH-DT, IgG of higher antibody titers could be induced. They also possessed various binding affinities for SSEA-3 (Gb5) and SSEA-4 antigens, both of which are specifically expressed on the surface of breast cancer stem cells.

To further compare the effect of different glycolipid adjuvants in a Globo H vaccine, we immunized seven groups of mice with GH-KLH. The results suggested that mice vaccinated with glycolipids induce higher levels of anti-Globo H antibodies (FIG. 8). Although MF59 is a potent adjuvant, it has not been able to cooperate with GH-KLH to induce antibodies to Globo H. AlPO ₄ (aluminum phosphate) also failed to show a clear effect in the induction of antibodies. On the other hand, GH-KLH with C34 exhibited excellent immunogenicity after the first and second vaccination, but did not show a significant difference with C1 after the third vaccination. Ultimately, these observations suggest efficacy as novel adjuvants for carbohydrate-based vaccines of new glycolipid derivatives.

The properties of cellular and humoral immune responses are affected not only by antigen and adjuvant combinations, but also by carrier and immunization pathways. DT-CRM 197 induces antigen-specific T cell proliferation and increased IL-2, IFN-γ and IL-6 production in splenocytes, as described by Cesar Dick et al. , Suggesting its role in the Th1 drive path (Miyaji EN et al . (2001) Infect Immun 69: 869-874; Godefroy S, et al . (2005) Infect Immun 73: 4803-4809; Stickings P, et al . (2008) Infect Immun 76: 1766-1773). Despite the fact that the cytokine profile was predominantly Th1, the subclassification of the anti-CRM 197 antibody was IgG1 and there was no detectable IgG2a suggesting a mixed Th1 / Th2 response. These results recall the evaluation of the antibody isotype profile of the Globo H vaccine and this study shows that GH-DT or GH-KLH in combination with the galangal adjuvant mainly induces IgG1 antibody with a small amount of IgG2a ).

No antibody class switch (IgG2a) was observed, despite the fact that the galangal adjuvant improves Th1-biased cytokine secretion when administered intravenously (i.v.) alone. Collectively, glycolipids play a pivotal role in promoting both cellular and humoral immune responses.

Globo  H, SSEA -3 and SSEA -4 Cancer vaccine

DT and conjugated SSEA-3 (Gb5) and SSEA-4 were synthesized and tested. After the third vaccination, the antibody titers of IgM and IgG were compared, and SSEA-3-DT and SSEA-4-DT also induced significantly higher titers of IgG than IgM (FIG. 10).

The specificity of the SSEA-3-DT and SSEA-4-DT vaccines in the presence of the adjuvant was tested against a series of 24 glycans as GH-DT and C34 induce antibodies recognizing Globo H, Gb5 and SSEA- (Fig. 11).

As shown in Fig. 12, mice immunized with Globo H-DT and C34 adjuvant induced antibodies capable of recognizing Globo H, SSEA-3 (Gb5) and SSEA-4 with high selectivity, and Adjuvant MF59 And the vaccine SSEA-3-DT induced a high immune response with low selectivity. On the other hand, SSEA-3-DT in combination with Adjuvant C34 induced only antibodies against Globo H, SSEA-3 and SSEA-4.

Interestingly, SSEA-4-DT (sialyl-Gb5) in the presence or absence of azubantin specifically recognizes SSEA-4 and its truncated structure (SSEA-4 with head lactose deletion) IgG and IgM antibodies. Without being bound by theory, it is believed that sialic acid is highly immunogenic and induces a highly specific immune response.

Immunization of mice with SSEA-3-DT-C34 induced antibodies reactive with Globo H, SSEA-3 and SSEA-4, indicating that the Globo H-based vaccine expressed Globo H, SSEA-3 and SSEA- Suggesting that tumor cells and breast cancer stem cells can be targeted.

Immunization of the mouse with Globo H-DT-C34 induced antibodies reactive with Globo H, SSEA-3 and SSEA-4, indicating that the Globo H-based vaccine was able to inhibit the expression of Globo H, SSEA-3 and SSEA- Suggesting that cell and breast cancer stem cells can be targeted.

Immunization of SSEA-4-DT in mice induced antibodies reactive with SSEA-4, indicating that SSEA-4-DT-based vaccines can target SSEA-4 expressing tumor cells and breast cancer stem cells It suggests.

Tumor size reduction by cancer vaccine

To directly assess the efficacy of the synthetic glyco conjugate vaccine, the tumor size was measured three times per week, as shown in Fig. In general, tumors were grown for 2 weeks after injection of 4T1, a breast cancer cell line with Globo H. The group vaccinated with all glycolipids exhibited relatively less tumor progression on day 24 compared to the single GH-DT and PBS controls. This data suggests that vaccination with GH-DT and glutamate adjuvant delay tumor progression in vivo to some extent.

Breast cancer and BCSC In SSEA -3 and SSEA -4 expression

A lower frequency of non-BCSC, but higher expression of Globo H in BCSC and a higher frequency of expression of Globo H in breast cancer and BCSC has been described (see herein incorporated by reference in its entirety [Chang WW. Et al., (2008) Proc. Natl Acad Sci USA 105 (33): 11667-11672).

Table 2 summarizes the clinical characteristics of 35 breast cancer patients whose SSEA-3 or SSEA-4 expression levels were measured. The median age was 48 years (31 to 82 years). They are composed of 1 group of 0, 10 of I group, 19 of II group and 5 of III group. Most of the tumor specimens had lesions of invasive ductal carcinoma (80.0%), 51.4% were positive for ER, and 65.7% were positive for lymph node involvement. In Table 2, the expression range of SSEA-3 or SSEA-4 was expressed as a percentage of benign cells in total cancer cells. T- test was used for statistical analysis of SSEA-3 or SSEA-4 expression or lymph node involvement status relative to HER-2. HER-2 expression was determined by immunohistochemistry. (SSEA-4: P = 0.3498; SSEA-3: P = 0.9311) or HER-2 (SSEA-4: P = 0.0142; SSEA-3 :, P = 0.O128) (Table 2).

Clinical characteristics of breast cancer patients Characteristic Number of people % SSEA4
Cell percentage (range) with intermediate expression P value SSEA3
Cell percentage (range) with intermediate expression P value Participating patient
age
middle
range 35

48
31-82 100














Tumor type
Invasive ductal carcinoma
Invasive lobular carcinoma
Ductal carcinoma
Adenocarcinoma
Atypical subcutaneous carcinoma
Hydrological carcinoma
Inflammatory carcinoma
28
One
One
One
One
2
One
80.0
2.8
2.8
2.8
2.8
6.0
2.8
























step
0
I
II
III
One
10
19
5
2.9
28.6
54.3
14.2
33.1 (33.1)
41.4 (0.5-69.1)
39.3 (0.0-77.1)
49.8 (7.7-70.7) 0.7880




1.4 (1.4)
36.4 (0.0-55.9)
30.9 (0.0-66.4)
32.3 (0.0-36.1) 0.9311



Lymph node involvement
voice
positivity
23
12
65.7
34.3
37.8 (0.0-69.1)
49.1 (17.4-77.1) 0.0322


30.9 (0.0-66.4)
35.8 (0.0-60.7) 0.4925

ER
voice
positivity
18
17
51.4
48.6
36.2 (0.5-60.3)
48.5 (0.0-77.1) 0.0142


29.7 (0.0-38.6)
40.0 (0.0-66.4) 0.0128

Primary tumor cells isolated from patients participating in enzymatic degradation were stained with specific antibodies against CD45, CD24, and CD44, and CD45 ⁺ cells were first removed to remove leukocytes. To compare SSEA-3 or SSEA-4 expression between BCSC and non-BCSC, CD45 ^- tumor cells were further separated into BCSC and non-BCSC based on expression of their surface markers. BCSCs were identified as CD45 ^- / CD24 ^- / CD44 ⁺ cells; The remainder of the CD45 ^- population was considered non-BCSC.

Using this approach, the expression of SSEA-3 or SSEA-4 in BCSC and non-BCSC was evaluated in 35 tumor tests. Overall, SSEA-4 was detected in 34 out of 35 tumors (97.1%) and SSEA-3 in 27 out of 35 tumors (77.1%) (Table 3). SSEA4 or SSEA3 expression was determined by flow cytometry. BCSC was defined as CD45 ^- CD24 ^- CD44 ⁺ cells, and non ^- BCSC was defined as the remaining population of CD45 ^- cells. The range was calculated as the percentage of benign cells in total cells.

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

Among 34 of the 35 (97.1%) samples expressing SSEA-4, the percentage of positive cells ranged from 0.5% to 77.1%. Non-BCSCs isolated from 32 of 35 tumors expressed SSEA-3 and the percentage of positive cells ranged from 24.0% to 78.1%. In comparison, BCSC from 31 (88.6%) of 35 tumors showed positive staining for SSEA-4, and the percentage of positive cells ranged from 5.6% to 83.6%.

BCSC And non- BCSC In SSEA4  And SSEA3  Comparison of Expression Glycane population Number of patients positivity Number Cell percentage (range) with intermediate expression of everything % SSEA-4 all 35 34 41.4 (0.5-77.1) 97.1 Non-BCSC 35 32 43.7 (4.0-78.1) 91.4 BCSC 35 31 37.1 (5.6-83.6) 88.6 SSEA-3 all 35 27 36.4 (1.4-66.4) 77.1 Non-BCSC 35 25 40.5 (24.3-70.4) 71.4 BCSC 35 23 24.3 (5.0-58.4) 65.7

BCSC In SSEA -4 expression

To compare the expression of SSEA-4 between BCSC and non-BCSC, CD45 ^- tumor cells were further separated into BCSC and non-BCSC based on expression of their surface markers. BCSCs were identified as CD45 ^- / CD24 ^- / CD44 ⁺ cells; The remainder of the CD45 ^- population was considered non-BCSC. The expression of SSEA-4 in each of these two gated populations varies in tumor samples as shown in Fig. For example, BCSC of patient BC0264, which accounts for 5.7% of all isolated tumor cells, was negative for SSEA-4, but 60.3% of non-BCSCs expressed SSEA-4. For patient BC0266, SSEA-4 expression was detected in 59.4% of non-BCSC and 55.7% of BCSC. For patient BCO313, SSEA-4 expression was detected in 32.4% of non-BCSC and 83.6% of BCSC. Overall, SSEA-4 was detected in 34 out of 35 (97.1%) of the tested samples and the percentage of positive cells ranged from 0.5% to 77.1% (Table 3).

In normal tissue SSEA -3 and SSEA -4 expression

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

In normal tissue SSEA -4 expression Normal organization
antigen SSEA4 brain 0/5 bone 0/5 Lymph node 0/5 E C breast 1/5 0/5 colon* 2/4 0/4 esophagus 0/5 0/5 chapter 5/5 0/5 kidney 2/5 0/5 liver 0/5 0/5 lungs 1/5 0/5 ovary 1/5 0/5 Pancreas 1/5 0/5 prostate 0/5 0/5 rectal 5/5 0/5 skin 0/5 0/5 spleen 0/5 0/5 top 4/5 0/5 Testimony 4/5 0/5 Thymus 1/5 0/5 Cervix 1/5 0/5

SSEA-4 is expressed in epithelial cells of several gland tissues such as breast, colon, gastrointestinal tract, kidney, lung, ovary, pancreas, rectum, stomach, testis, thymus and cervix (Table 4). Also, in a manner similar to Globo H and SSEA-3 (Chang WW. et al., (2008) Proc Natl Acad Sci USA 105 (33): 11667-11672), SSEA-4 expression is confined mainly to the apical surface of cytoplasm or epithelial cells, which is essentially inaccessible to the immune system,

In comparison, Globo H is expressed on epithelial cells of several gland tissues such as breast, gastrointestinal tract, pancreas, prostate and cervix. The distribution of SSEA3 is similar to the distribution of Globo H except for the absence of it in normal breast tissue and the presence of negative for Globo H in the kidney, rectum, testis and thymus (Chang WW. Et al., (2008 ) Proc Natl Acad Sci USA 105 (33): 11667-11672).

Example

The following examples are included to illustrate preferred embodiments of the present invention. It should be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that function well in the practice of the invention as discovered by the inventors and thus can be considered to constitute a preferred way of practicing the same. However, those skilled in the art should, in light of the present disclosure, appreciate that many modifications may be made to the specific embodiments described and still obtain a like or similar result without departing from the spirit and scope of the invention.

General methods, materials and equipment

material

Conventional solvents and reagents were used as received without further purification and were purchased from Sigma-Aldrich, Acros, Merck, Echo chemical and Senn Chemical Respectively. Monoclonal antibody Mbr1 was purchased from ALEXIS biochemicals and Cy3-conjugated anti-mouse IgG (IgG, IgG1 and IgG2a) and IgM antibodies were purchased from Jackson ImmunoResearch ( Jackson Immuno Research ). DT-CRM 197 protein and tetanus toxoid were purchased from Merck and Adimmune, respectively. Aluminum phosphate gels adjuvant (AlPO ₄ ) was purchased from Brenntag Biosector. Bambus virus and the VK9 monoclonal antibody were prepared from the laboratories of Dr. Lin and Yu, respectively. A glycolipid derivative was synthesized and received by Dr. Wong's laboratory.

General method

The molecular sieve (MS, AW-300) used for saccharification was crushed and activated before use. The reaction was visualized by monitoring with an analytical TLC plate (PLC silica gel-60, F ₂₅₄ , 2 mm, Merck) and visualizing under UV (254 nm) or staining with p-anisaldehyde. Flash column chromatography was performed on silica gel (40-63 mu m) or LiChroprep RP18 (40-63 mu m). Dialysis membrane was washed with ddH ₂ O prior to use (cellulose ester, MCCO = 10,000).

device

The proton nuclear magnetic resonance ( ¹ H NMR) spectrum and carbon nuclear magnetic resonance ( ¹³ C NMR) spectra were recorded on a Bruker Advance 600 (600 MHz / 150 MHz) NMR spectrometer. Chemical shifts to protons were recorded in ppm (delta scale) and tetramethylsilane (delta = 0) was referenced. Chemical shifts to carbon were also recorded in parts per million (ppm, δ scale). DEPT 135 (distortion-less enhancement by polarization polarization) was used for the determination of multiplicity. The data are expressed as follows: Coupling constants in terms of chemical shift, multiplicity (s = single, d = 2, t = 3, q = 4, m = multiple, br = broad) ( J ). High resolution mass spectra were obtained with BioTOF III and MALDI-TOF MS was used by Ultraflex II TOF / TOF 200.

Example  1: different carrier  Protein-conjugated Globo  Synthesis of H

Globo H (1; see FIG. 11) and its fragments (2-10) were synthesized using a one-pot strategy that can be programmed (Huang CY, et al. (2006) Proc Natl Acad Sci USA 103: 15-20). The effective homologous in anhydrous DMF solution to one of the reaction was carried out at room temperature with a bifunctional (homobifunctional) linker (Wu X, et al (2004 ) Org Lett 6:. 4407-4410; Wu X, Bundle DR (2005) J Org Chem 70: 7381-7388). The reaction was easily monitored by TLC. When loss of free amine occurs with a larger R _f product, the reaction mixture is evaporated to remove DMF and washed with dichloromethane and water to remove excess linker. Finally, the product was purified by reverse phase (C18) column chromatography and eluted slowly with water containing 1% acetic acid to 40% methanol in water. The solution was then lyophilized to give a light yellow product (12). Finally, for the protein conjugate, the purified Globo H half ester 12 (30-40 equivalents) was incubated with the individual carrier proteins in phosphate buffer (10 mM, pH 7.2) for 24 hours at room temperature (Figure 14) . Importantly, the protein concentration should be adjusted to ~ 5 mg / ml to maximize the coupling of the lysine residue to the Globo H half ester. Then, after 24 hours, the glycoconjugate was diluted and dialyzed against deionized water to remove any remaining p-nitrophenyl groups. The solution was then lyophilized to a white powder to provide the glycoconjugates 13, 14 and 15.

The Globo H-protein conjugate was characterized by MALDI-TOF analysis to determine the number of Globo H molecules on each carrier protein. The average Globo H incorporation numbers are listed in Table 1 above.

The glycoconjugates 13, 14 and 15 were dissolved in ddH ₂ O to give a final concentration of about 1 pmol / μl. Sinapic acid was selected as the matrix and was made fresh by mixing with acetonitrile and deionized water (1: 1 v / v) to produce a final matrix concentration of 10 mg / mL containing 0.1% TFA. Each sample was detected under linear positive mode to obtain an m / z spectrum. The molecular weight of each glycoconjugate was determined by m / z . The glycoconjugate 14 showed heterogeneity, indicating an average incorporation of 2-4. The GH-KLH conjugate showed the highest number of Globo H incorporation, mainly due to the larger size of KLH and more Lys residues. In addition, the same coupling procedure using a p-nitrophenyl linker was also applied to the bamboo mosaic virus containing over 100,000 lysine residues on the virus envelope. However, the instability of the virus during the reaction in sodium phosphate buffer (pH = 7.2) at 4 ° C is a major concern for further development. In addition, GH-BaMV (16) is limited in its detection by its MALDI-TOF analysis because of its large size. Finally, the lyophilized glycon conjugate was stored at -30 [deg.] C and reconstituted with sterile water prior to immunization.

Example  2: Glycann Microarray  Production and verification:

Synthetic Globo H and cleaved fragments (Figure 1) were attached to pentylamine linkers at the reducing end and covalently immobilized on NHS-coated glass slides. Nine of the 11 oligosaccharides were selected for printing on the microarray. Continuous oligosaccharide concentrations (1, 5, 10, 20, 40, 50, 80, 100 μM) were tested to optimize binding affinity and fluorescence intensity. 50 μM of each of 9 Globo H analogues (SSEA-4, GH, Gb5, Gb4, Gb3, Gb2, BB4, BB3 and BB2) was spotted on each microarray slide in 12 batches. After reaction in an 80% humidity atmosphere, the slides were stored at room temperature in a desiccator prior to use.

To demonstrate the carbohydrate on the microarray, a mouse monoclonal antibody (VK9 and Mbr1 for Globo H, and anti-SSEA-3) was used and each secondary antibody (goat anti-mouse IgG and IgM) The binding specificity was tested and the results are shown in Figures 2A-2C. The data suggest that MBrl recognizes BB3 slightly, but both VK9 and Mbr1 recognize Globo H and the external tetracycline BB4 (Gilewski T. El al . (2001) Proc Natl Acad Sci USA 98: 3270-3275; Huang CY, et al . (2006) Proc Natl Acad Sci USA 103: 15-20). In addition, the anti-SSEA-3 antibody specifically recognized the SSEA-3 antigen (Gb5) without any cross-reactivity. The results indicate that the Globo H microarray can be used to profile the specificity and efficacy of polyclonal antibodies from immunized mice.

Example  3: Mouse immunization

In this study, a group of mice was immunized subcutaneously with 1 ug of a synthetic Globo H (GH) -conjugate with or without a gingival adjuvant, alpha-GalCer (C1). Ten days after three doses of biweekly vaccination, mouse sera were collected and then introduced into glycan microarray analysis to assess antibody levels. As summarized in Figure 3A, GH-KLH, GH-DT and GH-BV are the most effective immunogens for IgM induction, followed by GH-TT and GH-BSA, It has been observed that it can stimulate an immune response to induce antibodies. A similar trend was also observed with mouse IgG antibody (Figure 3B) and the relative level of IgG was higher than the IgM level. In summary, despite the lower carbohydrate density of the synthetic glycoconjugates, GH-DT had similar immunogenicity as GH-KLH, and azuban-α-GalCer was observed to enhance the immune response.

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

Groups of mice were immunized twice weekly with 1.6 ug of GH-DT and GH-BV intramuscularly, with or without 2 ug of glycolipid. Serum was obtained two weeks after the third vaccination and was introduced into the glycan microarray assay. In general, mouse anti-Globo H IgG reverse transcripts increased as immunization progressed, but IgM levels were hardly affected by the number of vaccinations (FIG. 5). In the GH-BV vaccine group, there was no significant difference in IgM levels between the glycolipid-vaccine treatment and the single vaccine. The results suggest that GH-BV in combination with glycolipid is not an effective immunotherapy, but low immunogenicity can be attributed to the unstable nature of BaMV. Nevertheless, the α-GalCer analog, particularly 7DW8-5, cooperated well with GH-DT to elicit a mouse immune response.

Interestingly, mouse polyclonal IgG antibodies generated by GH-DT and various glycolipid adjuvants not only neutralize Globo H, but also cross-react with Gb5, SSEA-4 and Gb4, suggesting that C34 is the most effective 6). In order to find a novel vaccine composition capable of inducing IgGs with a much higher potency than IgM, the Globo H-DT conjugate and glycogen C1 or C34 and the commercially available adjuvant AlPO ₄ (aluminum phosphate) or MF59 were tested Respectively. Surprisingly, Globo H-DT was able to induce almost IgG antibodies after the third vaccination with glycolipid C34 (Figure 7). In summary, the novel glycolipid Adjuvant 7DW8-5 in combination with the GH-DT conjugate was able to enhance both anti-Globo H IgG and IgM antibodies, and the glycolipid adjuvant C34, in combination with GH-DT, IgG of higher antibody titers could be induced. They also possessed various binding affinities for Gb5 and SSEA-4 antigens, both of which are specifically expressed on the surface of breast cancer stem cells.

Seven groups of mice were immunized with GH-KLH in order to further compare the effect of different glycolipid adjuvants in the Globo H vaccine. The results suggest that mice vaccinated with glycolipids induce higher levels of anti-Globo H antibodies (Figure 8). Although MF59 is a potent adjuvant, it has not been able to cooperate with GH-KLH to induce antibodies to Globo H. AlPO ₄ (aluminum phosphate) also failed to show a clear effect in the induction of antibodies. On the other hand, GH-KLH with C34 exhibited excellent immunogenicity after the first and second vaccination, but did not show a significant difference with C1 after the third vaccination.

DT-CRM 197 induces antigen-specific T cell proliferation and increases splenocyte IL-2, IFN-γ and IL-6 production, and its role in the Th1 driving pathway (Miyaji EN et al . (2001) Infect Immun 69: 869-874; Godefroy S, et al . (2005) Infect Immun 73: 4803-4809; Stickings P, et al . (2008) Infect Immun 76: 1766-1773). Despite the fact that the cytokine profile was predominantly Th1, the subclassification of the anti-CRM 197 antibody was IgG1 and there was no detectable IgG2a suggesting a mixed Th1 / Th2 response. These results recall the evaluation of the antibody isotype profile of the Globo H vaccine and this study shows that GH-DT or GH-KLH in combination with the galangal adjuvant mainly induces IgG1 antibodies and trace amounts of IgG2a ).

Antibody class conversion (IgG2a) was not observed despite the fact that the galangal adjuvant improves Th1 biased cytokine secretion when administered intravenously (i.v.) alone. Taken together, glycolipids play a pivotal role in promoting both cellular and humoral immune responses.

In addition, Gb5 and SSEA-4 conjugated with DT were synthesized by the same strategy. After the third vaccination, the antibody titers of IgM and IgG were compared, and Gb5-DT and SSEA-4-DT also induced significantly higher titers of IgG than IgM (FIG. 10).

Example  4: Specificity studies of antibodies induced by different vaccine compositions

The specificity of the SSEA-3-DT and SSEA-4-DT vaccines in the presence of adjuvant was compared to that of the SSEA-4-DT vaccine as a series of Using 24 glycans, the following tests were performed focusing on the study of IgG (Fig. 11).

As shown in Fig. 12, mice immunized with Globo H-DT and C34 adjuvant induced antibodies capable of recognizing Globo H, SSEA-3 (Gb5) and SSEA-4 with high selectivity, and Adjuvant MF59 And the vaccine SSEA-3-DT induced a high immune response with low selectivity. On the other hand, SSEA-3-DT in combination with Adjuvant C34 induced only antibodies against Globo H, SSEA-3 and SSEA-4.

Interestingly, SSEA-4-DT in the presence or absence of azubantin induced IgG and IgM antibodies that specifically recognize SSEA-4 and its truncated structure (SSEA-4 with head lactose deletion). However, the origin of selectivity is unclear.

To directly assess the efficacy of the synthetic glyco conjugate vaccine, the tumor size was measured three times per week as shown in FIG. In general, tumors were grown for 2 weeks after injection of 4T1, a breast cancer cell line with Globo H. All the groups vaccinated with the carbohydrate Adjuvant showed relatively less tumor progression on day 24 compared to the single GH-DT and PBS controls. Preliminary data suggest that vaccination with GH-DT and galangal adjuvant may delay tumor progression in vivo to some extent.

Example  5: Globo  Preparation of H half ester

The GloboH half ester was prepared as follows:

Globo H amine (1) (5 mg, 4.54 μmol) was dissolved in anhydrous DMF solution. Then, p-nitrophenyl ester linker (8.8 mg, 22.7 μmol) was added and stirred at room temperature for 1-3 hours. The reaction was monitored by TLC (1% AcOH in methanol) and Ninhydrin test. The disappearance of the free amine with the larger R _f product indicates the completion of the reaction. The reaction mixture was evaporated under reduced pressure without heating to remove DMF and then extracted twice with water containing CH ₂ Cl ₂ and 1% acetic acid. The aqueous solution was concentrated, purified by reverse phase (C18) column chromatography and eluted slowly with H ₂ O to MeOH: H ₂ O = 4: 6 containing 1% acetic acid. The solution was then lyophilized to give a light yellow solid product 12 (5.4 mg, 88% yield). ¹ H NMR (600 MHz, D ₂ O)? 8.25 (d, 2H, J = 9.0 Hz) 7.28 (d, 2H, J = 9.0 ㎐), 5.12 (d, 1H, J = 3.9 ㎐), 4.79 (d, 1H, J = 3.7 ㎐), 4.51 (d, 1H, J = 7.7 ㎐), 4.44 ( d, 1H, J = 7.7 ㎐ ), 4.39 (d, 1H, J = 7.7 ㎐), 4.31-4.28 (t, 2H, J = 7.7 ㎐), 4.15-4.11 (m, 2H), 3.99 (d, 1H , J = 2.0 ㎐), 3.92 (d, 1H, J = 2.8 ㎐), 3.89-3.44 (m, 33H), 3.16 (t, 1H, J = 8.6 ㎐), 3.10 (t, 2H, J = 6.7 ㎐ 2H), 2.62 (t, 2H, J = 6.9 Hz), 2.20 (t, 2H, J = 6.6 Hz), 1.93 (s, 3H), 1.62-1.49 (m, 4H) (M, 2H), 1.11 (d, 3H, J = 6.5 Hz) ¹³ C NMR (150 MHz, D ₂ O)? 178.0, 176.1, 176.0, 156.9, 77.1, 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: C ₅₅ H ₈₇ N ₃ O ₃₅ Na [M + Na] ⁺ Calculated: 1372.5018; Found: 1372.5016.

Example  6: General Procedure for Glycoconjugate Production

The glycoconjugate was made as follows:

BSA, DT-CRM 197 and tetanus toxoid (Adygon, Taiwan) were dissolved in 100 mM phosphate buffer pH 7.2 (~ 5 mg / ml) and 30 to 40 equivalents of Globo H half ester (35) Respectively. The mixture was stirred gently at room temperature for 24 hours. The mixture was then diluted with deionized water and dialyzed against 5 times exchanged deionized water. The solution was then lyophilized to a white powder. The resulting Globo H-protein conjugate can be characterized by MALDI-TOF analysis to determine the carbohydrate incorporation ratio. 41 (GH-BSA) MALDI-TOF found: 76029, 42 (GH-DT-CRM 197) found: 62138, 43 (GH-TT) found: 162902, 44 (GH-BaMV).

Example  7: Glycoconjugate on  About MALDI - TOF MS  analysis

The glycoconjugates (41, 42, 43) and the major carrier protein were reconstituted with ddH ₂ O (~ 1 μg / μl). The matrix cininic acid was freshly prepared with acetonitrile and deionized water (1: 1 v / v) to yield a final matrix concentration of 10 mg / mL containing 0.1% TFA. Carefully loaded, the matrix solution and glyco conjugate were mixed, and the plate was air dried. Be sure to calibrate using bovine serum albumin before the measurement. The glycoconjugate and major protein samples were each detected under linear positive mode. The average molecular weight enables the calculation of the average number of carbohydrates incorporated on the carrier protein.

Example  8: Glycann Microarray  making

The microarray was incubated with various concentrations of amine-containing glycans to 0.7 nL of robotic pins in printing buffer (containing 300 mM phosphate buffer, pH 8.5, 0.005% Tween-20) from 96 wells to NHS-coated glass slides (TeleChem International Inc., SMP3, USA) deposition (BioDot, Cartesian Technologies, U.S.A.). < / RTI > Each microarray slide was spotted in 12 batches with 50 μM of each of 9 Globo H analogues (SSEA-4, GH, Gb5, Gb4, Gb3, Gb2, BB4, BB3 and BB2). The printed slides were allowed to react in an 80% humidity atmosphere for 1 hour and then allowed to dry overnight. These slides were stored in a desiccator at room temperature before use.

Example  9: Serological analysis Glycann Microarray )

Mouse serum was diluted 1: 60 using 0.05% Tween 20 in 3% BSA / PBS buffer (pH 7.4) as a preliminary screening. The glycan microarray was blocked with 50 mM ethanolamine for 1 hour and washed twice with ddH ₂ O and PBS buffer before use. The serum dilution was then introduced into the Globo H microarray and incubated at room temperature for 1 hour. The microarray slides were further washed three times with PBST (0.05% Tween-20 in PBS buffer) and PBS buffer, respectively. Next, Cy3-affiniPure goat anti-mouse IgG (H + L), IgG1, IgG2a or anti-mouse IgM was added to the microarray slides and then sealed and incubated at room temperature for 1 hour. Finally, the slides were then washed three times with PBST, PBS and ddH ₂ O. The microarray slides were dried and then scanned at 532 nm with a microarray fluorescent chip reader (Genepix 4000B). Data was analyzed with software GenepixPro 6.0 (Axon Instruments, Union City, CA). To obtain accurate measurements, the PMT gain was adjusted to 400 to prevent fluorescence saturation. At each glycan spot, the background of the local area was subtracted from the signal. Spots with obvious defective or undetectable signals are omitted. The final fluorescence intensity was defined as the average of "F532 nm - B532 nm intermediate" from the repeating spot.

Example  10: Serological analysis (enzyme-linked immunosorbent assay)

0.2 [mu] g of Globo-H ceramides in 100 [mu] l carbonate bicarbonate buffer (pH 10) were coated overnight at 4 [deg.] C in 96-well plates (NUNC). Washed with PBS, and blocked with 3% bovine serum albumin for 30 minutes at room temperature. Serial dilutions of mouse serum were added to each well, incubated at room temperature for 1 hour, and then rinsed with DPBST (Dulbecco's Phosphate Buffered Saline, 0.05% Tween 20). Goat anti-mouse IgG-AP (1: 200, Southern Biotech. USA) was added and incubated at room temperature for 45 minutes. The plate was washed 5 times with PBST and then incubated with p-nitrophenylphosphate (Sigma), an alkaline phosphatase substrate, at 37 DEG C for 8 minutes. After incubation, the reaction was stopped by adding 3 M NaOH solution and the plate was read at 405 nm on an ELISA reader (SpectraMax, Molecular Devices). The reversal is defined as the highest dilution producing an optical density of greater than 0.1.

Example  11: Capacity and immunization

(1) GH-KLH (1) was administered to three mouse groups (6-week-old female C57BL / 6 mice, BioLASCO, Taiwan) with or without glycated adjuvant C1 or 7DW8-5 GH-BSA, GH-TT, GH-CRM 197 and GH-BaMV were subcutaneously administered to the abdomen. Each vaccine contained 1 μg of Globo H with or without 2 μg of glycolipid adjuvant. Control mice were injected with phosphate buffered saline (PBS) alone. Blood was collected from the mice before the first immunization (immunoprecipitation) and 10 days after the third immunization. (2) GH-BaMV or GH-CRM 197 with or without C1, C23 or 7DW8-5, respectively, into muscle in 3 mouse groups (8 week old female Balb / c mice, And immunized three times at weekly intervals. Each vaccine contained 1.6 μg of Globo H with or without 2 μg of adjuvant. Control mice were injected with phosphate buffered saline (PBS). Blood was drawn from the mice before immunization and two weeks after each vaccination. 3 of 3 mice as a group (2) as set forth in (8-week-old female Balb / c mouse, Bio raseuko Taiwan material), adjuvants C1, C17, 7DW8-5, C30, AlPO _4, MF59 (1: 1 mixture) with or without GH-CRM 197 or GH-KLH. All sera were obtained by centrifugation for 10 min under 4000 g. Serological responses were analyzed by glycan microarray or compared by conventional ELISA assays.

Example  12: Xenotransplantation model

1, five groups of immunized female Balb / c mice in (PBS, GH-CRM 197 alone, or with C1, C23 and 7DW8-5 each) 2 × 10 ⁵ metastatic mouse mammary tumor cell line, 4T1 (sterile in PBS ) Were injected subcutaneously 8 weeks after the final vaccination. (2) 2 × 10 ⁵ metastatic mouse mammary tumor cell line, 4T1 Seven groups of immunized female Balb / c mice (GH-KLH alone or respectively C1, C17, 8-5, C30, AlPO 4 , and with the MF59) (In sterile PBS) was injected subcutaneously 6 weeks after the final vaccination. Mouse anti-Globo H serum was monitored before and after tumor xenotransplantation. The mouse tumor size was measured three times per week with a vernier caliper and defined as (length × height × width) / 2 (㎣).

Example  13: Human breast cancer From  Isolation of Primary Tumor Cells

A human tumor cancer test was obtained from a patient who underwent initial surgery at the Tri-Service General Hospital in Taipei, Taiwan. The samples were fully encrypted to protect patient confidentiality and were placed under protocol approved by the Institutional Review Board of the Human Subjects Research Ethics Committee of Academia Sinica, Taipei, Taiwan Respectively. Tumor specimens were sliced into 1 mm 2 square pieces and incubated at 37 ° C for 2 hours with collagenase (1000 U / ml), hyaluronidase (300 U / ml) and DNase I (100 μg / And enzymatically degraded by incubation in RPMI 1640 medium. Primary breast tumor cells were collected after filtration through a 100-μm cell strainer (BD Biosciences) and resuspended in RPMI 1640 medium supplemented with 5% FBS.

Example  14: Flow cell  analysis

Containing from ₃ to primary breast cancer cells, 2% FBS and 0.1% NaN was placed in a 50 ㎕ 1 × 10 ⁵ cells in PBS on. Cells were labeled with anti-CD24-PE, anti-CD44-APC and anti-CD45-PerCP-Cy5.5 antibody mixtures (1 μl each). Globo H expression was detected by staining with Alexa 488 conjugated monoclonal anti-Globo H antibody (VK-9). Analysis was performed on a FACSCanto flow cytometer (Becton Dickinson). BCSC was defined as CD45 ^- / CD24 ^- / CD44 ⁺ cells, and non ^- BCSC was defined as the remaining population of CD45 ^- cells. Globo H expression was further analyzed in the gated region.

Example  15: Cell classification

Cells harvested from human breast tumors transplanted into mice were stained with anti-CD24-PE, anti-CD44-APC and anti-H2K ^d- FITC antibody mixtures (Vidi Biosciences). Fluorescence activated sorting of antibody-labeled cells was performed on a FACSAria cell sorter (Becton Dickinson). H2Kd ^- / CD24 ^- / CD44 ⁺ cells were classified as BCSC, and the remaining population of H2Kd ^- cells were classified as non ^- BCSC. Typical purity of BCSC and non-BCSC were > 85% and > 90%, respectively.

Example  16: Immunohistochemistry

For expression of SSEA-4 in normal tissues, tissue microarray slides (Biomax) containing 20 different organs derived from 5 organs were used. Following standard histological procedures, the slides were dried overnight at 56 ° C, dewaxed in xylenes, rehydrated, and then resuspended in AR-10 solution pH 9.0 (BioGenex Laboratories) Were used for antigen retrieval. SSEA-4 expression was determined with the use of anti-SSEA-4 antibody (eBioscience). Staining for SSEA-4 was detected using anti-rat IgM as secondary antibody and developed with DAB substrate. The slides were contrast dyed with hematoxylin. Tumor xenografts from primary breast tumor BCO145 and NOD / SCID mice were fixed in 10% phosphoric acid-buffered formalin and embedded in paraffin. The paraffin sections were cut to a thickness of 2 mu m and mounted on SuperFrost Plus microscopy slides (Menzel-Glaser) and dried at 55 ° C overnight. The sections were dewaxed in xylene, rehydrated according to standard histopathological procedures, and then stained with hematoxylin and eosin (H & E). Prior to immunostaining, the slides were first placed in a solution of 10 mmol / l citrate buffer (pH 6.0) and microwaved for 15 min. The slides were then incubated overnight with anti-ER or anti-PR antibodies. Immunodetection was performed using a Super Sensitive Polymer-HRP IHC Detection System (Biogenex).

All publications and patent applications mentioned in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art, in light of the teachings of the invention, should be able to ascertain readily that any changes and modifications may be made therein without departing from the spirit or scope of the appended claims. It will be readily understood that the invention may be practiced.

The abstract is designed to help readers quickly identify the nature and substance of the technical disclosure. Provided in accordance with § 1.72 (b). The summary is submitted with the understanding that this will not be used to interpret or limit the scope or meaning of the claims.

Claims (57)

(SSEA-3, Gb5), a step-specific embryo antigen-4 (SSEA-4, sialyl Gb5), a step-specific embryonic antigen- Bb4 or an immunogenic fragment thereof, or a combination of any of these glycans or a dendrimer containing at least one of the glycans, each conjugated to a carrier protein through a linker conjugated to said at least one glycan; And
(b) the following structure:

(CH ₂ ) ₁₀ PhFh, (CH ₂ ) ₁₀ PhF, or (CH ₂ ) ₇ PhF, (CH ₂ ) ₁₀ PhOPhF, and _{2) 24} CH _3,
An immunogenic composition that induces an immune response that produces a relatively higher level of IgG isotype antibody compared to an IgM isotype antibody. The immunogenic composition of claim 1, wherein said linker is a p -nitrophenyl linker. 2. The immunogenic composition of claim 1, wherein the carrier protein is a toxin protein. 4. The method of claim 3, wherein the toxin protein is selected from the group consisting of diphtheria toxin cross-reactive material 197 (DT-CRM 197), diphtheria toxoid, tetanus toxoid, bovine serum albumin (BSA) meningococcal OMP). The immunogenic composition of claim 1, wherein the carrier protein is a diphtheria toxin cross-reacting substance 197 (DT-CRM 197), wherein the adjuvant is a material of the following structure.

2. The immunogenic composition of claim 1, wherein the immune response comprises a humoral and cellular response. The method of claim 1, wherein said antibody is selected from Globo H, grobotetraose (Gb4), step-specific embryonic antigen-3 (SSEA-3), or step- specific embryonic antigen- ≪ / RTI > wherein the immunizing composition neutralizes at least one antigen. (SSEA-3, Gb5), a step-specific embryo antigen-4 (SSEA-4, sialyl Gb5), a step-specific embryonic antigen- Bb4 or an immunogenic fragment thereof, or a combination of any of these glycans or a dendrimer containing at least one of the glycans, each conjugated to a carrier protein through a linker conjugated, at least one glycan; And
(b) the following structure:

(CH ₂ ) ₁₀ PhFh, (CH ₂ ) ₁₀ PhF, or (CH ₂ ) ₇ PhF, (CH ₂ ) ₁₀ PhOPhF, and _{2) 24} CH _3,
A pharmaceutical composition for inhibiting tumor growth comprising an immunogenic composition that induces an immune response that produces a relatively higher level of IgG isotype antibody relative to an IgM isotype antibody. 9. The method of claim 8, wherein the tumor is selected from the group consisting of breast cancer, lung cancer, esophageal cancer, rectal cancer,
Cancer, ovarian cancer, cervical cancer, endometrial cancer, pancreatic cancer, testicular cancer, bladder cancer, head and neck cancer, oral cancer, neuroendocrine carcinoma, adrenal carcinoma, adrenal cancer, Thyroid cancer, bone cancer, skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma or brain tumor. 9. The pharmaceutical composition of claim 8, wherein the linker is a p -nitrophenyl linker. 9. The method of claim 8, wherein said antibody is selected from Globo H, grobotetraose (Gb4), step-specific embryonic antigen-3 (SSEA-3), or step- specific embryonic antigen- ≪ / RTI > wherein the at least one antigen is neutralized. 9. The pharmaceutical composition according to claim 8, wherein the carrier protein is a toxin protein. 13. The method of claim 12, wherein the toxin protein is selected from the group consisting of diphtheria toxin cross-reactive material 197 (DT-CRM 197), diphtheria toxoid, tetanus toxoid, bovine serum albumin (BSA) meningococcal OMP). (SSEA-3, Gb5), step-specific embryonic antigen-4 (SSEA-4, sialyl Gb5), Bb4 Or an immunogenic fragment thereof, or a combination of any of these glycans or a dendrimer containing at least one of the glycans, each conjugated via a linker to a carrier protein ), Said one or more glycans and
The following structure:

(CH ₂ ) ₁₀ PhFh, (CH ₂ ) ₁₀ PhF, or (CH ₂ ) ₇ PhF, (CH ₂ ) ₁₀ PhOPhF, and _{2) 24} CH _3,
An immunogenic composition that elicits an immune response that produces a relatively higher level of IgG isotype antibody relative to an IgM isotype antibody; And
(b) a pharmaceutically acceptable excipient
≪ / RTI > 15. The cancer vaccine according to claim 14, wherein the carrier protein is a toxin protein. 16. The method of claim 15, wherein the toxin protein is selected from the group consisting of diphtheria toxin cross-reactive material 197 (DT-CRM 197), diphtheria toxoid, tetanus toxoid, bovine serum albumin (BSA) meningococcal OMP). 15. The cancer vaccine according to claim 14, wherein the linker is a p -nitrophenyl linker. 15. The method of claim 14, wherein said antibody is selected from Globo H, glbotetraose (Gb4), step-specific embryonic antigen-3 (SSEA-3), or step- specific embryonic antigen- Lt; RTI ID = 0.0 > 1, < / RTI > 15. The method of claim 14, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, esophageal cancer, rectal cancer, biliary cancer, hepatoma, buccal cancer, gastric cancer, colon cancer, nasopharyngeal carcinoma, renal cancer, prostate cancer, ovarian cancer, Endometrial cancer, pancreatic cancer, testicular cancer, bladder cancer, head and neck cancer, oral cancer, neuroendocrine cancer, adrenal cancer, thyroid cancer, bone cancer, skin cancer, basal cell cancer, squamous cell cancer, melanoma or brain tumor. Globo H conjugated to a carrier protein of diphtheria toxin cross-reacting substance 197 (DT-CRM 197) through a linker comprising an aliphatic chain having 9 to 13 carbon chain lengths; And
The following structure:

(? -GalCer) adjuvant having an? -Galactosyl-ceramide (? 21. The therapeutic agent according to claim 20, wherein administration of the therapeutic agent to a subject induces production of an antibody recognizing an antigen expressed on breast cancer stem cell (BCSC). delete delete delete delete delete delete delete delete delete delete delete 22. The therapeutic agent according to claim 21, wherein the antigen is selected from Globo H, step-specific embryonic antigen-3 (SSEA-3), step-specific embryonic antigen-4 (SSEA-4), or Bb4. Conjugated with a carrier protein of diphtheria toxin cross-reacting substance 197 (DT-CRM 197) through a linker comprising an aliphatic chain having 3 to 25 carbon chain lengths with or without one or more aromatic moieties Embryonic antigen-3 (SSEA-3) and
The following structure:

(? -GalCer) adjuvant having an? -Galactosyl-ceramide (? 35. The therapeutic agent according to claim 34, wherein administration of the therapeutic agent to a subject induces production of an antibody recognizing an antigen expressed on breast cancer stem cell (BCSC). 36. The therapeutic agent according to claim 35, wherein the antigen is selected from Globo H, step-specific embryonic antigen-3 (SSEA-3), step-specific embryonic antigen-4 (SSEA-4), or Bb4. Conjugated with a carrier protein of diphtheria toxin cross-reacting substance 197 (DT-CRM 197) through a linker comprising an aliphatic chain having 3 to 25 carbon chain lengths with or without one or more aromatic moieties Embryonic antigen-4 (SSEA-4) and
The following structure:

(? -GalCer) adjuvant having an? -Galactosyl-ceramide (? 38. The therapeutic agent according to claim 37, wherein administration of the therapeutic agent to a subject induces production of an antibody recognizing an antigen expressed on breast cancer stem cell (BCSC). 39. The therapeutic agent of claim 38, wherein said antigen is selected from Globo H, step-specific embryonic antigen-3 (SSEA-3), step-specific embryonic antigen-4 (SSEA-4), or Bb4. Use of a therapeutic agent according to any one of claims 20, 21, 33 to 39 for the manufacture of a medicament for the treatment of tumor growth. 40. The method of claim 40, wherein the tumor is selected from the group consisting of breast cancer, lung cancer, esophageal cancer, rectal cancer, biliary cancer, hepatoma, buccal cancer, stomach cancer, colon cancer, nasopharyngeal carcinoma, renal cancer, prostate cancer, ovarian cancer, Wherein the cancer is selected from the group consisting of endometrial cancer, pancreatic cancer, testicular cancer, bladder cancer, head and neck cancer, oral cancer, neuroendocrine cancer, adrenal carcinoma, thyroid cancer, bone cancer, skin cancer, basal cell carcinoma, squamous cell carcinoma or melanoma or brain tumor. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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