Cancer markers and methods of use thereof

Abstract

The present disclosure relates to methods and compositions which can modulate the globoseries glycosphingolipid synthesis. Particularly, the present disclosure is directed to glycoenzyme inhibitor compound and compositions and methods of use thereof that can modulate the synthesis of globoseries glycosphingolipid SSEA-3 / SSEA-4 / GloboH in the biosynthetic pathway; par- ticularly, the glycoenzyme inhibitors target the alpha-4GalT; beta-4GalNAcT-I; or beta-3GalT-V enzymes in the globoseries syn- thetic pathway. Additionally, the present disclosure is also directed to vaccines, antibodies, and / or immunogenic conjugate composi- tions targeting the SSEA-3 / SSEA-4 / GLOBO H associated epitopes (natural and modified) which elicit antibodies and / or binding fragment production useful for modulating the globoseries glycosphingolipid synthesis. Moreover, the present disclosure is also dir- ected to the method of using the compositions described herein for the treatment or detection of hyperproliferative diseases and / or conditions. Furthermore, the instant disclosure also relates to cancer stem cell biomarkers for disgnostic and therapeutic uses.

Description

CANCER MARKERS AND METHODS OF USE THEREOF RELATED APPLICATIONS

[0001] This application claims the benefit of priority to USSN 62 / 107378 filed January 24, 2015 and USSN 62 / 266514, filed December 11th, 2015. FIELD

[0002] The present disclosure relates to methods and compositions useful for modulating the globoseries glycosphingolipid synthesis as well as markers useful for selecting cancer stem cells. Particularly, the present disclosure is directed to glycoenzyme inhibitor compound and compositions and methods of use thereof that can modulate the synthesis of globoseries glycosphingolipid SSEA-3 / SSEA-4 / GloboH in the biosynthetic pathway; particularly, the glycoenzyme inhibitors target the alpha-4GalT; beta-4GalNAcT-I; or beta-3GalT-V enzymes in the globoseries synthetic pathway. Additionally, the present disclosure is also directed to vaccines, antibodies, and / or immunogenic conjugate compositions targeting the SSEA- 3 / SSEA-4 / Globo H associated epitopes (natural and modified) which can elicit antibodies and / or binding fragment production useful for modulating the globoseries glycosphingolipid synthesis. Moreover, the present disclosure is also directed to the method of using the compositions described herein for the treatment or detection of hyperproliferative diseases and / or conditions. Furthermore the present disclosure is also directed to markers useful for selecting cancer stem cells in diagnostic and / or therapeutic applications. BACKGROUND OF THE INVENTION

[0003] The carbohydrate antigens Globo H, stage-specific embryonic antigen-3 (SSEA-3), and stage-specific embryonic antigen-4 (SSEA-4) are closely related to one another in either structure or in function. Globo H, SSEA-3 and SSEA-4 are globoseries glycosphingolipids, <com:Sup>1-3< / com:Sup> with SSEA-3 being the non-fucosylated pentasaccharide precursor structure of Globo H, SSEA-4 is sialylated SSEA-3 with sialic acid <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>2-3 links to the non-reducing end of galactose of SSEA-3.

[0004] Stage-specific embryonic antigen-3 (SSEA-3) was first identified and defined by the reactivity of an IgM monoclonal antibody generated in a rat immunized with 4- to 8-cell stage mouse embryos. This monoclonal antibody reacted with all mouse preimplantation embryos from oocytes up to the early blastocyst stage where its expression became more restricted, in the primitive endoderm after implantation. The SSEA-3 antigenic determinant was determined to be a carbohydrate present on glycolipids and glycoproteins; it was also found on human teratocarcinoma cells and human erythrocytes.<com:Sup>4< / com:Sup> In a panel of structures isolated from the 2102Ep human teratocarcinoma cell line, the SSEA-3 antibody had the highest affinity for <com:Math><semantics>Galβ(1−3)GalNAcβ(1−3)Galα(1−4)Galβ(1−4)Glcβ(1)Cer.5<annotation encoding="application / x-tex">Gal\beta(1-3)GalNAc\beta(1-3)Gal\alpha(1-4)Gal\beta(1-4)Glc\beta(1)Cer.^5< / annotation>< / semantics>< / com:Math> This structure is also known as Gb5, galactosyl-globoside, or globopentaosylceramide.

[0005] Synthesis of SSEA-3 occurs when \(\beta 1,3\)-galactosyltransferase V (\(\beta 3GalT-V\)) transfers galactose to the GalNAc of globoside to form Gb5 or galactosyl-globoside. In more recent studies, attempts were made to determine if SSEA-3 could be used as a marker to identify stem cells in umbilical cord blood. It was determined that SSEA-3 was not expressed in hematopoietic or mesenchymal stem cells and therefore was not a good marker of multipotent cells. Schrump et al. immortalized lymph node lymphocytes from primary lung cancer patients, generated hybridomas, and selected for antibody secreting clones. Monoclonal antibodies were then generated from two of these clones--J309 and D579, which recognized the SSEA-3 antigenic determinant. The antibodies recognized SSEA-3 on several tumor cell lines including lung and breast cancer cell lines, and a teratocarcinoma cell line; in an immune adherence assay, rodent monoclonal SSEA-3 antibody, also referred to as MC631, reacted against the same cell lines as the J309 and D579 antibodies. SSEA-3 has also been found on testicular germ cell tumors, <com:Sup>9< / com:Sup> as well as in breast cancer and in BCSCs (breast cancer stem cells).

[0006] Chang et al. looked at SSEA-3 expression on normal tissues using a tissue microarray because its location outside of cancer and development was largely unknown. The group found SSEA-3 to be expressed on normal epithelium of colon, esophagus, small intestine, kidney, prostate, rectum, skin, testis, thymus, and uterine cervix. Expression was located only on the apical surfaces of epithelial cells or in the cytoplasm, which are considered immune system restricted or inaccessible sites. In an experiment using a KLH conjugated Globo H monovalent vaccine in mice, an antibody response was made to only the Globo H antigen. When α-GalCer was added as an adjuvant, the amount of overall antibody production increased and the mice made polyclonal antibodies to both the Globo H, the SSEA-3 and the SSEA-4 antigen structures, which vaccination was unable to generate in the absence of the adjuvant. This result showed that SSEA-3, Globo H and SSEA-4 could make promising targets for cancer vaccines and could be targeted simultaneously.

[0007] However, most tumor associated carbohydarte antigens have poor immunogenicity and many approaches have been developed to increase the immune response of carbohydrate- based vaccines, including conjugation with a carrier protein administration with an immunologic adjuvant using unnatural glycosidic linkage, clustered antigens, unimolecular polyvalent vaccine or hetero-glycan multivalent vaccine. Using these strategies, a few carbohydrate-based vaccines that could elicit significant immune responses to target glycan structures were designed for cancer therapy and entered clinical trials. Among them, the clinical trials of Theratope and GMK with adjuvant QS-21 failed to produce statistically significant difference between time-to-disease and overall survival rate. Probably these two vaccines could not elicit robust T cell-dependent immune response in patients. Specifically, Theratope and GMK induced a higher level of IgM in patients but could not induce a strong immune IgG response, which is a major problem in carbohydrate-based vaccine development.

[0008] Previous studies showed that modification of carbohydrate antigen structures (MCAS) could effectively elicit a higher level of immune response. For example, in the modification study of the capsular polysaccharide of group B meningococci, the N-acetyl groups of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-(2,8)- linked polysialic acid (PSA) was replaced with the N-propinoyl group and such a modification elicited a high antibody response to recognize not only the N-propinoyl PSA, but also the nature N-acetyl PSA.<com:Sup>20< / com:Sup> Similar approaches were applied to STn<com:Sup>21< / com:Sup> and GM3<com:Sup>22< / com:Sup> antigens to produce high antibody titers against modified and nature forms. The results indicated that N- phenylacetyl, N-fluoroacetyl or N-difluoroacetyl modifications on glycan antigens could improve the immunogenicity. Moreover, the Schultz group reported that incorporation of a p- nitrophenylalanine into the tumor necrosis factor-<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> (TNF-<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>) could break immune tolerance and induce more antibody response to TNF-α.<com:Sup>23< / com:Sup> Using glycans as antigens, although some progress has been achieved, most cases are the N-modification of disaccharide (STn), trisaccharide (GM3) and polysialic acid (PSA) and some are based on fluorinated MUC1 glycopeptide antigens.

[0009] The discovery of cancer stem cells (CSCs), which are responsible for self-renewal and tumor-growth in heterogeneous cancer tissues, has stimulated interests in developing new cancer therapies and early diagnosis. The markers currently used for isolation of CSCs, however, are often not selective enough to enrich CSCs for the study of this special cell population. SUMMARY OF THE INVENTION

[0010] The present disclosure relates to the rapeutic methods which are based on the use of an agent that specifically targets tumor-associated carbohydrate antigen(s) and / or pathways regulating those targets, for the treatment of cancers comprising cancer stem cells and related diseases. Also provided are diagnostic and prognostic methods using one or more tumor- associated carbohydrate antigens as markers for cancer stem cells.

[0011] In one aspect, a binding agent is provided which specifically binds to one or more tumor-associated carbohydrate antigens for use in the treatment of cancers comprising cancer stem cells expressing said one or more tumor-associated carbohydrate antigens. Also provided is a pharmaceutical composition, comprising a respective binding agent for use in the treatment of cancers comprising cancer stem cells expressing said one or more tumor- associated carbohydrate antigens. Bindign agents include agents targeting SSEA3, SSEA4, and GloboH antigens, any combinations thereof and / or agents targeting related pathway targets.

[0012] In a second aspect, a method is provided for identifying a cancer comprising cancer stem cells that is susceptible to treatment with one or more binding agents each of which specifically binds to one or more tumor-associated carbohydrate antigens, wherein said treatment effects the cancer stem cells, the method comprising determining whether a cancer sample obtained from a patient comprises cancer stem cells that express one or more tumor- associated carbohydrate antigens the one or more binding agents is specific for, wherein the presence of said tumor-associated carbohydrate antigen(s) on cancer stem cells indicates that the cancer is susceptible to treatment with the binding agent(s) that specifically binds said tumor-associated carbohydrate antigen and wherein said treatment effects the cancer stem cells. In some embodiments, the tumor-associated carbohydrate antigens are selected from the list consisting of SSEA3, SSEA4, and GloboH antigens.

[0013] In a third aspect, a method is provided for identifying a population of cancer stem cells, the method comprising a) providing a starting population of cancer cells, b) determining the expression level of one or more tumor-associated carbohydrate antigens, c) selecting a population of cells whose expression level of said one or more tumor-associated carbohydrate antigens as determined in step b) is increased when compared to control cells, wherein said selected population of cells are cancer stem cells and d) optionally isolating and / or enriching for said population of cells selected in step c), wherein said control cells are cells from the same starting cancer cell population which do not express, or express lower levels of said one or more tumor-associated carbohydrate antigens. In some embodiments, the tumor-associated carbohydrate antigens are selected from the list consisting of SSEA3, SSEA4, and GloboH antigens. In some embodiments, the determining step in b) further includes determining the expression level of one or more additional tumor associated antigens, and the selection and isolating steps in c) and d), respectively, also includes consideration of the expression level of said one or more additional tumor associated antigens. In some embodiments, the one or more tumor-associated antigens include, but are not limited by CD24, CD44, PROCR, ESA, CD176, CD175, CD175s, CD174, CD173 and CA19-9 antigens. In some embodiments, the determining step in b) and / or the isolation step in d) is carried out using FACS. In some embodiments, the level of expression of said one or more tumor-associated carbohydrate antigens is high or highly increased, when compared to control cells. In some embodiments, the level of expression of said one or more tumor- associated carbohydrate antigens is low or lowly increased, when compared to control cells.

[0014] In a fourth aspect, a method for diagnosing, staging and / or prognosing cancer and / or monitoring the susceptibility to treatment is provided, comprising the step of analyzing the expression of one or more tumor-associated carbohydrate antigens on cells in a sample isolated from a patient, wherein the presence of cells expressing the one or more tumor- associated carbohydrate antigens indicates the presence of cancer stem cells in said sample.

[0015] In another aspect, a kit for use in a method according to the present invention is provided, comprising a binding agent which specifically binds to one or more tumor- associated carbohydrate antigens and instructions for use in a method according to the present invention.

[0016] In another aspect, a method is provided for screening a candidate therapeutic agent, e.g. a chemotherapeutic agent or another anti-cancer drug, for effectiveness against a cancer comprising cells that express one or more tumor-associated carbohydrate antigens as described herein, the method comprising: a. providing a cancer sample comprising cells that express one or more tumor-associated carbohydrate antigens, b. contacting said agent with the cells, and c. determining the effectiveness of said agent against said tumor-associated carbohydrate antigen positive cancer cells.

[0017] The present disclosure also relates to methods and compositions which can modulate the globoseries glycosphingolipid synthesis. Particularly, the present disclosure is directed to glycoenzyme inhibitor compound and compositions and methods of use thereof that can modulate the synthesis of globoseries glycosphingolipid SSEA-3 / SSEA-4 / GloboH in the biosynthetic pathway; particularly, the glycoenzyme inhibitors target the alpha-4GalT; beta- 4GalNAcT-I; or beta-3GalT-V enzymes in the globoseries synthetic pathway. Additionally, the present disclosure is also directed to vaccines, antibodies, and / or immunogenic conjugate compositions targeting the SSEA-3 / SSEA-4 / Globo H associated epitopes (natural and modified) which can elicit antibodies and / or binding fragment production useful for modulating the globoseries glycosphingolipid synthesis. Moreover, the present disclosure is also directed to the method of using the compositions described herein for the treatment or detection of hyperproliferative diseases and / or conditions. In addition, the disclosure is also directed to cancer stem cell markers useful for selecting cancer stem cells (e.g. breast cancer) in diagnostic and / or therapeutic applications.

[0018] The present disclosure is also based on a surprising discovery is that the inhibition or silencing the galactosyl transferase (beta3GalT5) for the biosynthesis of SSEA3 abolishes the grow of cancer stem cell. This finding imply the stage-specific embryonic antigen SSEA-3 can serve as a target for development of therapeutics and vaccines. Moreover, the 3 enzymes alpha4GalT, beta4GalNAcT-I, and beta3GalT-V those involved in the biosynthesis of SSEA- 3 can be targets for inhibitors development.

[0019] The present disclosure is also based on the discovery that the modification of the stage-specific embryonic antigens (SSEA3 and SSEA4) with certain groups disclosed herein elicited robust IgG antibody response to specifically recognize SSEA3 and SSEA4, respectively. The antibodies induced by an immunogenic composition comprising such unnatural glycan moiety are able to mediate the complement-dependent cell cytotoxicity against tumor cells.

[0020] Accordingly, the present invention features the design of antibodies against SSEA-3 for treating cancers. The present invention also features novel compounds consisting of the modified carbohydrate antigens (SSEA3, SSEA4), glycan conjugates comprising such, and immmunogenic compositions and vaccines thereof.

[0021] Present disclosure provides inhibitor compound which modulates the globoseries synthetic pathway and optionally at least one pharmaceutically acceptable carrier for the treatment of a proliferative disease, especially a proliferative disease in which the globoseries pathway is concomitantly dysregulated; a pharmaceutical composition comprising such a composition; the use of such a composition for the preparation of a medicament for the treatment of a proliferative disease; a commercial package or product comprising such a preparation; and to a method of treatment of a warm-blooded animal, especially a human.

[0022] The present disclosure is based on a surprising discovery that the inhibition or silencing the glycoenzymes such as galactosyl transferase (β3GalT5), alpha-4GalT, and beta- 4GalNAcT-I for the biosynthesis of SSEA3 abolishes the growth of cancer cells and cancer stem cell. This finding imply the stage-specific embryonic antigen SSEA-3 and / or SSEA4 and / or Globo-H can serve as a target for development of therapeutics and vaccines.

[0023] The present disclosure is also based on the discovery that the modification of the stage-specific embryonic antigens (SSEA3 and SSEA4) with certain groups disclosed herein elicited robust IgG antibody response to specifically recognize SSEA3 and SSEA4, respectively. The antibodies induced by an immmunogenic composition comprising such unnatural glycan moiety are able to mediate the complement-dependent cell cytotoxicity against tumor cells.

[0024] Accordingly, the present invention features the design of antibodies against SSEA-3 and / or SSEA4 for treating cancers. The present invention also features novel compounds consisting of the modified carbohydrate antigens (SSEA3 and SSEA4), glycan conjugates comprising such, and immunogenic compositions and vaccines thereof.

[0025] In one aspect, the present invention provides a compound of formula (I): [Image disponible dans le document PDF, Image available in the PDF document] ), or a salt thereof, wherein <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math>, R<com:Sup>1< / com:Sup>, R<com:Sup>2< / com:Sup>, R<com:Sup>3< / com:Sup>, R<com:Sup>4< / com:Sup>, R<com:Sup>5< / com:Sup>, R<com:Sup>6< / com:Sup> and L are as described herein. In certain embodiments, a compound of Formula (I) is useful for making an immunogenic composition for treating cancers.

[0001] In another aspect, the present invention provides a compound of Formula (II): [Image disponible dans le document PDF, Image available in the PDF document] (III), or a salt thereof, wherein <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R1<annotation encoding="application / x-tex">R^1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R3<annotation encoding="application / x-tex">R^3< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R8<annotation encoding="application / x-tex">R^8< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R9<annotation encoding="application / x-tex">R^9< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R10<annotation encoding="application / x-tex">R^{10}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R11<annotation encoding="application / x-tex">R^{11}< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>RN<annotation encoding="application / x-tex">R_N< / annotation>< / semantics>< / com:Math> are as described herein. In certain embodiments, a compound of Formula (II) is useful for making an immunogenic composition for treating cancers.

[0002] In another aspect, the present invention provides an immunogenic composition, comprising (a) a glycan conjugate including a carrier and one or more glycans, and optionally (b) an adjuvant, wherein: each of the one or more glycans is conjugated with the carrier through a linker, having the formula (III) or (IV): [Image disponible dans le document PDF, Image available in the PDF document] (III); [Image disponible dans le document PDF, Image available in the PDF document] (IV) wherein <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R1<annotation encoding="application / x-tex">R^1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R3<annotation encoding="application / x-tex">R^3< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R4<annotation encoding="application / x-tex">R^4< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R5<annotation encoding="application / x-tex">R^5< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R6<annotation encoding="application / x-tex">R^6< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R8<annotation encoding="application / x-tex">R^8< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R9<annotation encoding="application / x-tex">R^9< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R10<annotation encoding="application / x-tex">R^{10}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R11<annotation encoding="application / x-tex">R^{11}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>L<annotation encoding="application / x-tex">L< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>RN<annotation encoding="application / x-tex">R_N< / annotation>< / semantics>< / com:Math> are as described herein. In certain aspects, it is contemplated that any construct of vaccine containing a combination of any one or more of the three glycans (SSEA3, SSEA4 and Globo-H) and analogs thereof in any ratio can be linked to a carrier. [Image disponible dans le document PDF, Image available in the PDF document] wherein n can be an integer from 1 to 10; wherein Glycan can be selected from the group consisting of Formulas I, II, III, and IV; wherein if n is 2 or more, each Glycan can be the same as another Glycan on the aspartyl peptide or a difference Glycan on the aspartyl peptide. In some embodiments, Glycan can be selected from the group consisting of SSEA-3, SSEA-4, and Globo-5. In some embodiments, the exemplary multivalent construct can be: [Image disponible dans le document PDF, Image available in the PDF document] [Image disponible dans le document PDF, Image available in the PDF document] ---- [Image disponible dans le document PDF, Image available in the PDF document] Wherein R<com:Sup>1< / com:Sup>, R<com:Sup>2< / com:Sup>, R<com:Sup>3< / com:Sup>, R<com:Sup>4< / com:Sup>, R<com:Sup>5< / com:Sup>, R<com:Sup>6< / com:Sup>, and L on each Glycan moiety can be the same or different.

[0003] In certain embodiments, the immunogenic composition of the invention comprises an adjuvant. Adjuvants suitable for the invention are as described herein.

[0004] In certain embodiments, the immunogenic composition is capable of eliciting an immune response against a cancer cell in a subject. In certain embodiments, the cancer cell is selected from the group consisting of a brain cancer cell, a lung cancer cell, a breast cancer cell, an oral cancer cell, an esophagus cancer cell, a stomach cancer cell, a liver cancer cell, a bile duct cancer cell, a pancreas cancer cell, a colon cancer cell, a kidney cancer cell, a bone cancer cell, a skin cancer cell, a cervix cancer cell, an ovary cancer cell, and a prostate cancer cell.

[0005] In certain embodiments, the immune response includes generation of antibodies that specifically bind to one or more of the antigens selected from the group consisting of Globo H, SSEA-3 and SSEA-4. In certain embodiments, the antibodies are developed to neutralize one or more of Globo H, SSEA-3 and SSEA-4 expressed on the surface of cancer cells or cancer stem cells. In certain embodiments, the antibodies predominantly include IgG antibodies. In certain embodiments, the immunogenic compositions provided herein mainly induce IgG1, IgG2b, IgG2c and IgG3.

[0006] Further, the present disclosure features monoclonal antibodies and binding fragments raised against the immunogenic composition described herein.

[0007] In one embodiment, the antibody is a human antibody.

[0008] In one embodiment, the antibody is a humanized antibody.

[0009] In one embodiment, the antibody is specifically targeted against one or more of SSEA4, SSEA3, or Globo-H.

[0010] In one embodiment, the antibody is specifically targeted against SSEA3.

[0011] In one embodiment, the antibody is specifically targeted against SSEA4.

[0012] In one embodiment, the antibody is a homogeneous antibody having the biantennary glycan terminated by two sialic acid in alpha-2,6-linkage.

[0013] In one aspect, the present disclosure provides a pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment specifically targeted against one or more of SSEA4, SSEA3, or Globo-H and a pharmaceutically acceptable carrier

[0014] In one embodiment, the pharmaceutical composition comprises a combination of antibodies and / or binding fragment thereof each independently targeting one or more of the SSEA4, SSEA3, or Globo-H glycans.

[0015] In one embodiment, the pharmaceutical composition is useful for the treatment of cancer, infectious diseases, and / or anti-inflammatory diseases,

[0016] In one embodiment, the pharmaceutical composition comprises antibodies or binding fragments thereof having universal biantennary n-glycan terminated with sialic acid in alpha-2,6-linkage.

[0017] In another aspect, the present invention provides a cancer vaccine comprising an immmunogenic composition described herein and a pharmaceutically acceptable excipient.

[0018] In another aspect, the present invention provides methods for treating and / or reducing the risk for cancer in a subject comprising administering to a subject in need thereof a therapeutically effective amount of an immunogenic composition or a cancer vaccine as described herein.

[0019] The treatment results in reduction of tumor size, elimination of malignant cells, prevention of metastasis, prevention of relapse, reduction or killing of disseminated cancer, prolongation of survival and / or prolongation of time to tumor cancer progression.

[0020] In some embodiments, the treatment further comprises administering an additional therapy to said subject prior to, during or subsequent to said administering of the immunogenic composition or the cancer vaccine described herein. In some embodiments, the additional therapy is treatment with a chemotherapeutic agent. In some embodiments, the additional therapy is radiation therapy.

[0021] Another aspect of the present disclosure features a method of vaccinating a mammal against cancers, comprising administering to the mammal a pharmacologically effective amount of an immunogenic composition or a cancer vaccine as described herein.

[0022] In some embodiments, the mammal is a human. In some embodiments, the immunogenic composition or the cancer vaccine described herein is administered subcutaneously.

[0023] Examples of the cancer include, but are not limited to, brain cancer, lung cancer, breast cancer, oral cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, pancreas cancer, colon cancer, kidney cancer, cervix cancer, ovary cancer and prostate cancer. In some embodiments, the cancer is brain cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, or pancreas cancer.

[0024] In another aspect, the present invention provides methods of synthesizing the compounds of the invention as described herein.

[0025] In yet another aspect, the present disclosure features the process for making an immunogenic composition or a cancer vaccine as described herein. [0025A] In another aspect, the present disclosure provides a method of detecting a ESA<com:Sup>hi< / com:Sup>PROCR<com:Sup>hi< / com:Sup>SSEA-3<com:Sup>+< / com:Sup> or CD44<com:Sup>+< / com:Sup>CD24<com:Sup>- / lo< / com:Sup>SSEA-3<com:Sup>+< / com:Sup> breast cancer stem cell in a patient, the method comprising: contacting a sample that has been isolated from the patient with antibodies that bind to cancer stem cell markers SSEA-3, ESA, and PROCR, or antibodies that bind to cancer stem cell markers SSEA-3, CD24, and CD44 on the breast cancer stem cell; and detecting a specific binding of the antibodies that bind to the cancer stem cell markers on the breast cancer stem cell, thereby detecting the ESA<com:Sup>hi< / com:Sup>PROCR<com:Sup>hi< / com:Sup>SSEA-3<com:Sup>+< / com:Sup> or CD44<com:Sup>+< / com:Sup>CD24<com:Sup>- / lo< / com:Sup>SSEA-3<com:Sup>+< / com:Sup> breast cancer stem cell. [0025B] In another aspect, the present disclosure provides a method of detecting the presence of ESA<com:Sup>hi< / com:Sup>PROCR<com:Sup>hi< / com:Sup>SSEA-3<com:Sup>+< / com:Sup> or CD44<com:Sup>+< / com:Sup>CD24<com:Sup>- / lo< / com:Sup>SSEA-3<com:Sup>+< / com:Sup> breast cancer stem cells in a patient, the method comprising: analyzing an expression of cancer stem cell markers on cells in a sample isolated from the patient, wherein the cancer stem cell markers are i) SSEA-3, CD24, and CD44; or ii) SSEA-3, PROCR, and ESA, wherein the presence of cells expressing ESA<com:Sup>hi< / com:Sup>PROCR<com:Sup>hi< / com:Sup>SSEA-3<com:Sup>+< / com:Sup> or CD44<com:Sup>+< / com:Sup>CD24<com:Sup>-< / com:Sup> <com:Sup> / lo< / com:Sup>SSEA-3<com:Sup>+< / com:Sup> indicates the presence of the ESA<com:Sup>hi< / com:Sup>PROCR<com:Sup>hi< / com:Sup>SSEA-3<com:Sup>+< / com:Sup> or CD44<com:Sup>+< / com:Sup>CD24<com:Sup>- / lo< / com:Sup>SSEA-3<com:Sup>+< / com:Sup> breast cancer stem cells. [0025C] In another aspect, the present disclosure provides a method for identifying whether a breast cancer is present in a patient, comprising: determining whether a sample obtained from the patient comprises stem cells that overexpress <com:Math><semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics>< / com:Math>-1,3-galactosyltransferase 5 (<com:Math><semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics>< / com:Math>3GalT5); and assaying for the presence of cancer stem cell markers in the sample obtained from the patient, wherein the cancer stem cell markers are i) SSEA-3, CD24, and CD44; or ii) SSEA-3, PROCR, and ESA, wherein the presence of stem cells expressing ESA<com:Sup>hi< / com:Sup>PROCR<com:Sup>hi< / com:Sup>SSEA-3<com:Sup>+< / com:Sup> or CD44<com:Sup>+< / com:Sup>CD24<com:Sup>- / lo< / com:Sup>SSEA-3<com:Sup>+< / com:Sup>, and overexpressing <com:Math><semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics>< / com:Math>-1,3-galactosyltransferase 5 (<com:Math><semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics>< / com:Math>3GalT5) indicates that the breast cancer is present. [0025D] In another aspect, the present disclosure provides a method for identifying a population of breast cancer stem cells, the method comprising: a) providing a starting population of cancer cells, b) determining the expression level of cancer stem cell markers i) SSEA-3, CD24, and CD44; or ii) SSEA-3, PROCR, and ESA, in the starting population of cancer cells, c) selecting a population of cells expressing ESA<com:Sup>hi< / com:Sup>PROCR<com:Sup>hi< / com:Sup>SSEA-3<com:Sup>+< / com:Sup> or CD44<com:Sup>+< / com:Sup>CD24<com:Sup>- / lo< / com:Sup>SSEA-3<com:Sup>+< / com:Sup>, wherein said selected population of cells are breast cancer stem cells, and d) isolating and / or enriching for said population of cells selected in step c).

[0026] The details of certain embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the Detailed Description, the Figures, the Examples, and the Claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 the tumorigenicity of cells carrying conventional markers and SSEA-3 was higher than other subpopulations. A, C Percentage of cell colony or mammosphere formation of the subpopulation isolated by selected marker(s) for suspension culture or soft agar assay in MCF-7 or MDA-MB-231 respectively. Graphs are the triple samples from one representative experiment. B, D The number of tumor formed in mammary gland of NS injected with selected marker-expressing cell subpopulations from breast cancer cell lines MDA-MB-231 and MCF-7. The corresponding limiting dilution assay was done in vivo. E, F The tumor volume from different subpopulations of MDA-MB- 231 (2500 cells / injection), and MCF-7 (500 cells / injection) was monitored and compared (n=4 tumors per group). #the percentage of SSEA-3<com:Sup>+< / com:Sup> cells sorted in the total cell population. Data represent the mean and standard deviation (S.D.). Asterisks indicate statistical significance, <com:Math><semantics>p<0.05<annotation encoding="application / x-tex">p < 0.05< / annotation>< / semantics>< / com:Math>.

[0028] Figure 2 Knockdown or overexpression of β3GalT5 in MCF-7 and MDA-MB-231 cell culture reduced or increased the level of SSEA-3 on cell surface and stemness properties by FACS analysis. The expression of cancer stem cell markers and SSEA-3 in parental cells. The level of SSEA-3 in subpopulations CD44<com:Sup>+< / com:Sup>CD24<com:Sup>- / lo< / com:Sup> and ESA<com:Sup>hi< / com:Sup> PROCR<com:Sup>hi< / com:Sup> gated in overexpressed β3GalT5 group was also determined. A The expression of CD24, CD44 and SSEA-3 in MCF-7 with overexpression, knocked down of β3GalT5 or their corresponding vector control. B The expression of ESA, PROCR and SSEA-3 in MDA-MB-231 with overexpression, knockdown down of beta-3GalT5 or their corresponding vector control. All of experiments are the representative sample from triplicate.

[0029] Figure 3- the induction of apoptosis in β3GalT5 knockdown cell lines. A The relative percentage of gene expression (β3GalT5 in MCF-7, β3GalT5, FUT1, FUT2 and ST3Gal2 in MDA-MB-231) after knocking down the respective genes. The percentage of gene expression in vector control cells were normalized to 100. B. The mean percentage of apoptosis in breast normal (hTERT-HME1, MCF-10A) and cancer (MCF-7, MDA-MB-231) cell lines from three experiments. C Flow cytometric analysis of the apoptosis percentage in breast cancer cell lines MDA-MB-231, MCF-7, and breast non-cancer lines hTERT-HME1 and MCF-10A was examined after knockdown of beta3GalT5 for 4 days. The apoptotic cells were compared with unstained cells and gated. D The percentage of apoptosis in MDA-MB- 231 cells with knockdown of gene FUT1, FUT2, ST3Gal2 or beta3GalT5 and vector control. The mean of apoptotic cells is from three experiments. Asterisks indicate statistical significance, <com:Math><semantics>p<0.05<annotation encoding="application / x-tex">p < 0.05< / annotation>< / semantics>< / com:Math>; n.s. not significant.

[0030] Figure 4- Knockdown of β3GalT5 caused reduced proliferation rate and increased apoptosis in cancer cell culture but no effect on normal breast cell culture. A-D The rate of proliferation in cancer cell culture MCF-7 and MDA-231, as well as breast normal cell culture MCF-10A and hTERT-HME1. Proliferation rate, in terms of absorbance (A450nm-A690nm), is the triplicate from a representative sample. E MDA-MB-231 cells infected with shRNA beta3GalT5 or shRNA vector were lysed and whole-cell extract, cytoplasmic and nuclear fractions were prepared. Top, western-blot analysis of anti-caspase-3 antibody; middle, that of cleaved caspase-3 antibody; bottom, that of <com:Math><semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics>< / com:Math>-actin (served as a loading control) F, G The percentage of apoptotic MDA-MB-231 cells with β3GalT5 knockdown. MDA-MB-231 cells were treated with caspase-3 inhibitors Z-DEVD in different concentrations or the inhibitors for caspase-8, 9 or 12. Data represent here is the mean and standard deviation (S.D.) from triplicated sample. Asterisks indicate statistical significance, <com:Math><semantics>p<0.05<annotation encoding="application / x-tex">p < 0.05< / annotation>< / semantics>< / com:Math>; n.s. not significant.

[0031] Figure 5- the comparison of the abundance of globo-series epitopes in cell lines by flow cytometry and mass spectrometry. A The scheme of extraction of glycan from glycolipid on cells for fluorescent labeling and LC-MS analysis. B-G The relative abundance of globo- series epitopes SSEA-3, SSEA-4 and globo-H in breast cancer cell lines MCF-7 and MDA- MB-231, normal cell lines hTERT-HME1 and MCF-10A, embryonic stem cells (ESC) as well as induced pluripotent stem cells (iPSC) was detected by FACS and mass spectrometry. For figures of flow cytometry, histograms of cells stained with anti-glycan antibodies (in red, blue or green) and their corresponding antibody isotype controls (in gray) were shown. Geometric mean of fluorescence was shown in the bracket. For MS, the retention times of m / z (SSEA- 3=1008.3667, SSEA-4=1299.4621 and globo-H=1154.4246) were shown in graphs.

[0032] Figure 6. (Fig. S1)- the subpopulations in cell lines obtained by sorting for in vitro and in vivo assays. Subpopulations including A CD44<com:Sup>+< / com:Sup> CD24<com:Sup>hi< / com:Sup>, CD44<com:Sup>+< / com:Sup> CD24<com:Sup>- / lo< / com:Sup>, CD44<com:Sup>+< / com:Sup> CD24<com:Sup>- / lo< / com:Sup> SSEA-3<com:Sup>+< / com:Sup>, CD44<com:Sup>+< / com:Sup> CD24<com:Sup> / lo< / com:Sup> SSEA-3<com:Sup>-< / com:Sup>, various percentages of SSEA-3<com:Sup>+< / com:Sup> (top 1, 5, 10%), and SSEA-3 in MCF-7, as well as B ESAloPROCRlo, ESAliPROCRli, ESAliPROCRliSSEA- 3<com:Sup>+< / com:Sup>, ESA<com:Sup>hi< / com:Sup>PROCR<com:Sup>hi< / com:Sup>SSEA-3<com:Sup>-< / com:Sup>, various percentages of SSEA-3<com:Sup>+< / com:Sup> (top 1, 5, 10%), and SSEA-3<com:Sup>-< / com:Sup> in MDA-MB-231 were enriched by cell staining (as the methods and materials) and flow cytometry for the further analysis.

[0033] Figure 7 (Fig. S2)- BCSCs were not enriched with globo-series epitopes SSEA-4 and globo-H by in vitro assays. A-D Percentage of cell colony formation of unsorted cells or selected marker (known marker set CD24 / CD44, or ESA / PROCR, along with SSEA-4 or globo-H) expressing cell subpopulation from breast cancer cell lines MCF-7 and MDA-MB- 231. Graphs are the triple samples from one representative experiment. Data represent the mean and standard deviation (S.D.). Asterisks indicate statistical significance, <com:Math><semantics>p<0.05<annotation encoding="application / x-tex">p < 0.05< / annotation>< / semantics>< / com:Math>; n.s. not significant.

[0034] Figure 8 (Fig. S3)- the globo-series pathway in human. The biosynthetic pathway of globo-series epitopes SSEA-3, SSEA-4 and globo-H from Gb4 with corresponding glycotransferases.

[0035] Figure 9 (Fig. S4)- the characterization of iPSC5. A Immunofluorescent staining of stem cell proteins TRA1-60 and Nanog. Nuclei were stained with DAPI. B qPCR of stem genes OCT4, SOX2, NANOG and c-Myc. C in vitro differentiation capability of iPSC5 to three germ layer lineages. D H&E staining of iPSC5 to three germ layer lineages in teratoma.

[0036] Figure 10 (Fig S5)- the mRNA level of beta3GalT5 in breast cancer cell culture is higher than that of normal cell culture. GAPDH-normalized qPCR level of beta3GalT5 gene in normal cell culture MCF-10A and hTERT-HME1, as well as in breast cancer cell culture MCF-7 and MDA-MB-231. Triplicated samples from one representative experiment are shown. Data represent the mean and standard deviation (S.D.). Asterisks indicate statistical significance, <com:Math><semantics>p<0.05<annotation encoding="application / x-tex">p < 0.05< / annotation>< / semantics>< / com:Math>; n.s. not significant.

[0037] Figure 11. Biosynthetic Pathway of Globo Series of Glycosphingolipids.

[0038] Figure 12. The induced GH-IgG collected from different epitope ratios of SSE4-DT or SSEA4-Gc-DT immunization DETAILED DESCRIPTIONS

[0039] The present invention is based on the finding that tumor-associated carbohydrate antigens are suitable cancer stem cell markers.

[0040] The present disclosure is based on the surprising discovery that tumor-associated carbohydrate antigens are expressed on cancer stem cells. Thus, these tumor-associated carbohydrate antigens are suitable markers for cancer stem cells and furthermore, provide suitable therapeutic targets for a therapy that attacks cancer stem cells.

[0041] Both normal stem cells and tumorigenic cells have extensive proliferative potential and the ability to give rise to new (normal or abnormal) tissues. Tumorigenic cells can be thought of as cancer stem cells (CSC) or cancer initiating cells (CIC - the terms CSC and CIC are used as synonyms herein) that undergo an aberrant and poorly regulated process of organogenesis analogous to what normal stem cells do. Both tumors and normal tissues are composed of heterogeneous combinations of cells, with different phenotypic characteristics and different proliferative potentials.

[0042] Cancer stem cells are believed to be a certain fraction of tumor cells with stem cell- like properties, which initiate and maintain neoplastic clones. These cells have the ability to self-renew, but also give rise to progenitors that yield phenotypically diverse cancer cells but with lower tumorigenic potential. This subpopulation of stem cell-like cells are the ones that are efficient at tumor formation and metastatic tumor spread as compared to tumor cells that are not cancer stem cells.

[0043] Cancer stem cells (CSCs) have now been identified in a wide variety of cancers including leukemias, glioblastomas, medulloblastomas, and almost all types of epithelial tumors (carcinomas). Cancer stem cells can be characterized based on the investigation of distinct surface marker patterns within primary tumors. CD44 was reported as a robust marker of cancer stem cells. A single CD44+ cell from a colorectal tumor could form a sphere in vitro and was able to generate a xenograft tumor resembling the properties of the primary tumor. CD133 is also a marker of cancer stem cells.

[0044] The presence of cancer stem cells has profound implications for cancer therapy. Existing therapies have been developed largely against the bulk population of tumor cells, because the therapies are identified by their ability to shrink the tumor mass. However, cancer stem cells are often resistant to chemotherapy and can account for chemotherapy failure. To design novel therapeutic agents that (also) target cancer-initiating cells (also referred herein as cancer stem cells), it will be desirable to seek molecular targets of cancer stem cells that are preferably absent on benign tumors and / or normal non-tumor cells, and at the same time are specifically directed against cancer stem cells. Such agents are anticipated to result in more durable responses and cures of tumors, and especially of metastatic tumors. Therefore, new cancer stem cell markers are wanted to provide novel therapeutic targets to improve therapy. Most of the known stem-cell markers are proteins. Many of them have also been found to be normal stem cell markers and are thus expressed on non-tumor stem cells. This makes them not or at least less suitable as therapeutic target. At present, there is no clear cut distinction between normal and cancer stem cell markers.

[0045] A "binding agent" may be any compound or complex of compounds which is capable of binding a target substance such as a tumor-associated carbohydrate antigen and / or combination of carbohydrate and non-carbohydrate specific antigens, individually or in a combination (such as a panel). Preferably, the binding agent is capable of specifically binding the target substance. Suitable binding agents may be obtained by screening a binding agent library in order to identify / obtain binding agents that bind to the target substance. Examples for respective binding agent includes glycoantibodies. Such as antibodies against SSEA3, SSEA4, and / or globoH. The binding agents may have any structure, as long as they are able to specifically recognize and bind the target substance, here a tumor-associated carbohydrate antigen. Binding agents may be selected from the group consisting of antibodies, antigen- binding fragments or derivatives thereof or binding agents having a protein scaffold providing a binding function such as for example anticalins or lectins. Binding agents may also be peptides or fusion proteins providing a binding function. An overview of binding agents which have a similar binding function as antibodies is given in Hey, et al. (Hey et al. (2005) "Artificial, non-antibody binding proteins for pharmaceutical and industrial application", Trends in Biotechnology 23(10), 514-522). An antibody derivative also includes antibodies or antibody fragments having the same binding function but e.g. an altered amino acid sequence.

[0046] As used herein, exemplary binding agents targeting the SSEA3 marker is reported in USSN 14 / 599,174.

[0047] According to the invention, "staging" of a cancer preferably refers to the classification of the progression and extent of a cancer. A preferred cancer staging system is the TNM classification of malignant tumors, wherein T describes the size of the tumor and whether it has invaded nearby tissue, N describes regional lymph nodes that are involved, and M describes distant metastasis. Each of these parameters is given a particular value depending on the situation in the patient, wherein generally a higher number indicates a more severe situation (T(0-4), N(0-3), M(0 / 1)). Additionally, for a more detailed classification further parameters can be determined and / or prefixes can be used. Furthermore, the TNM classification may be summarized in a cancer staging system according to the UICC, referring to cancer of from stage 0 to stage IV.

[0048] According to the invention, a "sample" in particular refers to but is not limited to a tissue sample, a body fluid and / or a cellular sample and may be obtained by conventional manners such as by tissue biopsy, including punch biopsy or by taking blood, bronchial aspirate, sputum, urine, feces or other body fluids or tissue sections, slides, etc. containing or suspected of containing cancer cells. According to the invention, the term "sample" also includes fractions or components of respective samples.

[0049] The terms "cell proliferation" and "to proliferate" as used herein in particular refer to the amplification of the cell by cell division. The term "cancer stem cells" in particular relates but is not limited to cells capable of generating aggregates of undifferentiated cells, so called tumor spheres, under suitable conditions in vitro. The cells that form spheres are capable of self-renewal; when they are dissociated and grown under the same conditions, they will form spheres again. In vivo, cancer stem cells are characterized by their potential to form metastases and the expression of stem cell markers such as, e.g., CD44. They may also provide drug resistance. The terms "cancer stem cells" and "cancer initiating cells" are used as synonyms herein.

[0050] The term "tumor-associated carbohydrate antigen" in particular refers to a carbohydrate antigen that is expressed on cancer and / or tumor cells, in particular on malignant cancer and / or malignant tumor cells.

[0051] The term "tumor-specific carbohydrate antigen" in particular refers to a carbohydrate antigen that is predominantly or even exclusively expressed on cancer and / or tumor cells and thus not or only to a low extent on non-cancer respectively non-tumor cells. Preferably, the term "tumor-specific carbohydrate antigen" refers to a carbohydrate antigen that is predominantly or preferably exclusively expressed on malignant cancer and / or malignant tumor cells and thus not or only to a low extent on non-cancer respectively non-tumor cells, on benign cancer and / or benign tumor cells and / or on healthy tissue of the same patient. Preferentially, the tumor-specific carbohydrate antigen is not expressed on most normal cells, even more preferred it is expressed only on few normal cells or cell types, even more preferred the expression on these normal cells has a special localization, e.g. strictly apical or in between the tight junctions, so that a binding molecule administered systemically, and especially i.v., can not or barely reach the antigen on these normal cells, even more preferred it is not expressed on normal epithelial cells, most preferred it is not expressed on normal cells. In certain embodiments, the tumor-specific carbohydrate antigen may be attached to a carrier molecule when expressed. Such carrier molecule may in particular be a protein, peptide or carbohydrate.

[0052] "CD44" is an adherence molecule (H-CAM, Pgp-1) of varying molecular weight. It is a cell surface hyaluronan receptor, interacts with matrix metalloproteinases, and plays a key role in cell migration. CD44 has been described as a cancer stem cell marker in breast, ovarian, pancreatic, prostate, colon, gastric, and other cancer types (see Li et al, 2007, and Takaishi et al, 2009). It is also a marker of normal pluripotent stem cells.

[0053] The present inventors have shown that several tumor-associated antigens such as CD24, CD44, SSEA3, PROCR, ESA are expressed on cancer stem cells and thus, are novel cancer stem cell markers. The identification of tumor-associated carbohydrate antigens as cancer stem cell markers provides novel therapeutic applications for agents specifically binding to one or more of said tumor-associated carbohydrate antigens. Agents specifically binding to one or more respective tumor-associated carbohydrate antigen can now be therapeutically used for targeting cancer stem cells that express said tumor- associated carbohydrate antigen. This provides the opportunity of therapeutic treatments that target and preferably kill cancer stem cells. The respective therapeutic agents can for example be used to target and thus destroy cancer stem cells which are resistant to regular chemotherapy. Thereby, improved cancer therapies are provided with the present invention. According to one embodiment, the tumor-associated carbohydrate antigen is expressed predominantly or even exclusively on breast cancer stem cells. According to another embodiment, the tumor- associated carbohydrate antigen is expressed on the cancer stem cells as well as on cancer cells that are not cancer stem cells. If the tumor-associated carbohydrate antigen is expressed on both cell populations, this has the advantage that treatment with the binding agent specifically binding the tumor-associated carbohydrate antigen targets both cell populations.

[0054] According to one embodiment, the binding agent specifically binding to the one or more tumor-associated carbohydrate antigens is therapeutically active. One example of a respective embodiment is the use of a therapeutically active antibody or antigen-binding fragment or derivative thereof as binding agent. A therapeutically active antibody or antigen- binding fragment or derivative thereof preferably is capable of inducing complement- dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC), which preferably results in lysis of the target cell, in particular the cancer stem cell expressing the tumor-associated carbohydrate antigen. According to a further embodiment, the binding agent specifically binding the tumor-associated carbohydrate antigen functions as a targeting molecule and is coupled to at least one therapeutic agent. Coupling can be achieved by covalent or non-covalent means. When the binding agent specifically binding the tumor- associated carbohydrate antigen functions as a targeting molecule, it can be itself therapeutically active or it may not be therapeutically active. In case it is not therapeutically active, it basically functions as a molecular carrier which brings the actual therapeutic agent (e.g. a radiopharmaceutical, chemotherapeutic agent or a toxin) to the desired target side of action, namely the cancer stem cells expressing the tumor-associated carbohydrate antigen. The therapeutic agent coupled to the binding agent specifically binding the tumor-associated carbohydrate antigen can be for example a chemotherapeutic agent or other anti-cancer drug. Said coupled therapeutic agent preferably destroys or kills the targeted cancer stem cells or inhibits proliferation thereof. This can either be achieved directly by the coupled therapeutic agent or indirectly via induction of suitable biological mechanisms of the targeted cancer stem cells and / or of the subject to be treated.

[0055] In certain embodiments, the tumor-associated carbohydrate antigen as described herein can be further combined with any one or more of the markers selected from the group consisting of CD24, CD44, PROCR, ESA, CD176, CD175, CD175s, CD174, CD173 and CA19-9.

[0056] According to a further aspect of the present invention, a method is provided for identifying a cancer comprising cancer stem cells that is susceptible to treatment with a binding agent that specifically binds a tumor-associated carbohydrate antigen wherein said treatment effects the cancer stem cells, comprising determining whether a cancer sample obtained from a patient comprises cancer stem cells that express the tumor-associated carbohydrate antigen the binding agent is specific for, wherein the presence of said tumor- associated carbohydrate antigen on cancer stem cells indicates that the cancer is susceptible to treatment with the binding agent that specifically binds a tumor- associated carbohydrate antigen and wherein said treatment also effects the cancer stem cells.

[0057] This method according to the present invention allows to test whether the cancer stem cells of a cancer are susceptible to treatment with a binding agent that specifically binds a tumor-associated carbohydrate antigen. The results of the method provide valuable diagnostic information to the physician. E.g. in case the cancer would comprise cancer stem cells which do not express said tumor-associated carbohydrate antigen, treatment with a binding agent specifically binding said tumor-associated carbohydrate antigen would not affect the cancer stem cells and accordingly, would be useless against the cancer stem cells. However, in case it is shown by said method that the cancer stem cells express said tumor- associated carbohydrate antigen the binding agent is specific for, chances are good that treatment with said binding agent will also target and accordingly affect the cancer stem cells. Thus, the method according to the present invention provides valuable aid to the physician for choosing the best therapy for the patient and to estimate whether a certain treatment will affect the cancer stem cells of a cancer.

[0058] According to a related diagnostic aspect, a method is provided for diagnosing, staging and / or prognosing cancer and / or monitoring the susceptibility to treatment, comprising the step of analyzing the expression of a tumor-associated carbohydrate antigen on cells in a sample isolated from a patient, wherein the presence of cells expressing the tumor-associated carbohydrate antigen indicates the presence of cancer stem cells in said sample.

[0059] According to a further aspect of the present invention, a method is provided for identifying a population of cancer stem cells, the method comprising: a) providing a starting population of cancer cells, b) determining the expression level of one or more tumor- associated carbohydrate antigens, c) selecting a population of cells whose expression level of said one or more tumor-associated carbohydrate antigens as determined in step b) is increased when compared to control cells, wherein said selected population of cells are cancer stem cells and d) optionally isolating and / or enriching for said population of cells selected in step c), wherein said control cells are cells from the same starting cancer cell population which do not express, or express lower levels of said one or more tumor-associated carbohydrate antigens. In some embodiments, the tumor-associated carbohydrate antigens are selected from the list consisting of SSEA3, SSEA4, and GloboH antigens. In some embodiments, the determining step in b) further includes determining the expression level of one or more additional tumor associated antigens, and the selection and isolating steps in c) and d), respectively, also includes consideration of the expression level of said one or more additional tumor associated antigens. In some embodiments, the one or more tumor-associated antigens include, but are not limited by CD24, CD44, PROCR, ESA, CD176, CD175, CD175s, CD174, CD173 and CA19-9 antigens. In some embodiments, the determining step in b) and / or the isolation step in d) is carried out using FACS. In some embodiments, the level of expression of said one or more tumor-associated carbohydrate antigens is high or highly increased, when compared to control cells. In some embodiments, the level of expression of said one or more tumor- associated carbohydrate antigens is low or lowly increased, when compared to control cells.

[0060] The presence of cancer stem cells in a patient sample can be indicative of the stage of a cancer. In addition, detection of cancer stem cells can be used to monitor response to therapy and to aid in prognosis. The information obtained by the methods according to the present invention is useful in prognosis and diagnosis, including analysing the susceptibility to acceleration of the disease, the analysis by active monitoring of the disease wherein it is analysed whether the cancer progresses and e.g. needs treatment, the status of a disease state, the response to changes in the environment, such as the passage of time, the treatment with a chosen therapeutic agent, in particular a binding agent as described above, or other modalities. By analysing whether cells contained in the sample express a tumor-associated carbohydrate antigen and accordingly, the sample comprises cancer stem cells, the cells can also be classified as to their ability to respond to the rapeutic agents and treatments. Furthermore, the information derived is useful in determining and / or predicting the metastatic behavior of a cancer.

[0061] According to one aspect of the diagnostic methods according to the present invention, the binding agents according to the present invention which specifically bind to one or more tumor-associated carbohydrate antigens expressed on cancer stem cells are used for in vivo diagnostic, in particular in vivo imaging. A respective method is also useful for diagnostic purposes. E.g. it can be determined, whether cancer cells expressing the tumor- associated carbohydrate antigen expressed on cancer stem cells can be identified and / or located in the patient. If this is the case, there is a risk that there are cancer stem cells. As is described above and below, it is preferred that a second stem cell marker is additionally detected to confirm and / or determine the nature of the cancer stem cells. Furthermore, the response to therapy can be monitored as it can be determined e.g. whether the tumor size descreases or whether metases develop. Furthermore, a respective method is advantageous to identify the suitable dosage for a patient. According to one embodiment, the binding agent is labelled, e.g. being a radiopharmaceutical comprising a radionuclide. However, the binding agent may also be coupled to other agents / compounds such as e.g. a PET tracer that allow in vivo imaging. Suitable compounds are known in the prior art and thus, do not need further description here. Details regarding the binding agents, the tumor-associated carbohydrate antigens, further cancer stem cell markers and cancer types are described above and below and also apply to the in vivo imaging embodiment. It is referred to the respective disclosure.

[0062] According to one embodiment, samples, containing or suspected of containing cancer cells, are contacted and preferably stained with at least one agent specifically binding the tumor-associated, preferably tumor-specific carbohydrate antigen and thus the carbohydrate cancer stem cell marker and optionally at least one further agent specifically binding at least one second cancer stem cell marker, such as CD44. This allows to detect the presence of cancer stem cells in the sample. According to one embodiment, binding of the binding agent against the tumor-associated carbohydrate antigen and preferably the binding of the binding agent against the second cancer stem cell marker is detected by appropriate detection methods as known in the art and as described herein. Suitable detection methods are for example ELISA, FACS, fluorescence microscopy and the like.

[0063] Samples to be analysed by the methods of the invention may be obtained from a variety of sources, particularly from a biopsy sample. Cells of such samples can be separated by centrifugation, elutriation, density gradient separation, apheresis, affinity selection, panning, FACS, centrifugation with Hypaque, etc. prior to analysis. Once a sample is obtained, it can be used directly, frozen, or maintained in appropriate culture medium for short periods of time, or fixed in a suited fixation solution, or fixed and embedded in a medium suited for histological or immunohistological examination. Various media can be employed to maintain cells. The samples may be obtained by any convenient procedure, such as biopsy, or from surgical specimen. Usually a sample will comprise at least about 10<com:Sup>2< / com:Sup> cells, more usually at least about 10<com:Sup>3< / com:Sup> cells, and preferable 10<com:Sup>4< / com:Sup>, 10<com:Sup>5< / com:Sup> or more cells. In one embodiment, the sample comprises 10 cells. In one embodiment, the sample comprises any number of cells between 10 and 100 cells. In one embodiment, the sample comprises any number of cells between 10 and 1000 cells. In one embodiment, the sample comprises any number of cells between 1 and 10 cells. Typically the samples will be from human patients, although animal models may find use, e.g. equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc.

[0064] The samples may be frozen, embedded, fixed, present in a tissue microarray, and the like. The agents used for binding, detecting and in particular staining the tumor-associated carbohydrate antigen and, optionally, a further cancer stem cell marker can be e.g. binding agents specifically binding the cancer stem cell markers such as e.g. antibodies. Suitable examples are described above. These agents may be detectably labelled, or may be indirectly labelled in the staining procedure. According to one embodiment, the label can also be used for separating the tumor- associated carbohydrate antigen positive cells. Suitable labels as well as staining procedures are known in the prior art and accordingly, do not need further description here even though some examples are described herein. A standard procedure for analyis may include a histological fixation of the sample (e.g. by formalin) and subsequent staining as is e.g. described in the examples. The obtained data allows to determine the number and distribution of cancer stem cells in the sample.

[0065] Methods suitable for detecting and / or quantifying cells expressing the tumor- associated carbohydrate antigen include, for example, immunologic assays such ELISA, RIA, Western blot and immunohistochemistry, flow cytometry, immunohistochemistry or the like.

[0066] In screening assays for candidate therapeutic agents usually a culture comprising cancer stem cells expressing the tumor-associated carbohydrate antigen of interest is contacted with the binding agent of interest, and the effect of the agent assessed by monitoring output parameters, such as expression of markers, cell viability, and the like. The screening may also involve determining modulation of growth, proliferation, viability, and / or differentiation status of the cell in the presence of the candidate therapeutic agent as compared to the growth, proliferation, viability, and / or differentiation status of the cell in the absence of the candidate therapeutic agent.

[0067] Isolated cell populations that are highly enriched for cancer stem cells expressing one or more tumor-associated carbohydrate antigens can be isolated and enriched / purified using these markers. In some embodiments, said cancer stem cell populations are isolated from the circulation, such as from the blood. In some embodiments, said cancer stem cell populations are isolated from the a tumor sample, or from pleural effusions or other fluids obtained from a cancer patient. Chemical definitions

[0068] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987. Moreover, exemplary glycan and antibody methodologies are described in Wong et al, US20100136042, US20090317837, and US20140051127.

[0069] Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and / or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The invention additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0070] When a range of values is listed, it is intended to encompass each value and sub- range within the range. For example "<com:Math><semantics>C1−6<annotation encoding="application / x-tex">C_{1-6}< / annotation>< / semantics>< / com:Math>" is intended to encompass <com:Math><semantics>C1<annotation encoding="application / x-tex">C_1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C2<annotation encoding="application / x-tex">C_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C3<annotation encoding="application / x-tex">C_3< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C4<annotation encoding="application / x-tex">C_4< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C5<annotation encoding="application / x-tex">C_5< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C6<annotation encoding="application / x-tex">C_6< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C1−6<annotation encoding="application / x-tex">C_{1-6}< / annotation>< / semantics>< / com:Math>" <com:Math><semantics>6<annotation encoding="application / x-tex">6< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C1−5<annotation encoding="application / x-tex">C_{1-5}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C1−4<annotation encoding="application / x-tex">C_{1-4}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C1−3<annotation encoding="application / x-tex">C_{1-3}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C1−2<annotation encoding="application / x-tex">C_{1-2}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C2−6<annotation encoding="application / x-tex">C_{2-6}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C2−5<annotation encoding="application / x-tex">C_{2-5}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C2−4<annotation encoding="application / x-tex">C_{2-4}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C2−3<annotation encoding="application / x-tex">C_{2-3}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C3−6<annotation encoding="application / x-tex">C_{3-6}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C3−5<annotation encoding="application / x-tex">C_{3-5}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C3−4<annotation encoding="application / x-tex">C_{3-4}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C4−6<annotation encoding="application / x-tex">C_{4-6}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C4−5<annotation encoding="application / x-tex">C_{4-5}< / annotation>< / semantics>< / com:Math>, and <com:Math><semantics>C5−6<annotation encoding="application / x-tex">C_{5-6}< / annotation>< / semantics>< / com:Math>.

[0071] "Alkyl" refers to a radical of a straight—chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms ("<com:Math><semantics>C1−20<annotation encoding="application / x-tex">C_{1-20}< / annotation>< / semantics>< / com:Math> alkyl"). In some embodiments, an alkyl group has 1 to 10 carbon atoms ("<com:Math><semantics>C1−10<annotation encoding="application / x-tex">C_{1-10}< / annotation>< / semantics>< / com:Math> alkyl"). In some embodiments, an alkyl group has 1 to 9 carbon atoms ("C<com:Sub>1-9< / com:Sub> alkyl"). In some embodiments, an alkyl group has 1 to 8 carbon atoms ("<com:Math><semantics>C1−8<annotation encoding="application / x-tex">C_{1-8}< / annotation>< / semantics>< / com:Math> alkyl"). In some embodiments, an alkyl group has 1 to 7 carbon atoms ("<com:Math><semantics>C1−7<annotation encoding="application / x-tex">C_{1-7}< / annotation>< / semantics>< / com:Math> alkyl"). In some embodiments, an alkyl group has 1 to 6 carbon atoms ("<com:Math><semantics>C1−6<annotation encoding="application / x-tex">C_{1-6}< / annotation>< / semantics>< / com:Math> alkyl"). In some embodiments, an alkyl group has 1 to 5 carbon atoms ("<com:Math><semantics>C1−5<annotation encoding="application / x-tex">C_{1-5}< / annotation>< / semantics>< / com:Math> alkyl"). In some embodiments, an alkyl group has 1 to 4 carbon atoms ("<com:Math><semantics>C1−4<annotation encoding="application / x-tex">C_{1-4}< / annotation>< / semantics>< / com:Math> alkyl"). In some embodiments, an alkyl group has 1 to 3 carbon atoms ("<com:Math><semantics>C1−3<annotation encoding="application / x-tex">C_{1-3}< / annotation>< / semantics>< / com:Math> alkyl"). In some embodiments, an alkyl group has 1 to 2 carbon atoms ("<com:Math><semantics>C1−2<annotation encoding="application / x-tex">C_{1-2}< / annotation>< / semantics>< / com:Math> alkyl"). In some embodiments, an alkyl group has 1 carbon atom ("<com:Math><semantics>C1<annotation encoding="application / x-tex">C_1< / annotation>< / semantics>< / com:Math> alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C<com:Sub>2-6< / com:Sub> alkyl"). Examples of <com:Math><semantics>C1−6<annotation encoding="application / x-tex">C_{1-6}< / annotation>< / semantics>< / com:Math> alkyl groups include methyl <com:Math><semantics>(C1)<annotation encoding="application / x-tex">(C_1)< / annotation>< / semantics>< / com:Math>, ethyl <com:Math><semantics>(C2)<annotation encoding="application / x-tex">(C_2)< / annotation>< / semantics>< / com:Math>, n-propyl <com:Math><semantics>(C3)<annotation encoding="application / x-tex">(C_3)< / annotation>< / semantics>< / com:Math>, iso-propyl <com:Math><semantics>(C3)<annotation encoding="application / x-tex">(C_3)< / annotation>< / semantics>< / com:Math>, n-butyl (C<com:Sub>4< / com:Sub>), tert-butyl (C<com:Sub>4< / com:Sub>), sec-butyl (C<com:Sub>4< / com:Sub>), iso-butyl (C<com:Sub>4< / com:Sub>), n-pentyl (C<com:Sub>5< / com:Sub>), 3-pentanyl (C<com:Sub>5< / com:Sub>), amyl (<com:Math><semantics>C5<annotation encoding="application / x-tex">C_5< / annotation>< / semantics>< / com:Math>), neopentyl (<com:Math><semantics>C5<annotation encoding="application / x-tex">C_5< / annotation>< / semantics>< / com:Math>), 3-methyl-2-butanyl (<com:Math><semantics>C5<annotation encoding="application / x-tex">C_5< / annotation>< / semantics>< / com:Math>), tertiary amyl (<com:Math><semantics>C5<annotation encoding="application / x-tex">C_5< / annotation>< / semantics>< / com:Math>), and n-hexyl (<com:Math><semantics>C6<annotation encoding="application / x-tex">C_6< / annotation>< / semantics>< / com:Math>). Additional examples of alkyl groups include <com:Math><semantics>n<annotation encoding="application / x-tex">n< / annotation>< / semantics>< / com:Math>-heptyl (<com:Math><semantics>C7<annotation encoding="application / x-tex">C_7< / annotation>< / semantics>< / com:Math>), <com:Math><semantics>n<annotation encoding="application / x-tex">n< / annotation>< / semantics>< / com:Math>-octyl (<com:Math><semantics>C8<annotation encoding="application / x-tex">C_8< / annotation>< / semantics>< / com:Math>) and the like. Unless otherwise specified, each instance of an alkyl group is independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") with one or more substituents. In certain embodiments, the alkyl group is unsubstituted <com:Math><semantics>C1−10<annotation encoding="application / x-tex">C_{1-10}< / annotation>< / semantics>< / com:Math> alkyl (e.g., - CH<com:Sub>3< / com:Sub>). In certain embodiments, the alkyl group is substituted <com:Math><semantics>C1−10<annotation encoding="application / x-tex">C_{1-10}< / annotation>< / semantics>< / com:Math> alkyl.

[0072] "Alkenyl" refers to a radical of a straight—chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon—carbon double bonds, and no triple bonds ("<com:Math><semantics>C2−20<annotation encoding="application / x-tex">C_{2-20}< / annotation>< / semantics>< / com:Math> alkenyl"). In some embodiments, an alkenyl group has 2 to 10 carbon atoms ("<com:Math><semantics>C2−10<annotation encoding="application / x-tex">C_{2-10}< / annotation>< / semantics>< / com:Math> alkenyl"). In some embodiments, an alkenyl group has 2 to 9 carbon atoms ("<com:Math><semantics>C2−9<annotation encoding="application / x-tex">C_{2-9}< / annotation>< / semantics>< / com:Math> alkenyl"). In some embodiments, an alkenyl group has 2 to 8 carbon atoms ("<com:Math><semantics>C2−8<annotation encoding="application / x-tex">C_{2-8}< / annotation>< / semantics>< / com:Math> alkenyl"). In some embodiments, an alkenyl group has 2 to 7 carbon atoms ("<com:Math><semantics>C2−7<annotation encoding="application / x-tex">C_{2-7}< / annotation>< / semantics>< / com:Math> alkenyl"). In some embodiments, an alkenyl group has 2 to 6 carbon atoms ("<com:Math><semantics>C2−6<annotation encoding="application / x-tex">C_{2-6}< / annotation>< / semantics>< / com:Math> alkenyl"). In some embodiments, an alkenyl group has 2 to 5 carbon atoms ("<com:Math><semantics>C2−5<annotation encoding="application / x-tex">C_{2-5}< / annotation>< / semantics>< / com:Math> alkenyl"). In some embodiments, an alkenyl group has 2 to 4 carbon atoms ("<com:Math><semantics>C2−4<annotation encoding="application / x-tex">C_{2-4}< / annotation>< / semantics>< / com:Math> alkenyl"). In some embodiments, an alkenyl group has 2 to 3 carbon atoms ("<com:Math><semantics>C2−3<annotation encoding="application / x-tex">C_{2-3}< / annotation>< / semantics>< / com:Math> alkenyl"). In some embodiments, an alkenyl group has 2 carbon atoms ("C<com:Sub>2< / com:Sub> alkenyl"). The one or more carbon— carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of <com:Math><semantics>C2−4<annotation encoding="application / x-tex">C_{2-4}< / annotation>< / semantics>< / com:Math> alkenyl groups include ethenyl (<com:Math><semantics>C2<annotation encoding="application / x-tex">C_2< / annotation>< / semantics>< / com:Math>), 1-propenyl (<com:Math><semantics>C3<annotation encoding="application / x-tex">C_3< / annotation>< / semantics>< / com:Math>), 2-propenyl (<com:Math><semantics>C3<annotation encoding="application / x-tex">C_3< / annotation>< / semantics>< / com:Math>), 1- butenyl <com:Math><semantics>(C4)<annotation encoding="application / x-tex">(C_4)< / annotation>< / semantics>< / com:Math>, 2-butenyl <com:Math><semantics>(C4)<annotation encoding="application / x-tex">(C_4)< / annotation>< / semantics>< / com:Math>, butadienyl <com:Math><semantics>(C4)<annotation encoding="application / x-tex">(C_4)< / annotation>< / semantics>< / com:Math>, and the like. Examples of <com:Math><semantics>C2−6<annotation encoding="application / x-tex">C_{2-6}< / annotation>< / semantics>< / com:Math> alkenyl groups include the aforementioned <com:Math><semantics>C2−4<annotation encoding="application / x-tex">C_{2-4}< / annotation>< / semantics>< / com:Math> alkenyl groups as well as pentenyl (<com:Math><semantics>C5<annotation encoding="application / x-tex">C_5< / annotation>< / semantics>< / com:Math>), pentadienyl (<com:Math><semantics>C5<annotation encoding="application / x-tex">C_5< / annotation>< / semantics>< / com:Math>), hexenyl (<com:Math><semantics>C6<annotation encoding="application / x-tex">C_6< / annotation>< / semantics>< / com:Math>), and the like. Additional examples of alkenyl include heptenyl (<com:Math><semantics>C7<annotation encoding="application / x-tex">C_7< / annotation>< / semantics>< / com:Math>), octenyl (<com:Math><semantics>C8<annotation encoding="application / x-tex">C_8< / annotation>< / semantics>< / com:Math>), octatrienyl (C<com:Sub>8< / com:Sub>), and the like. Unless otherwise specified, each instance of an alkenyl group is independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkenyl") or substituted (a "substituted alkenyl") with one or more substituents. In certain embodiments, the alkenyl group is unsubstituted <com:Math><semantics>C2−10<annotation encoding="application / x-tex">C_{2-10}< / annotation>< / semantics>< / com:Math> alkenyl. In certain embodiments, the alkenyl group is substituted <com:Math><semantics>C2−10<annotation encoding="application / x-tex">C_{2-10}< / annotation>< / semantics>< / com:Math> alkenyl.

[0073] "Alkynyl" refers to a radical of a straight—chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon–carbon triple bonds, and optionally one or more double bonds ("<com:Math><semantics>C2−20<annotation encoding="application / x-tex">C_{2-20}< / annotation>< / semantics>< / com:Math> alkynyl"). In some embodiments, an alkynyl group has 2 to 10 carbon atoms ("<com:Math><semantics>C2−10<annotation encoding="application / x-tex">C_{2-10}< / annotation>< / semantics>< / com:Math> alkynyl"). In some embodiments, an alkynyl group has 2 to 9 carbon atoms ("<com:Math><semantics>C2−9<annotation encoding="application / x-tex">C_{2-9}< / annotation>< / semantics>< / com:Math> alkynyl"). In some embodiments, an alkynyl group has 2 to 8 carbon atoms ("<com:Math><semantics>C2−9<annotation encoding="application / x-tex">C_{2-9}< / annotation>< / semantics>< / com:Math> 8 alkynyl"). In some embodiments, an alkynyl group has 2 to 7 carbon atoms ("<com:Math><semantics>C2−7<annotation encoding="application / x-tex">C_{2-7}< / annotation>< / semantics>< / com:Math> alkynyl"). In some embodiments, an alkynyl group has 2 to 6 carbon atoms ("<com:Math><semantics>C2−6<annotation encoding="application / x-tex">C_{2-6}< / annotation>< / semantics>< / com:Math> alkynyl"). In some embodiments, an alkynyl group has 2 to 5 carbon atoms ("<com:Math><semantics>C2−5<annotation encoding="application / x-tex">C_{2-5}< / annotation>< / semantics>< / com:Math> alkynyl"). In some embodiments, an alkynyl group has 2 to 4 carbon atoms ("C<com:Sub>2-4< / com:Sub> alkynyl"). In some embodiments, an alkynyl group has 2 to 3 carbon atoms ("C<com:Sub>2-3< / com:Sub> alkynyl"). In some embodiments, an alkynyl group has 2 carbon atoms ("C<com:Sub>2< / com:Sub> alkynyl"). The one or more carbon— carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of <com:Math><semantics>C2−4<annotation encoding="application / x-tex">C_{2-4}< / annotation>< / semantics>< / com:Math> alkynyl groups include, without limitation, ethynyl (<com:Math><semantics>C2<annotation encoding="application / x-tex">C_2< / annotation>< / semantics>< / com:Math>), 1-propynyl (<com:Math><semantics>C3<annotation encoding="application / x-tex">C_3< / annotation>< / semantics>< / com:Math>), 2-propynyl (<com:Math><semantics>C3<annotation encoding="application / x-tex">C_3< / annotation>< / semantics>< / com:Math>), 1-butynyl (<com:Math><semantics>C4<annotation encoding="application / x-tex">C_4< / annotation>< / semantics>< / com:Math>), 2-butynyl (<com:Math><semantics>C4<annotation encoding="application / x-tex">C_4< / annotation>< / semantics>< / com:Math>), and the like. Examples of <com:Math><semantics>C2−6<annotation encoding="application / x-tex">C_{2-6}< / annotation>< / semantics>< / com:Math> alkenyl groups include the aforementioned <com:Math><semantics>C2−4<annotation encoding="application / x-tex">C_{2-4}< / annotation>< / semantics>< / com:Math> alkynyl groups as well as pentynyl (<com:Math><semantics>C5<annotation encoding="application / x-tex">C_5< / annotation>< / semantics>< / com:Math>), hexynyl (<com:Math><semantics>C6<annotation encoding="application / x-tex">C_6< / annotation>< / semantics>< / com:Math>), and the like. Additional examples of alkynyl include heptynyl <com:Math><semantics>(C7)<annotation encoding="application / x-tex">(C_7)< / annotation>< / semantics>< / com:Math>, octynyl <com:Math><semantics>(C8)<annotation encoding="application / x-tex">(C_8)< / annotation>< / semantics>< / com:Math>, and the like. Unless otherwise specified, each instance of an alkynyl group is independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkynyl") or substituted (a "substituted alkynyl") with one or more substituents. In certain embodiments, the alkynyl group is unsubstituted <com:Math><semantics>C2−10<annotation encoding="application / x-tex">C_{2-10}< / annotation>< / semantics>< / com:Math> alkynyl. In certain embodiments, the alkynyl group is substituted <com:Math><semantics>C2−10<annotation encoding="application / x-tex">C_{2-10}< / annotation>< / semantics>< / com:Math> alkynyl.

[0074] "Heterocyclyl" or "heterocyclic" refers to a radical of a 3– to 10–membered non– aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3–10 membered heterocyclyl"). In certain embodiments, the heteroatom is independently selected from nitrogen, sulfur, and oxygen. In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic ("monocyclic heterocyclyl") or a fused, bridged or spiro ring system such as a bicyclic system ("bicyclic heterocyclyl"), and can be saturated or partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems wherein the heterocyclic ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclic ring, or ring systems wherein the heterocyclic ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclic ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclic ring system. Unless otherwise specified, each instance of heterocyclyl is independently optionally substituted, i.e., unsubstituted (an "unsubstituted heterocyclyl") or substituted (a "substituted heterocyclyl") with one or more substituents. In certain embodiments, the heterocyclyl group is unsubstituted 3–10 membered heterocyclyl. In certain embodiments, the heterocyclyl group is substituted 3–10 membered heterocyclyl.

[0075] "Aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) <com:Math><semantics>4n+2<annotation encoding="application / x-tex">4n+2< / annotation>< / semantics>< / com:Math> aromatic ring system (e.g., having 6, 10, or 14 <com:Math><semantics>π<annotation encoding="application / x-tex">\pi< / annotation>< / semantics>< / com:Math> electrons shared in a cyclic array) having 6–14 ring carbon atoms and zero heteroatoms in the aromatic ring system ("<com:Math><semantics>C6−14<annotation encoding="application / x-tex">C_{6-14}< / annotation>< / semantics>< / com:Math> aryl"). In some embodiments, an aryl group has six ring carbon atoms ("C<com:Sub>6< / com:Sub> aryl"; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms ("C<com:Sub>10< / com:Sub> aryl"; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms ("C<com:Sub>14< / com:Sub> aryl"; e.g., anthracyl). "Aryl" also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently optionally substituted, i.e., unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl") with one or more substituents. In certain embodiments, the aryl group is unsubstituted <com:Math><semantics>C6−14<annotation encoding="application / x-tex">C_{6-14}< / annotation>< / semantics>< / com:Math> aryl. In certain embodiments, the aryl group is substituted <com:Math><semantics>C6−14<annotation encoding="application / x-tex">C_{6-14}< / annotation>< / semantics>< / com:Math> aryl.

[0076] Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, which are divalent bridging groups are further referred to using the suffix – ene, e.g., alkylene, alkenylene, alkynylene, carbocyclylene, heterocyclylene, arylene, and heteroarylene.

[0077] The term "alkoxy" or "alkyloxy" refers to an -O-alkyl radical, wherein alkyl is optionally substituted alkyl as defined herein. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, and tert- butoxy.

[0078] The term "aryloxy" refers to an -O-aryl, wherein aryl is optionally substituted aryl as defined herein.

[0079] As used herein, the term "optionally substituted" refers to a substituted or unsubstituted moiety.

[0800] Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted (e.g., "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" carbocyclyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl or "substituted" or "unsubstituted" heteroaryl group). In general, the term "substituted", whether preceded by the term "optionally" or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term "substituted" is contemplated to include substitution with all permissible substituents of organic compounds, any of the substituents described herein that results in the formation of a stable compound. The present invention contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.

[0081] "Halo" or "halogen" refers to fluorine (fluoro, –F), chlorine (chloro, –Cl), bromine (bromo, –Br), or iodine (iodo, –I).

[0082] "Acyl" as used herein refers to a moiety selected from the group consisting of – <com:Math><semantics>C(=O)Raa<annotation encoding="application / x-tex">C(=O)R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−CHO<annotation encoding="application / x-tex">-CHO< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−CO2Raa<annotation encoding="application / x-tex">-CO_2R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=O)N(Rbb)2<annotation encoding="application / x-tex">-C(=O)N(R^{bb})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=NRbb)Raa<annotation encoding="application / x-tex">-C(=NR^{bb})R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=NRbb)ORaa<annotation encoding="application / x-tex">-C(=NR^{bb})OR^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=NRbb)ORaa<annotation encoding="application / x-tex">-C(=NR^{bb})OR^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=NRbb)ORaa<annotation encoding="application / x-tex">-C(=NR^{bb})OR^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=NRbb)ORaa<annotation encoding="application / x-tex">-C(=NR^{bb})OR^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=NRbb)ORaa<annotation encoding="application / x-tex">-C(=NR^{bb})OR^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=NRbb)ORaa<annotation encoding="application / x-tex">-C(=NR^{bb})OR^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−<annotation encoding="application / x-tex">-< / annotation>< / semantics>< / com:Math> <com:Math><semantics>C(=NRbb)N(Rbb)2<annotation encoding="application / x-tex">C(=NR^{bb})N(R^{bb})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=O)NRbbSO2Raa<annotation encoding="application / x-tex">-C(=O)NR^{bb}SO_2R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=S)N(Rbb)2<annotation encoding="application / x-tex">-C(=S)N(R^{bb})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=O)SRaa<annotation encoding="application / x-tex">-C(=O)SR^{aa}< / annotation>< / semantics>< / com:Math>, and <com:Math><semantics>−C(=S)SRaa<annotation encoding="application / x-tex">-C(=S)SR^{aa}< / annotation>< / semantics>< / com:Math>, wherein R<com:Sup>aa< / com:Sup> and R<com:Sup>bb< / com:Sup> are as defined herein.

[0083] Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quarternary nitrogen atoms. Exemplary nitrogen atom substituents include, but are not limited to, hydrogen, -OH, -OR<com:Sup>aa< / com:Sup>, -N(R<com:Sup>cc< / com:Sup>)<com:Sub>2< / com:Sub>, -CN, -C(=O)R<com:Sup>aa< / com:Sup>, -C(=O)N(R<com:Sup>cc< / com:Sup>)<com:Sub>2< / com:Sub>, -CO<com:Sub>2< / com:Sub>R<com:Sup>aa< / com:Sup>, -SO<com:Sub>2< / com:Sub>R<com:Sup>aa< / com:Sup>, -C(=NR<com:Sup>bb< / com:Sup>)R<com:Sup>aa< / com:Sup>, -C(=NR<com:Sup>cc< / com:Sup>)OR<com:Sup>aa< / com:Sup>, -C(=NR<com:Sup>cc< / com:Sup>)N(R<com:Sup>cc< / com:Sup>)<com:Sub>2< / com:Sub>, - <com:Math><semantics>SO2N(Rcc)2<annotation encoding="application / x-tex">SO_2N(R^{cc})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SO2Rcc<annotation encoding="application / x-tex">-SO_2R^{cc}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SO2ORcc<annotation encoding="application / x-tex">-SO_2OR^{cc}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SORaa<annotation encoding="application / x-tex">-SOR^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=S)N(Rcc)2<annotation encoding="application / x-tex">-C(=S)N(R^{cc})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=O)SRcc<annotation encoding="application / x-tex">-C(=O)SR^{cc}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=S)SRcc<annotation encoding="application / x-tex">-C(=S)SR^{cc}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=S)SRcc<annotation encoding="application / x-tex">-C(=S)SR^{cc}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=S)SRcc<annotation encoding="application / x-tex">-C(=S)SR^{cc}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=S)SRcc<annotation encoding="application / x-tex">-C(=S)SR^{cc}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=S)SRcc<annotation encoding="application / x-tex">-C(=S)SR^{cc}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=S)SRcc<annotation encoding="application / x-tex">-C(=S)SR^{cc}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=S)SRcc<annotation encoding="application / x-tex">-C(=S)SR^{cc}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>P(=O)2Raa<annotation encoding="application / x-tex">P(=O)_2R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−P(=O)(Raa)2<annotation encoding="application / x-tex">-P(=O)(R^{aa})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−P(=O)2N(Rcc)2<annotation encoding="application / x-tex">-P(=O)_2N(R^{cc})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−P(=O)(NRcc)2<annotation encoding="application / x-tex">-P(=O)(NR^{cc})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>C1−10<annotation encoding="application / x-tex">C_{1-10}< / annotation>< / semantics>< / com:Math> alkyl, <com:Math><semantics>C1−10<annotation encoding="application / x-tex">C_{1-10}< / annotation>< / semantics>< / com:Math> perhaloalkyl, <com:Math><semantics>C2−10<annotation encoding="application / x-tex">C_{2-10}< / annotation>< / semantics>< / com:Math> alkenyl, <com:Math><semantics>C2−10<annotation encoding="application / x-tex">C_{2-10}< / annotation>< / semantics>< / com:Math> alkynyl, <com:Math><semantics>C3−10<annotation encoding="application / x-tex">C_{3-10}< / annotation>< / semantics>< / com:Math> carbocyclyl, 3–14 membered heterocyclyl, <com:Math><semantics>C6−14<annotation encoding="application / x-tex">C_{6-14}< / annotation>< / semantics>< / com:Math> aryl, and 5–14 membered heteroaryl, or two R<com:Sup>cc< / com:Sup> groups attached to a nitrogen atom are joined to form a 3–14 membered heterocyclyl or 5–14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R<com:Sup>dd< / com:Sup> groups, and wherein R<com:Sup>aa< / com:Sup>, R<com:Sup>bb< / com:Sup>, R<com:Sup>cc< / com:Sup>, and R<com:Sup>dd< / com:Sup> are as defined above.

[0084] In certain embodiments, the substituent present on an oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group). Oxygen protecting groups include, but are not limited to, <com:Math><semantics>−Raa<annotation encoding="application / x-tex">-R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−N(Rbb)2<annotation encoding="application / x-tex">-N(R^{bb})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=O)SRaa<annotation encoding="application / x-tex">-C(=O)SR^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=O)Raa<annotation encoding="application / x-tex">-C(=O)R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−CO2Raa<annotation encoding="application / x-tex">-CO_2R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−CO3Raa<annotation encoding="application / x-tex">-CO_3R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−CO3Raa<annotation encoding="application / x-tex">-CO_3R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−CO3Raa<annotation encoding="application / x-tex">-CO_3R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−CO3Raa<annotation encoding="application / x-tex">-CO_3R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−CO3Raa<annotation encoding="application / x-tex">-CO_3R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−CO3Raa<annotation encoding="application / x-tex">-CO_3R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−CO3Raa<annotation encoding="application / x-tex">-CO_3R^{aa}< / annotation>< / semantics>< / com:Math>, <Formule mathématique disponible dans le document PDF, Math available in the PDF document>-CO_3R^{ <com:Math><semantics>C(=O)N(Rbb)2<annotation encoding="application / x-tex">C(=O)N(R^{bb})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=NRbb)Raa<annotation encoding="application / x-tex">-C(=NR^{bb})R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=NRbb)ORaa<annotation encoding="application / x-tex">-C(=NR^{bb})OR^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=NRbb)N(Rbb)2<annotation encoding="application / x-tex">-C(=NR^{bb})N(R^{bb})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−S(=O)Raa<annotation encoding="application / x-tex">-S(=O)R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SO2Raa<annotation encoding="application / x-tex">-SO_2R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SO2Raa<annotation encoding="application / x-tex">-SO_2R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SO2Raa<annotation encoding="application / x-tex">-SO_2R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SO2Raa<annotation encoding="application / x-tex">-SO_2R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SO2Raa<annotation encoding="application / x-tex">-SO_2R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SO2Raa<annotation encoding="application / x-tex">-SO_2R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SO2Raa<annotation encoding="application / x-tex">-SO_2R^{aa}< / annotation>< / semantics>< / com:Math>, <Formule mathématique disponible dans le document PDF, Math available in the PDF document>$-SO_2R^$ <com:Math><semantics>Si(Raa)3<annotation encoding="application / x-tex">Si(R^{aa})_3< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−P(Rcc)2<annotation encoding="application / x-tex">-P(R^{cc})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−P(Rcc)3<annotation encoding="application / x-tex">-P(R^{cc})_3< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−P(=O)2Raa<annotation encoding="application / x-tex">-P(=O)_2R^{aa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−P(=O)(Raa)2<annotation encoding="application / x-tex">-P(=O)(R^{aa})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−P(=O)(ORcc)2<annotation encoding="application / x-tex">-P(=O)(OR^{cc})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−P(=O)2N(Rbb)2<annotation encoding="application / x-tex">-P(=O)_2N(R^{bb})_2< / annotation>< / semantics>< / com:Math>, and <com:Math><semantics>−P(=O)(Raa)2<annotation encoding="application / x-tex">-P(=O)(R^{aa})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−P(=O)(Raa)2<annotation encoding="application / x-tex">-P(=O)(R^{aa})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−P(=O)(Raa)2<annotation encoding="application / x-tex">-P(=O)(R^{aa})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−P(=O)(Raa)2<annotation encoding="application / x-tex">-P(=O)(R^{aa})_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−P(=O)<annotation encoding="application / x-tex">-P(=O)< / annotation>< / semantics>< / com:Math> P(=O)(NR<com:Sup>bb< / com:Sup>)<com:Sub>2< / com:Sub>, wherein R<com:Sup>aa< / com:Sup>, R<com:Sup>bb< / com:Sup>, and R<com:Sup>cc< / com:Sup> are as defined herein. Oxygen protecting groups are well known in the art and include those described in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3<com:Sup>rd< / com:Sup> edition, John Wiley & Sons, 1999.

[0085] Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t—butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3– bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4- methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4- methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4- methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1- methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2- (phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p- halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3- methyl–2–picolyl N–oxido, diphenylmethyl, p,p ’–dinitrobenzhydryl, 5–dibenzosuberyl, triphenylmethyl, <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-naphthyldiphenylmethyl, <com:Math><semantics>p<annotation encoding="application / x-tex">p< / annotation>< / semantics>< / com:Math>-methoxyphenyldiphenylmethyl, di<com:Math><semantics>(p<annotation encoding="application / x-tex">(p< / annotation>< / semantics>< / com:Math>- methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'- bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5- dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4"- tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1- bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4- oxopentanoate (levulinate), 4,4–(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6- trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate (BOC), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1- napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4- methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2- (methylthiomethoxy)ethyl, 4—(methylthiomethoxy)butyrate, 2— (methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro- 4–(1,1,3,3–tetramethylbutyl)phenoxyacetate, 2,4–bis(1,1–dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o- (methoxyacyl)benzoate, <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-naphthoate, nitrate, alkyl <com:Math><semantics>N,N,N′,N′<annotation encoding="application / x-tex">N,N,N',N'< / annotation>< / semantics>< / com:Math>- tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4—dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). Other definitions

[0086] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably.

[0087] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, 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 Lane s (Cold Spring Harbor Laboratory Press, 1988); and Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986).

[0088] As used herein, the term "glycan" refers to a polysaccharide, or oligosaccharide. Glycan is also used herein to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or a proteoglycan. Glycans usually consist solely of O-glycosidic linkages between monosaccharides. For example, cellulose is a glycan (or more specifically a glucan) composed of B-1,4-linked D-glucose, and chitin is a glycan composed of B-1,4-linked N- acetyl-D-glucosamine. Glycans can be homo or heteropolymers of monosaccharide residues, and can be linear or branched. Glycans can be found attached to proteins as in glycoproteins and proteoglycans. They are generally found on the exterior surface of cells. O- and N-linked glycans are very common in eukaryotes but may also be found, although less commonly, in prokaryotes. N-Linked glycans are found attached to the R-group nitrogen (N) of asparagine in the sequon. The sequon is a Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid except praline.

[0089] As used herein, the term "antigen" is defined as any substance capable of eliciting an immune response.

[0090] As used herein, the term "immunogenicity" refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.

[0091] As used herein, the term "CD1d" refers to a member of the CD1 (cluster of differentiation 1) family of glycoproteins expressed on the surface of various human antigen- presenting cells. CD1d presented lipid antigens activate natural killer T cells. CD1d has a deep antigen-binding groove into which glycolipid antigens bind. CD1d molecules expressed on dendritic cells can bind and present glycolipids, including <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-GalCer analogs such as C34.

[0092] As used herein, the term "epitope" is defined as the parts of an antigen molecule which contact the antigen binding site of an antibody or a T cell receptor.

[0093] As used herein, the term "vaccine" refers to a preparation that contains an antigen, consisting of whole disease-causing organisms (killed or weakened) or components of such organisms, such as proteins, peptides, or polysaccharides, that is used to confer immunity against the disease that the organisms cause. Vaccine preparations can be natural, synthetic or derived by recombinant DNA technology.

[0094] As used herein, the term "antigen specific" refers to a property of a cell population such that supply of a particular antigen, or a fragment of the antigen, results in specific cell proliferation.

[0095] As used herein, the term "specifically binding," refers to the interaction between binding pairs (e.g., an antibody and an antigen). In various instances, specifically binding can be embodied by an affinity constant of about 10<com:Sup>-6< / com:Sup> moles / liter, about 10<com:Sup>-7< / com:Sup> moles / liter, or about 10<com:Sup>-8< / com:Sup> moles / liter, or less.

[0096] As used herein, the term "Flow cytometry" or "FACS" means a technique for examining the physical and chemical properties of particles or cells suspended in a stream of fluid, through optical and electronic detection devices.

[0097] As used herein, the terms glycoenzymes refers to at least in part the enzymes in the globoseries biosynthetic pathway; exemplary glycoenzymes include alpha-4GalT; beta- 4GalNAcT-I; or beta-3GalT-V enzymes.

[0098] As used herein, the term "globoseries pathway" refers to a biochemical pathway described in Figure 1.

[0099] An "isolated" antibody is one which has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In one embodiment, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[00100] The phrase "substantially similar," "substantially the same", "equivalent", or "substantially equivalent", as used herein, denotes a sufficiently high degree of similarity between two numeric values (for example, one associated with a molecule and the other associated with a reference / comparator molecule) such that one of skill in the art would consider the difference between the two values to be of little or no biological and / or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values, anti-viral effects, etc.). The difference between said two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and / or less than about 10% as a function of the value for the reference / comparator molecule.

[00101] The phrase "substantially reduced," or "substantially different", as used herein, denotes a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference / comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50% as a function of the value for the reference / comparator molecule.

[00102] "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described in the following.

[00103] In one embodiment, the "Kd" or "Kd value" according to this invention is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay. Solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (125I)- labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (Chen, et al., (1999) J. Mol Biol 293:865-881). To establish conditions for the assay, microtiter plates (Dynex) are coated overnight with 5 μg / ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [125I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of an anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., 65 hours) to insure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% Tween-20 in PBS. When the plates have dried, 150 µl / well of scintillant (MicroScint-20; Packard) is added, and the plates are counted on a Topcount gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays. According to another embodiment the Kd or Kd value is measured by using surface plasmon resonance assays using a BIAcore<com:Sup>TM< / com:Sup>-2000 or a BIAcore<com:Sup>TM< / com:Sup>-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N -(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg / ml (~0.2 μM) before injection at a flow rate of 5 μl / minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. In each experiment, a spot was activated and ethanolamine blocked without immobilizing protein, to be used for reference subtraction. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 µl / min. Association rates (kon) and dissociation rates (koff) are calculated using a simple one- to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881. If the on-rate exceeds 106 M-1s-1 by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[00104] An "on-rate" or "rate of association" or "association rate" or "kon" according to this invention can also be determined with the same surface plasmon resonance technique described above using a BIAcore<com:Sup>TM< / com:Sup>-2000 or a BIAcore<com:Sup>TM< / com:Sup>-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at 10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N -(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg / ml (~0.2 μM) before injection at a flow rate of 5 μl / minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 µl / min. Association rates (kon) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgram. The equilibrium dissociation constant (Kd) was calculated as the ratio koff / kon. See, e.g., Chen, Y., et al., (1999)J. Mol Biol 293:865-881. However, if the on- rate exceeds 106 M-1s-1 by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[00105] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "recombinant vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector.

[00106] "Polynucleotide," or "nucleic acid," as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotides(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5 and 3 terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2 -O-methyl- , 2 -O-allyl, 2 -fluoro- or 2 -azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), "(O)NR2 ("amidate"), P(O)R, P(O)OR , CO or CH2 ( "formacetal" ), in which each R or R is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[00107] "Oligonucleotide," as used herein, generally refers to short, generally single- stranded, generally synthetic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.

[00108] "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which generally lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.

[00109] The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, monovalent, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein). An antibody can be chimeric, human, humanized and / or affinity matured.

[00110] The "variable region" or "variable domain" of an antibody refers to the amino- terminal domains of heavy or light chain of the antibody. These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[00111]

[00112] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[00113] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab 1)2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[00114] "Fv" is the minimum antibody fragment which contains a complete antigen- recognition and -binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single- chain Fv species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[00115] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab -SH is the designation herein for Fab in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab )2 antibody fragments originally were produced as pairs of Fab fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[00116] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>) and lambda (<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>), based on the amino acid sequences of their constant domains.

[00117] Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>δ<annotation encoding="application / x-tex">\delta< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ϵ<annotation encoding="application / x-tex">\epsilon< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>< / com:Math>, and <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[00118] The terms "full length antibody," "intact antibody" and "whole antibody" are used herein interchangeably, to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain the Fc region.

[00119] "Antibody fragments" comprise only a portion of an intact antibody, wherein the portion retains at least one, and as many as most or all, of the functions normally associated with that portion when present in an intact antibody. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment, for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half life modulation, ADCC function and complement binding. In one embodiment, an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody. For example, such an antibody fragment may comprise an antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

[00120] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. Such monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones. It should be understood that the selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler et al., Nature, 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display technologies (See, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO98 / 24893; WO96 / 34096; WO96 / 33735; WO91 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio. Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[00121] The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

[00122] Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and / or capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[00123] The term "hypervariable region", "HVR", or "HV", when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions are noted below.

[00124]

[00125] Loop Kabat AbM Chothia Contact

[00126] L1 L24-L34 L24-L34 L26-L32 L30-L36

[00127] L2 L50-L56 L50-L56 L50-L52 L46-L55

[00128] L3 L89-L97 L89-L97 L91-L96 L89-L96

[00129] H1 H31-H35B H26-H35B H26-H32 H30-H35B

[00130] (Kabat Numbering)

[00131] H1 H31-H35 H26-H35 H26-H32 H30-H35

[00132] (Chothia Numbering)

[00133] H2 H50-H65 H50-H58 H53-H55 H47-H58

[00134] H3 H95-H102 H95-H102 H96-H101 H93-H101

[00135] Hypervariable regions may comprise "extended hypervariable regions" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 or 49-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26- 35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[00136] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[00137] The term "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.

[00138] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[00139] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light- chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO93 / 1161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

[00140] A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[00141] An "affinity matured" antibody is one with one or more alterations in one or more HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In one embodiment, an affinity matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. Bio / Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described by: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[00142] A "blocking" antibody or an "antagonist" antibody is one which inhibits or reduces biological activity of the antigen it binds. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[00143] An "agonist antibody", as used herein, is an antibody which mimics at least one of the functional activities of a polypeptide of interest.

[00144] A "disorder" is any condition that would benefit from treatment with an antibody of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non- limiting examples of disorders to be treated herein include cancer.

[00145] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[00146] "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder" and "tumor" are not mutually exclusive as referred to herein.

[00147] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non- Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[00148] The term "muscular disorder" refers to or describes the physiological condition in muscle-containing animals that is typically characterized by deterioration or weakening of skeletal and / or smooth muscle such that normal muscular function is significantly reduced. Examples of muscular disorders include, but are not limited to, muscular dystrophy, multiple sclerosis, amyotrophic lateral sclerosis, Isaac's syndrome; stiff-person syndrome; familiar periodic paralyses, myopathy, myotonia, rhabdomyolyses, muscle atrophy, and various types of muscle weakness and muscle rigidity.

[00149] The term "globoseries -related disorder" refers to or describes a disorder that is typically characterized by or contributed to by aberrant functioning or presentation of the pathway. Examples of such disorders include, but are not limited to, hyperproliferative diseases, including cancer.

[00150] The terms "neurological disorder" or "neurological disease" refer to or describe a disease or disorder of the central and / or peripheral nervous system in mammals that is typically characterized by deterioration of nervous tissue or deterioration of communication between cells in nervous tissue. Examples of neurological disorders include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including, but not limited to, Alzheimer disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorders (including, but not limited to, Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (including, but not limited to, Pick's disease, and spinocerebellar ataxia.

[00151] The terms "inflammatory disorder" and "immune disorder" refer to or describe disorders caused by aberrant immunologic mechanisms and / or aberrant cytokine signaling. Examples of inflammatory and immune disorders include, but are not limited to, autoimmune diseases, immunologic deficiency syndromes, and hypersensitivity. An "autoimmune disease" herein is a non-malignant disease or disorder arising from and directed against an individual's own tissues. The autoimmune diseases herein specifically exclude malignant or cancerous diseases or conditions, especially excluding B cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hairy cell leukemia and chronic myeloblastic leukemia. Examples of autoimmune diseases or disorders include, but are not limited to, inflammatory responses such as inflammatory skin diseases including psoriasis and dermatitis (e.g. atopic dermatitis); systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions involving infiltration of T cells and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including but not limited to lupus nephritis, cutaneous lupus); diabetes mellitus (e.g. Type I diabetes mellitus or insulin dependent diabetes mellitus); multiple sclerosis; Reynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile onset diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T- lymphocytes typically found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; pernicious anemia (Addison's disease); diseases involving leukocyte diapedesis; central nervous system (CNS) inflammatory disorder; multiple organ injury syndrome; hemolytic anemia (including, but not limited to cryoglobinemia or Coombs positive anemia); myasthenia gravis; antigen-antibody complex mediated diseases; anti-glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert- Eaton myasthenic syndrome; pemphigoid bullous; pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-man syndrome; Behcet disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathies; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia, etc.

[00152] Examples of immunologic deficiency syndromes include, but are not limited to, ataxia telangiectasia, leukocyte-adhesion deficiency syndrome, lymphopenia, dysgammaglobulinemia, HIV or deltaretrovirus infections, common variable immunodeficiency, severe combined immunodeficiency, phagocyte bactericidal dysfunction, agammaglobulinemia, DiGeorge syndrome, and Wiskott-Aldrich syndrome. Examples of hypersensitivity include, but are not limited to, allergies, asthma, dermatitis, hives, anaphylaxis, Wissler's syndrome, and thrombocytopenic purpura.

[00153] As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing or decreasing inflammation and / or tissue / organ damage, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or disorder.

[00154] An "individual" or a "subject" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice and rats. In certain embodiments, the vertebrate is a human.

[00155] "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. In certain embodiments, the mammal is human.

[00156] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[00157] A "therapeutically effective amount" of a substance / molecule of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance / molecule are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.

[00158] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu), chemotherapeutic agents (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells.

[00159] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enedigne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2 -,2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol- Myers Squibb Oncology, Princeton, N.J.), ABRAXANE<com:Sup>TM< / com:Sup> Cremophor-free, albumin- engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN<com:Sup>TM< / com:Sup>) combined with 5-FU and leucovovin.

[00160] Also included in this definition are anti-hormonal agents that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves. Examples include anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® toremifene; anti-progesterones; estrogen receptor down-regulators (ERDs); agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON® and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid / zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[00161] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by 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 now described. Publications and patents specifically mentioned herein may describe and disclose chemicals, cell lines, vectors, animals, instruments, statistical analysis and methodologies which might be used in connection with the invention. All references cited in this specification are to be taken as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[00162] Most recently, Wong et al 25 attempts to improve the immunogenicity of the vaccine, the research team synthesized a variety of GH-derivatives containing modifications at either the reducing end or at the non-reducing end of the carbohydrate, and found that vaccines containing GH modified with a fluoro, azido, or phenyl group at the reducing end or an azido group at the non-reducing end could stimulate the production of GH, SSEA3, and SSEA4- targeting antibodies, with the latter vaccine eliciting an especially favorable high ratio of IgG:IgM antibodies not usually achieved in anticancer vaccines. Encouragingly, the antibodies produced in response to these vaccines mediated complement-dependent cytotoxicity towards cultured GH-positive human breast cancer cells.

[00163] The present disclosure is based on the surprising discovery that the modification of the stage-specific embryonic antigens (SSEA3 and SSEA4) with certain groups elicited robust IgG antibody response to specifically recognize SSEA3 and SSEA4, respectively.

[00164] In some examples, the modification of SSEA-3 comprises a fluoro, an azido or an O- phenyl group at the one or more positions of the glucose of SSEA-3. In some examples, the modification of SSEA-3 comprises a fluoro, an azido or an O-phenyl group at the one or more positions of the non-reducing end galactose. In some examples, the modification of SSEA-4 comprises a fluoro, an azido or an O-phenyl group at one or more positions of the glucose of SSEA-4. In some examples, the modification of SSEA-4 comprises a fluoro, an azido or an O- phenyl group at one or more positions of the sialic acid residue.

[00165] Described herein are SSEA3 and SSEA4 derivatives having the modification at the reducing and / or non-reducing end. Such SSEA3 and SSEA4 derivatives can elicit a stronger immune response (e.g., induction of IgG antibodies against SSEA3 and / or SSEA4) as compared to the native SSEA3 and SSEA4. The antibodies induced by an immunogenic composition comprising such unnatural glycan moiety are able to mediate the complement- dependent cell cytotoxicity against tumor cells. Compounds

[00166] Accordingly, the present invention also features novel compounds consisting of the modified carbohydrate antigens (SSEA3 and SSEA4), glycan conjugates comprising such, and immunogenic compositions and vaccines thereof.

[00167] In one aspect, the present invention provides a compound of formula (I): [Image disponible dans le document PDF, Image available in the PDF document] wherein: <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is -OR or -SR, wherein R is hydrogen, a oxygen or sulfur protecting group, optionally substituted <com:Math><semantics>C1−10<annotation encoding="application / x-tex">C_{1-10}< / annotation>< / semantics>< / com:Math> alkyl, optionally substituted aryl, optionally substituted acyl, or optionally substituted imidoyl; each instance of R<com:Sup>1< / com:Sup>, R<com:Sup>2< / com:Sup>, R<com:Sup>3< / com:Sup>, R<com:Sup>4< / com:Sup>, R<com:Sup>5< / com:Sup>, R<com:Sup>6< / com:Sup> and L is independently selected from hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, -N<com:Sub>3< / com:Sub>, -NO<com:Sub>2< / com:Sub>, <com:Math><semantics>−N(RB)2<annotation encoding="application / x-tex">-N(R^B)_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−N(RA)C(O)RA<annotation encoding="application / x-tex">-N(R^A)C(O)R^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−ORA<annotation encoding="application / x-tex">-OR^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−OC(O)RA<annotation encoding="application / x-tex">-OC(O)R^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SRA<annotation encoding="application / x-tex">-SR^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)N(RB)2<annotation encoding="application / x-tex">-C(O)N(R^B)_2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−CN<annotation encoding="application / x-tex">-CN< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)RA<annotation encoding="application / x-tex">-C(O)R^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)ORA<annotation encoding="application / x-tex">-C(O)OR^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)ORA<annotation encoding="application / x-tex">-C(O)OR^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)ORA<annotation encoding="application / x-tex">-C(O)OR^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)ORA<annotation encoding="application / x-tex">-C(O)OR^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)ORA<annotation encoding="application / x-tex">-C(O)OR^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)ORA<annotation encoding="application / x-tex">-C(O)OR^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)ORA<annotation encoding="application / x-tex">-C(O)OR^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)ORA<annotation encoding="application / x-tex">-C(O)OR^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)ORA<annotation encoding="application / x-tex">-C(O)OR^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)<annotation encoding="application / x-tex">-C(O)< / annotation>< / semantics>< / com:Math> <com:Math><semantics>S(O)RA<annotation encoding="application / x-tex">S(O)R^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SO2RA<annotation encoding="application / x-tex">-SO_2R^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SO2N(RB)2<annotation encoding="application / x-tex">-SO_2N(R^B)_2< / annotation>< / semantics>< / com:Math>, and <com:Math><semantics>−NHSO2RB<annotation encoding="application / x-tex">-NHSO_2R^B< / annotation>< / semantics>< / com:Math>; each instance of R<com:Sup>A< / com:Sup> is independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl; each instance of R<com:Sup>B< / com:Sup> is independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl; and provided the compound is not of the formula: [Image disponible dans le document PDF, Image available in the PDF document]

[00168] In certain embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is in the alpha configuration. In certain embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is in the beta configuration.

[00169] In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−ORA<annotation encoding="application / x-tex">-OR^A< / annotation>< / semantics>< / com:Math>. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−OH<annotation encoding="application / x-tex">-OH< / annotation>< / semantics>< / com:Math>. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−O(protecting group)<annotation encoding="application / x-tex">-O(\text{protecting group})< / annotation>< / semantics>< / com:Math>. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−ORA<annotation encoding="application / x-tex">-OR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is unsubstituted <com:Math><semantics>C1−10<annotation encoding="application / x-tex">C_{1-10}< / annotation>< / semantics>< / com:Math> alkyl. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−ORA<annotation encoding="application / x-tex">-OR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is substituted <com:Math><semantics>C1−10<annotation encoding="application / x-tex">C_{1-10}< / annotation>< / semantics>< / com:Math> alkyl. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−ORA<annotation encoding="application / x-tex">-OR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is unsubstituted aryl. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−ORA<annotation encoding="application / x-tex">-OR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is substituted aryl. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−<annotation encoding="application / x-tex">-< / annotation>< / semantics>< / com:Math> <com:Math><semantics>ORA<annotation encoding="application / x-tex">OR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is unsubstituted acyl. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−ORA<annotation encoding="application / x-tex">-OR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is substituted acyl. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−ORA<annotation encoding="application / x-tex">-OR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is unsubstituted imidoyl. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−ORA<annotation encoding="application / x-tex">-OR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is substituted imidoyl.

[00170] In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−SRA<annotation encoding="application / x-tex">-SR^A< / annotation>< / semantics>< / com:Math>. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−SH<annotation encoding="application / x-tex">-SH< / annotation>< / semantics>< / com:Math>. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−S(protecting group)<annotation encoding="application / x-tex">-S(\text{protecting group})< / annotation>< / semantics>< / com:Math>. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−SRA<annotation encoding="application / x-tex">-SR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is unsubstituted <com:Math><semantics>C1−10<annotation encoding="application / x-tex">C_{1-10}< / annotation>< / semantics>< / com:Math> alkyl. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−SRA<annotation encoding="application / x-tex">-SR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is substituted <com:Math><semantics>C1−10<annotation encoding="application / x-tex">C_{1-10}< / annotation>< / semantics>< / com:Math> alkyl. In certain embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−SCH3<annotation encoding="application / x-tex">-SCH_3< / annotation>< / semantics>< / com:Math>. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−SRA<annotation encoding="application / x-tex">-SR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is unsubstituted aryl. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−SRA<annotation encoding="application / x-tex">-SR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is substituted aryl. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−SRA<annotation encoding="application / x-tex">-SR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is unsubstituted acyl. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−SRA<annotation encoding="application / x-tex">-SR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is substituted acyl. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−SRA<annotation encoding="application / x-tex">-SR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is unsubstituted imidoyl. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−SRA<annotation encoding="application / x-tex">-SR^A< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RA<annotation encoding="application / x-tex">R^A< / annotation>< / semantics>< / com:Math> is substituted imidoyl.

[00171] In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>C1−10<annotation encoding="application / x-tex">C_{1-10}< / annotation>< / semantics>< / com:Math> alkoxy. In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>C1−3<annotation encoding="application / x-tex">C_{1-3}< / annotation>< / semantics>< / com:Math> alkoxy. In certain embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is methoxy. In certain embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is alpha-methoxy.

[00172] In some embodiments, <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> is selected from the group consisting of alpha-thiomethyl, beta-thiomethyl, alpha-thiocresyl, beta-thiocresyl, alpha-t-butyldiphenylsilyloxy, beta-t- butyldiphenylsilyloxy, and alpha-methoxy.

[00173] In some embodiments, <com:Math><semantics>R1<annotation encoding="application / x-tex">R^1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>1< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, <com:Math><semantics>R1<annotation encoding="application / x-tex">R^1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>1< / com:Sup> is -NH<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>1< / com:Sup> is - NHR<com:Sup>W< / com:Sup>, wherein R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>1< / com:Sup> is -N(R<com:Sup>W< / com:Sup>)<com:Sub>2< / com:Sub>, wherein each R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>1< / com:Sup> is selected from the group consisting of -N<com:Sub>3< / com:Sub>, -NH(Cbz), -NH(Boc), -NH(Fmoc), -NHC(O)CCl<com:Sub>3< / com:Sub>, - NHC(O)CH<com:Sub>3< / com:Sub>, and -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>1< / com:Sup> is -NH(Cbz). In certain embodiments, R<com:Sup>1< / com:Sup> is -NH(Fmoc). In certain embodiments, R<com:Sup>1< / com:Sup> is -NHC(O)CCl<com:Sub>3< / com:Sub>. In certain embodiments, <com:Math><semantics>R1<annotation encoding="application / x-tex">R^1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−NHC(O)CH3<annotation encoding="application / x-tex">-NHC(O)CH_3< / annotation>< / semantics>< / com:Math>. In certain embodiments, <com:Math><semantics>R1<annotation encoding="application / x-tex">R^1< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(C(O)CH3)2<annotation encoding="application / x-tex">-N(C(O)CH_3)_2< / annotation>< / semantics>< / com:Math>.

[00174] In some embodiments, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^2< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^2< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math>. In certain embodiments, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^2< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, <com:Math><semantics>ℝ2<annotation encoding="application / x-tex">\mathbb{R}^2< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−ℕH2<annotation encoding="application / x-tex">-\mathbb{N}H_2< / annotation>< / semantics>< / com:Math>. In certain embodiments, <com:Math><semantics>ℝ2<annotation encoding="application / x-tex">\mathbb{R}^2< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−ℕH2<annotation encoding="application / x-tex">-\mathbb{N}H_2< / annotation>< / semantics>< / com:Math>. <com:Math><semantics>NHRW<annotation encoding="application / x-tex">NHR^{W}< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RW<annotation encoding="application / x-tex">R^{W}< / annotation>< / semantics>< / com:Math> is a nitrogen protecting group. In certain embodiments, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^{2}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^{W})_{2}< / annotation>< / semantics>< / com:Math>, wherein each R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>2< / com:Sup> is selected from the group consisting of -N<com:Sub>3< / com:Sub>, -NH(Cbz), -NH(Boc), -NH(Fmoc), -NHC(O)CCl<com:Sub>3< / com:Sub>, - NHC(O)CH<com:Sub>3< / com:Sub>, and -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>. In certain embodiments, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^2< / annotation>< / semantics>< / com:Math> is -NH(Cbz). In certain embodiments, R<com:Sup>2< / com:Sup> is -NH(Fmoc). In certain embodiments, R<com:Sup>2< / com:Sup> is -NHC(O)CCl<com:Sub>3< / com:Sub>. In certain embodiments, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^2< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−NHC(O)CH3<annotation encoding="application / x-tex">-NHC(O)CH_3< / annotation>< / semantics>< / com:Math>. In certain embodiments, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^2< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(C(O)CH3)2<annotation encoding="application / x-tex">-N(C(O)CH_3)_2< / annotation>< / semantics>< / com:Math>.

[00175] In some embodiments, <com:Math><semantics>R3<annotation encoding="application / x-tex">R^3< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>3< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, <com:Math><semantics>R3<annotation encoding="application / x-tex">R^3< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>3< / com:Sup> is -NH<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>3< / com:Sup> is - NHR<com:Sup>W< / com:Sup>, wherein R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>3< / com:Sup> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>3< / com:Sup> is selected from the group consisting of -N<com:Sub>3< / com:Sub>, -NH(Cbz), -NH(Boc), -NH(Fmoc), -NHC(O)CCl<com:Sub>3< / com:Sub>, - NHC(O)CH<com:Sub>3< / com:Sub>, and -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>3< / com:Sup> is -NH(Cbz). In certain embodiments, R<com:Sup>3< / com:Sup> is -NH(Fmoc). In certain embodiments, R<com:Sup>3< / com:Sup> is -NHC(O)CCl<com:Sub>3< / com:Sub>. In certain embodiments, <com:Math><semantics>R3<annotation encoding="application / x-tex">R^3< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−NHC(O)CH3<annotation encoding="application / x-tex">-NHC(O)CH_3< / annotation>< / semantics>< / com:Math>. In certain embodiments, <com:Math><semantics>R3<annotation encoding="application / x-tex">R^3< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(C(O)CH3)2<annotation encoding="application / x-tex">-N(C(O)CH_3)_2< / annotation>< / semantics>< / com:Math>.

[00176] In some embodiments, <com:Math><semantics>R4<annotation encoding="application / x-tex">R^4< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>4< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, <com:Math><semantics>R4<annotation encoding="application / x-tex">R^4< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>4< / com:Sup> is -NH<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>4< / com:Sup> is - NHR<com:Sup>W< / com:Sup>, wherein R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>4< / com:Sup> is -N(R<com:Sup>W< / com:Sup>)<com:Sub>2< / com:Sub>, wherein each R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>4< / com:Sup> is selected from the group consisting of -N<com:Sub>3< / com:Sub>, -NH(Cbz), -NH(Boc), -NH(Fmoc), -NHC(O)CCl<com:Sub>3< / com:Sub>, - NHC(O)CH<com:Sub>3< / com:Sub>, and -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>4< / com:Sup> is -NH(Cbz). In certain embodiments, R<com:Sup>4< / com:Sup> is -NH(Fmoc). In certain embodiments, R<com:Sup>4< / com:Sup> is -NHC(O)CCl<com:Sub>3< / com:Sub>. In certain embodiments, R<com:Sup>4< / com:Sup> is -NHC(O)CH<com:Sub>3< / com:Sub>. In certain embodiments, R<com:Sup>4< / com:Sup> is -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>.

[00177] In some embodiments, <com:Math><semantics>R5<annotation encoding="application / x-tex">R^5< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, <com:Math><semantics>R5<annotation encoding="application / x-tex">R^5< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math>. In certain embodiments, <com:Math><semantics>R5<annotation encoding="application / x-tex">R^5< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>5< / com:Sup> is -NH<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>5< / com:Sup> is - <com:Math><semantics>NHRW<annotation encoding="application / x-tex">NHR^{W}< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RW<annotation encoding="application / x-tex">R^{W}< / annotation>< / semantics>< / com:Math> is a nitrogen protecting group. In certain embodiments, <com:Math><semantics>R5<annotation encoding="application / x-tex">R^{5}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^{W})_{2}< / annotation>< / semantics>< / com:Math>, wherein each R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>5< / com:Sup> is selected from the group consisting of -N<com:Sub>3< / com:Sub>, -NH(Cbz), -NH(Boc), -NH(Fmoc), -NHC(O)CCl<com:Sub>3< / com:Sub>, - NHC(O)CH<com:Sub>3< / com:Sub>, and -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>5< / com:Sup> is -NH(Cbz). In certain embodiments, R<com:Sup>5< / com:Sup> is -NH(Fmoc). In certain embodiments, R<com:Sup>5< / com:Sup> is -NHC(O)CCl<com:Sub>3< / com:Sub>. In certain embodiments, R<com:Sup>5< / com:Sup> is -NHC(O)CH<com:Sub>3< / com:Sub>. In certain embodiments, R<com:Sup>5< / com:Sup> is -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>.

[00178] In some embodiments, <com:Math><semantics>R6<annotation encoding="application / x-tex">R^6< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>6< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, <com:Math><semantics>R6<annotation encoding="application / x-tex">R^6< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>6< / com:Sup> is -NH<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>6< / com:Sup> is - NHR<com:Sup>W< / com:Sup>, wherein R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>6< / com:Sup> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>6< / com:Sup> is selected from the group consisting of -N<com:Sub>3< / com:Sub>, -NH(Cbz), -NH(Boc), -NH(Fmoc), -NHC(O)CCl<com:Sub>3< / com:Sub>, - NHC(O)CH<com:Sub>3< / com:Sub>, and -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>6< / com:Sup> is -NH(Cbz). In certain embodiments, R<com:Sup>6< / com:Sup> is -NH(Fmoc). In certain embodiments, R<com:Sup>6< / com:Sup> is -NHC(O)CCl<com:Sub>3< / com:Sub>. In certain embodiments, R<com:Sup>6< / com:Sup> is -NHC(O)CH<com:Sub>3< / com:Sub>. In certain embodiments, R<com:Sup>6< / com:Sup> is -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>.

[00179] In some embodiments, <com:Math><semantics>R1<annotation encoding="application / x-tex">R^1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^2< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>R3<annotation encoding="application / x-tex">R^3< / annotation>< / semantics>< / com:Math> are the same. In some embodiments, <com:Math><semantics>R1<annotation encoding="application / x-tex">R^1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^2< / annotation>< / semantics>< / com:Math> and R<com:Sup>3< / com:Sup> are -OH. In some embodiments, R<com:Sup>4< / com:Sup>, R<com:Sup>5< / com:Sup> and R<com:Sup>6< / com:Sup> are the same. In some embodiments, <com:Math><semantics>ℝ4<annotation encoding="application / x-tex">\mathbb{R}^4< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ℝ5<annotation encoding="application / x-tex">\mathbb{R}^5< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>ℝ6<annotation encoding="application / x-tex">\mathbb{R}^6< / annotation>< / semantics>< / com:Math> are -OH.

[00180] In certain embodiments, L is -OH.

[00181] In certain embodiments, L is -OH and R<com:Sup>1< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, L is -OH, <com:Math><semantics>ℝ1<annotation encoding="application / x-tex">\mathbb{R}^1< / annotation>< / semantics>< / com:Math> is -N<com:Sub>3< / com:Sub>, and each instance of <com:Math><semantics>ℝ2<annotation encoding="application / x-tex">\mathbb{R}^2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ℝ3<annotation encoding="application / x-tex">\mathbb{R}^3< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ℝ4<annotation encoding="application / x-tex">\mathbb{R}^4< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ℝ5<annotation encoding="application / x-tex">\mathbb{R}^5< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>ℝ6<annotation encoding="application / x-tex">\mathbb{R}^6< / annotation>< / semantics>< / com:Math> is -OH.

[00182] In certain embodiments, L is -OH and R<com:Sup>2< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, L is -OH, <com:Math><semantics>ℝ2<annotation encoding="application / x-tex">\mathbb{R}^2< / annotation>< / semantics>< / com:Math> is -N<com:Sub>3< / com:Sub>, and each instance of <com:Math><semantics>ℝ1<annotation encoding="application / x-tex">\mathbb{R}^1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ℝ3<annotation encoding="application / x-tex">\mathbb{R}^3< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ℝ4<annotation encoding="application / x-tex">\mathbb{R}^4< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ℝ5<annotation encoding="application / x-tex">\mathbb{R}^5< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>ℝ6<annotation encoding="application / x-tex">\mathbb{R}^6< / annotation>< / semantics>< / com:Math> is -OH.

[00183] In certain embodiments, L is -OH and R<com:Sup>3< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, L is -OH, <com:Math><semantics>R3<annotation encoding="application / x-tex">R^3< / annotation>< / semantics>< / com:Math> is -N<com:Sub>3< / com:Sub>, and each instance of <com:Math><semantics>R1<annotation encoding="application / x-tex">R^1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R4<annotation encoding="application / x-tex">R^4< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R5<annotation encoding="application / x-tex">R^5< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>R6<annotation encoding="application / x-tex">R^6< / annotation>< / semantics>< / com:Math> is -OH.

[00184] In certain embodiments, L is -OH and R<com:Sup>4< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, L is -OH, <com:Math><semantics>ℝ4<annotation encoding="application / x-tex">\mathbb{R}^4< / annotation>< / semantics>< / com:Math> is -N<com:Sub>3< / com:Sub>, and each instance of <com:Math><semantics>ℝ1<annotation encoding="application / x-tex">\mathbb{R}^1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ℝ2<annotation encoding="application / x-tex">\mathbb{R}^2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ℝ3<annotation encoding="application / x-tex">\mathbb{R}^3< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ℝ5<annotation encoding="application / x-tex">\mathbb{R}^5< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>ℝ6<annotation encoding="application / x-tex">\mathbb{R}^6< / annotation>< / semantics>< / com:Math> is -OH.

[00185] In certain embodiments, L is -OH and R<com:Sup>5< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, L is -OH, <com:Math><semantics>R5<annotation encoding="application / x-tex">R^5< / annotation>< / semantics>< / com:Math> is -N<com:Sub>3< / com:Sub>, and each instance of <com:Math><semantics>R1<annotation encoding="application / x-tex">R^1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R3<annotation encoding="application / x-tex">R^3< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R4<annotation encoding="application / x-tex">R^4< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>R6<annotation encoding="application / x-tex">R^6< / annotation>< / semantics>< / com:Math> is -OH.

[00186] In certain embodiments, L is -OH and R<com:Sup>6< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, L is -OH, <com:Math><semantics>ℝ6<annotation encoding="application / x-tex">\mathbb{R}^6< / annotation>< / semantics>< / com:Math> is -N<com:Sub>3< / com:Sub>, and each instance of <com:Math><semantics>ℝ1<annotation encoding="application / x-tex">\mathbb{R}^1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ℝ2<annotation encoding="application / x-tex">\mathbb{R}^2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ℝ3<annotation encoding="application / x-tex">\mathbb{R}^3< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ℝ4<annotation encoding="application / x-tex">\mathbb{R}^4< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>ℝ5<annotation encoding="application / x-tex">\mathbb{R}^5< / annotation>< / semantics>< / com:Math> is -OH.

[00187] In certain embodiments, each instance of R<com:Sup>1< / com:Sup>, R<com:Sup>2< / com:Sup>, R<com:Sup>3< / com:Sup>, R<com:Sup>4< / com:Sup>, R<com:Sup>5< / com:Sup>, R<com:Sup>6< / com:Sup> and L is –F. In certain embodiments, R<com:Sup>1< / com:Sup> is -F. In certain embodiments, R<com:Sup>2< / com:Sup> is -F. In certain embodiments, R<com:Sup>3< / com:Sup> is -F. In certain embodiments, R<com:Sup>4< / com:Sup> is -F. In certain embodiments, R<com:Sup>5< / com:Sup> is -F. In certain embodiments, R<com:Sup>6< / com:Sup> is -F. In certain embodiments, L is -F.

[00188] In certain embodiments, L is of the following structure: HOOC R<com:Sup>10< / com:Sup> ₽<com:Sup>8< / com:Sup> wherein: each instance of R<com:Sup>8< / com:Sup>, R<com:Sup>9< / com:Sup>, R<com:Sup>10< / com:Sup> and R<com:Sup>11< / com:Sup> is independently selected from hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, -N<com:Sub>3< / com:Sub>, -NO<com:Sub>2< / com:Sub>, -N(R<com:Sup>B< / com:Sup>)<com:Sub>2< / com:Sub>, - <com:Math><semantics>N(RA)C(O)RA<annotation encoding="application / x-tex">N(R^{A})C(O)R^{A}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−ORA<annotation encoding="application / x-tex">-OR^{A}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−OC(O)RA<annotation encoding="application / x-tex">-OC(O)R^{A}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SRA<annotation encoding="application / x-tex">-SR^{A}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)N(RB)2<annotation encoding="application / x-tex">-C(O)N(R^{B})_{2}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−CN<annotation encoding="application / x-tex">-CN< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)RA<annotation encoding="application / x-tex">-C(O)R^{A}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(O)ORA<annotation encoding="application / x-tex">-C(O)OR^{A}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−S(O)RA<annotation encoding="application / x-tex">-S(O)R^{A}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−S(O)RA<annotation encoding="application / x-tex">-S(O)R^{A}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−S(O)RA<annotation encoding="application / x-tex">-S(O)R^{A}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−S(O)RA<annotation encoding="application / x-tex">-S(O)R^{A}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−S(O)RA<annotation encoding="application / x-tex">-S(O)R^{A}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−S(O)RA<annotation encoding="application / x-tex">-S(O)R^{A}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−S(O)RA<annotation encoding="application / x-tex">-S(O)R^{A}< / annotation>< / semantics>< / com:Math>, <Formule mathématique disponible dans le document PDF, Math available in the PDF document>$-S(O)R^$ <com:Math><semantics>SO2RA<annotation encoding="application / x-tex">SO_2R^A< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−SO2N(RB)2<annotation encoding="application / x-tex">-SO_2N(R^B)_2< / annotation>< / semantics>< / com:Math>, and <com:Math><semantics>−NHSO2RB<annotation encoding="application / x-tex">-NHSO_2R^B< / annotation>< / semantics>< / com:Math>: [Image disponible dans le document PDF, Image available in the PDF document] each instance of R<com:Sup>A< / com:Sup> is independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl; and each instance of R<com:Sup>B< / com:Sup> is independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl.

[00189] In some embodiments, the compound is of Formula (II) [Image disponible dans le document PDF, Image available in the PDF document] wherein: <com:Math><semantics>R1<annotation encoding="application / x-tex">R^1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R3<annotation encoding="application / x-tex">R^3< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R8<annotation encoding="application / x-tex">R^8< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R9<annotation encoding="application / x-tex">R^9< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R10<annotation encoding="application / x-tex">R^{10}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R11<annotation encoding="application / x-tex">R^{11}< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>RN<annotation encoding="application / x-tex">R_N< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math> are as described herein, and provided the compound is not of the formula: [Image disponible dans le document PDF, Image available in the PDF document]

[00190] In some embodiments, <com:Math><semantics>R8<annotation encoding="application / x-tex">R^8< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>8< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, <com:Math><semantics>R8<annotation encoding="application / x-tex">R^8< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>8< / com:Sup> is -NH<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>8< / com:Sup> is - <com:Math><semantics>NHRW<annotation encoding="application / x-tex">NHR^{W}< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RW<annotation encoding="application / x-tex">R^{W}< / annotation>< / semantics>< / com:Math> is a nitrogen protecting group. In certain embodiments, <com:Math><semantics>R8<annotation encoding="application / x-tex">R^{8}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^{W})_{2}< / annotation>< / semantics>< / com:Math>, wherein each R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>8< / com:Sup> is selected from the group consisting of -N<com:Sub>3< / com:Sub>, -NH(Cbz), -NH(Boc), -NH(Fmoc), -NHC(O)CCl<com:Sub>3< / com:Sub>, - NHC(O)CH<com:Sub>3< / com:Sub>, and -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>8< / com:Sup> is -NH(Cbz). In certain embodiments, R<com:Sup>8< / com:Sup> is -NH(Fmoc). In certain embodiments, R<com:Sup>8< / com:Sup> is -NHC(O)CCl<com:Sub>3< / com:Sub>. In certain embodiments, R<com:Sup>8< / com:Sup> is -NHC(O)CH<com:Sub>3< / com:Sub>. In certain embodiments, R<com:Sup>8< / com:Sup> is -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>.

[00191] In some embodiments, <com:Math><semantics>R9<annotation encoding="application / x-tex">R^9< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>9< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, <com:Math><semantics>R9<annotation encoding="application / x-tex">R^9< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>9< / com:Sup> is -NH<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>9< / com:Sup> is - NHR<com:Sup>W< / com:Sup>, wherein R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>9< / com:Sup> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>9< / com:Sup> is selected from the group consisting of -N<com:Sub>3< / com:Sub>, -NH(Cbz), -NH(Boc), -NH(Fmoc), -NHC(O)CCl<com:Sub>3< / com:Sub>, - NHC(O)CH<com:Sub>3< / com:Sub>, and -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>. In certain embodiments, <com:Math><semantics>R9<annotation encoding="application / x-tex">R^9< / annotation>< / semantics>< / com:Math> is -NH(Cbz). In certain embodiments, R<com:Sup>9< / com:Sup> is -NH(Fmoc). In certain embodiments, R<com:Sup>9< / com:Sup> is -NHC(O)CCl<com:Sub>3< / com:Sub>. In certain embodiments, <com:Math><semantics>R9<annotation encoding="application / x-tex">R^9< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−NHC(O)CH3<annotation encoding="application / x-tex">-NHC(O)CH_3< / annotation>< / semantics>< / com:Math>. In certain embodiments, <com:Math><semantics>R9<annotation encoding="application / x-tex">R^9< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(C(O)CH3)2<annotation encoding="application / x-tex">-N(C(O)CH_3)_2< / annotation>< / semantics>< / com:Math>.

[00192] In some embodiments, <com:Math><semantics>R10<annotation encoding="application / x-tex">R^{10}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>10< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, R<com:Sup>10< / com:Sup> is -N(R<com:Sup>W< / com:Sup>)<com:Sub>2< / com:Sub>, wherein each R<com:Sup>W< / com:Sup> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>10< / com:Sup> is -NH<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>10< / com:Sup> is - NHR<com:Sup>W< / com:Sup>, wherein R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, <com:Math><semantics>R10<annotation encoding="application / x-tex">R^{10}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^{W})_{2}< / annotation>< / semantics>< / com:Math>, wherein each R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>10< / com:Sup> is selected from the group consisting of -N<com:Sub>3< / com:Sub>, -NH(Cbz), -NH(Boc), -NH(Fmoc), -NHC(O)CCl<com:Sub>3< / com:Sub>, - <com:Math><semantics>NHC(O)CH3<annotation encoding="application / x-tex">NHC(O)CH_3< / annotation>< / semantics>< / com:Math>, and <com:Math><semantics>−N(C(O)CH3)2<annotation encoding="application / x-tex">-N(C(O)CH_3)_2< / annotation>< / semantics>< / com:Math>. In certain embodiments, <com:Math><semantics>R10<annotation encoding="application / x-tex">R^{10}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−NH(Cbz)<annotation encoding="application / x-tex">-NH(Cbz)< / annotation>< / semantics>< / com:Math>. In certain embodiments, R<com:Sup>10< / com:Sup> is -NH(Fmoc). In certain embodiments, R<com:Sup>10< / com:Sup> is -NHC(O)CCl<com:Sub>3< / com:Sub>. In certain embodiments, <com:Math><semantics>R10<annotation encoding="application / x-tex">R^{10}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−NHC(O)CH3<annotation encoding="application / x-tex">-NHC(O)CH_3< / annotation>< / semantics>< / com:Math>. In certain embodiments, <com:Math><semantics>R10<annotation encoding="application / x-tex">R^{10}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(C(O)CH3)2<annotation encoding="application / x-tex">-N(C(O)CH_3)_2< / annotation>< / semantics>< / com:Math>.

[00193] In some embodiments, <com:Math><semantics>R11<annotation encoding="application / x-tex">R^{11}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>11< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, <com:Math><semantics>R11<annotation encoding="application / x-tex">R^{11}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^{W})_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^{W}< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>11< / com:Sup> is -NH<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>11< / com:Sup> is - <com:Math><semantics>NHRW<annotation encoding="application / x-tex">NHR^{W}< / annotation>< / semantics>< / com:Math>, wherein <com:Math><semantics>RW<annotation encoding="application / x-tex">R^{W}< / annotation>< / semantics>< / com:Math> is a nitrogen protecting group. In certain embodiments, <com:Math><semantics>R11<annotation encoding="application / x-tex">R^{11}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^{W})_{2}< / annotation>< / semantics>< / com:Math>, wherein each R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sup>11< / com:Sup> is selected from the group consisting of -N<com:Sub>3< / com:Sub>, -NH(Cbz), -NH(Boc), -NH(Fmoc), -NHC(O)CCl<com:Sub>3< / com:Sub>, - NHC(O)CH<com:Sub>3< / com:Sub>, and -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sup>11< / com:Sup> is -NH(Cbz). In certain embodiments, R<com:Sup>11< / com:Sup> is -NH(Fmoc). In certain embodiments, R<com:Sup>11< / com:Sup> is -NHC(O)CCl<com:Sub>3< / com:Sub>. In certain embodiments, R<com:Sup>11< / com:Sup> is -NHC(O)CH<com:Sub>3< / com:Sub>. In certain embodiments, R<com:Sup>11< / com:Sup> is -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>.

[00194] In some embodiments, <com:Math><semantics>R12<annotation encoding="application / x-tex">R^{12}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sup>12< / com:Sup> is -N<com:Sub>3< / com:Sub>. In certain embodiments, R<com:Sup>12< / com:Sup> is -N(R<com:Sup>W< / com:Sup>)<com:Sub>2< / com:Sub>, wherein each R<com:Sup>W< / com:Sup> is independently hydrogen or a nitrogen protecting group. In certain embodiments, <com:Math><semantics>R12<annotation encoding="application / x-tex">R^{12}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−NH2<annotation encoding="application / x-tex">-NH_2< / annotation>< / semantics>< / com:Math>. In certain embodiments, <com:Math><semantics>R12<annotation encoding="application / x-tex">R^{12}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−<annotation encoding="application / x-tex">-< / annotation>< / semantics>< / com:Math> NHR<com:Sup>W< / com:Sup>, wherein R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, <com:Math><semantics>R12<annotation encoding="application / x-tex">R^{12}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^{W})_2< / annotation>< / semantics>< / com:Math>, wherein each RW is a nitrogen protecting group. In certain embodiments, R12 is selected from the group consisting of -N<com:Sub>3< / com:Sub>, -NH(Cbz), -NH(Boc), -NH(Fmoc), -NHC(O)CCl<com:Sub>3< / com:Sub>, - <com:Math><semantics>NHC(O)CH3<annotation encoding="application / x-tex">NHC(O)CH_3< / annotation>< / semantics>< / com:Math>, and <com:Math><semantics>−N(C(O)CH3)2<annotation encoding="application / x-tex">-N(C(O)CH_3)_2< / annotation>< / semantics>< / com:Math>. In certain embodiments, <com:Math><semantics>R12<annotation encoding="application / x-tex">R^{12}< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−NH(Cbz)<annotation encoding="application / x-tex">-NH(Cbz)< / annotation>< / semantics>< / com:Math>. In certain embodiments, R<com:Sup>12< / com:Sup> is -NH(Fmoc). In certain embodiments, R<com:Sup>12< / com:Sup> is -NHC(O)CCl<com:Sub>3< / com:Sub>. In certain embodiments, R<com:Sup>12< / com:Sup> is -NHC(O)CH<com:Sub>3< / com:Sub>. In certain embodiments, R<com:Sup>12< / com:Sup> is -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>.

[00195] In some embodiments, <com:Math><semantics>RN<annotation encoding="application / x-tex">R_N< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, <com:Math><semantics>RN<annotation encoding="application / x-tex">R_N< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N3<annotation encoding="application / x-tex">-N_3< / annotation>< / semantics>< / com:Math>. In certain embodiments, <com:Math><semantics>RN<annotation encoding="application / x-tex">R_N< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each <com:Math><semantics>RW<annotation encoding="application / x-tex">R^W< / annotation>< / semantics>< / com:Math> is independently hydrogen or a nitrogen protecting group. In certain embodiments, R<com:Sub>N< / com:Sub> is -NH<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sub>N< / com:Sub> is - NHR<com:Sup>W< / com:Sup>, wherein R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, <com:Math><semantics>RN<annotation encoding="application / x-tex">R_N< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(RW)2<annotation encoding="application / x-tex">-N(R^W)_2< / annotation>< / semantics>< / com:Math>, wherein each R<com:Sup>W< / com:Sup> is a nitrogen protecting group. In certain embodiments, R<com:Sub>N< / com:Sub> is selected from the group consisting of -N<com:Sub>3< / com:Sub>, -NH(Cbz), -NH(Boc), -NH(Fmoc), -NHC(O)CCl<com:Sub>3< / com:Sub>, - NHC(O)CH<com:Sub>3< / com:Sub>, and -N(C(O)CH<com:Sub>3< / com:Sub>)<com:Sub>2< / com:Sub>. In certain embodiments, R<com:Sub>N< / com:Sub> is -NH(Cbz). In certain embodiments, R<com:Sub>N< / com:Sub> is -NH(Fmoc). In certain embodiments, R<com:Sub>N< / com:Sub> is -NHC(O)CCl<com:Sub>3< / com:Sub>. In certain embodiments, <com:Math><semantics>RN<annotation encoding="application / x-tex">R_N< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−NHC(O)CH3<annotation encoding="application / x-tex">-NHC(O)CH_3< / annotation>< / semantics>< / com:Math>. In certain embodiments, <com:Math><semantics>RN<annotation encoding="application / x-tex">R_N< / annotation>< / semantics>< / com:Math> is <com:Math><semantics>−N(C(O)CH3)2<annotation encoding="application / x-tex">-N(C(O)CH_3)_2< / annotation>< / semantics>< / com:Math>. Immunogenic Compositions

[00196] In another aspect, the present invention provides an immunogenic composition, comprising (a) a glycan conjugate including a carrier and one or more glycans, and optionally (b) an adjuvant, wherein: each of the one or more glycans is conjugated with the carrier through a linker, having the formula (III) or (IV): [Image disponible dans le document PDF, Image available in the PDF document] (III); [Image disponible dans le document PDF, Image available in the PDF document] wherein <com:Math><semantics>X1<annotation encoding="application / x-tex">X_1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R1<annotation encoding="application / x-tex">R^1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R2<annotation encoding="application / x-tex">R^2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R3<annotation encoding="application / x-tex">R^3< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R4<annotation encoding="application / x-tex">R^4< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R5<annotation encoding="application / x-tex">R^5< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R6<annotation encoding="application / x-tex">R^6< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R8<annotation encoding="application / x-tex">R^8< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R9<annotation encoding="application / x-tex">R^9< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R10<annotation encoding="application / x-tex">R^{10}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>R11<annotation encoding="application / x-tex">R^{11}< / annotation>< / semantics>< / com:Math>, L and <com:Math><semantics>RN<annotation encoding="application / x-tex">R_N< / annotation>< / semantics>< / com:Math> are as described herein.

[00197] In certain embodiments, the linker is a hetero- or homo-bifunctional linker.

[00198] In certain embodiments, the linker includes at least one sulfur atom, carboxylate group, amide group, carbamate group, carbonate group, thiocarbamate group, thiocarbonate group, thioether group, succinamide group, n-hydroxy succinamide group, or any combination thereof.

[00199] In certain embodiments, the linker is <com:Math><semantics>−L1−L2<annotation encoding="application / x-tex">-L^{1}-L^{2}< / annotation>< / semantics>< / com:Math>, wherein L<com:Sup>1< / com:Sup> is a bond, <com:Math><semantics>−O<annotation encoding="application / x-tex">-O< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−S<annotation encoding="application / x-tex">-S< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−<annotation encoding="application / x-tex">-< / annotation>< / semantics>< / com:Math> <com:Math><semantics>NRL1a<annotation encoding="application / x-tex">NR^{L1a}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=O)<annotation encoding="application / x-tex">-C(=O)< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−NRL1aC(=O)<annotation encoding="application / x-tex">-NR^{L1a}C(=O)< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−NRL1aC(=O)O<annotation encoding="application / x-tex">-NR^{L1a}C(=O)O< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=O)NRL1a<annotation encoding="application / x-tex">-C(=O)NR^{L1a}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−OC(=O)NRL1a<annotation encoding="application / x-tex">-OC(=O)NR^{L1a}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>−C(=O)NRL1a<annotation encoding="application / x-tex">-C(=O)NR^{L1a}< / annotation>< / semantics>< / com:Math> SC(=O)-, -C(=O)S-, -OC(=O)-, -C(=O)O-, -NR<com:Sup>L1a< / com:Sup>C(=S)-, -C(=S)NR<com:Sup>L1a< / com:Sup>-, trans- <com:Math><semantics>CRL1b=CRL1b−,cis−CRL1b=CRL1b−,−C=C−,−OC(RL1b)2−,−C(RL1b)2−,−NRL1aC(RL1b)2−,−C(RL1b)2−,−C(RL1b)2−,−C(RL1b)2−,−C(RL1b)2−,−C(RL1b)2−,−C(RL1b)2<annotation encoding="application / x-tex">CR^{L1b} = CR^{L1b} -, cis - CR^{L1b} = CR^{L1b} -, -C = C -, -OC(R^{L1b})_2 -, -C(R^{L1b})_2 -, -NR^{L1a}C(R^{L1b})_2 -, -C(R^{L1b})_2 -, -C(R^{L1b})_2 -, -C(R^{L1b})_2 -, -C(R^{L1b})_2 -, -C(R^{L1b})_2 -, -C(R^{L1b})_2< / annotation>< / semantics>< / com:Math> [Image disponible dans le document PDF, Image available in the PDF document] <com:Math><semantics>NRL1aS(=O)2−<annotation encoding="application / x-tex">NR^{L_{1a}}S(=O)_{2-}< / annotation>< / semantics>< / com:Math>, or an optionally substituted <com:Math><semantics>C1−20<annotation encoding="application / x-tex">C_{1-20}< / annotation>< / semantics>< / com:Math> hydrocarbon chain, optionally wherein one or more carbon units of the hydrocarbon chain is replaced with -O-, -S-, -NR<com:Sup>L1a< / com:Sup>-, -C(=O)-, <com:Math><semantics>NRL1aC(=O)−,−NRL1aC(=O)O−,−C(=O)NRL1a−,−OC(=O)NRL1a−,−SC(=O)−,−C(=O)S−,−C(=O)S−<annotation encoding="application / x-tex">NR^{L1a}C(=O)-, -NR^{L1a}C(=O)O-, -C(=O)NR^{L1a}-, -OC(=O)NR^{L1a}-, -SC(=O)-, -C(=O)S-, -C(=O)S-< / annotation>< / semantics>< / com:Math> OC(=O)-, -C(=O)O-, -NR<com:Sup>L1a< / com:Sup>C(=S)-, -C(=S)NR<com:Sup>L1a< / com:Sup>-, trans-CR<com:Sup>L1b< / com:Sup>=CR<com:Sup>L1b< / com:Sup>-, cis- <com:Math><semantics>CRL1b=CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−CRL1b−<annotation encoding="application / x-tex">CR^{L1b} = CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} - CR^{L1b} -< / annotation>< / semantics>< / com:Math> <com:Math><semantics>RL1a<annotation encoding="application / x-tex">R^{L1a}< / annotation>< / semantics>< / com:Math> is hydrogen, optionally substituted <com:Math><semantics>C1−6<annotation encoding="application / x-tex">C_{1-6}< / annotation>< / semantics>< / com:Math> alkyl, or a nitrogen protecting group, or <com:Math><semantics>RL1a<annotation encoding="application / x-tex">R^{L1a}< / annotation>< / semantics>< / com:Math> is joined with the adjacent carbon atom to form an optionally substituted heterocyclic ring, and wherein each occurrence of R<com:Sup>L1b< / com:Sup> is independently selected from the group consisting of hydrogen, halogen, optionally substituted <com:Math><semantics>C1−10<annotation encoding="application / x-tex">C_{1-10}< / annotation>< / semantics>< / com:Math> alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or R<com:Sup>L1b< / com:Sup> is joined with the adjacent carbon or nitrogen or oxygen atom to form an optionally substituted carbocyclic or heterocyclic ring, or two R<com:Sup>L1b< / com:Sup> groups are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring; and L<com:Sup>2< / com:Sup> is a moiety derived from a crosslinking reagent capable of crosslinking the carrier and L<com:Sup>1< / com:Sup>.

[00200] The carrier can be a protein, a lipid, a lipid, a lipolized protein, a virus, a peptide, or a dendrimer of glycopeptides. In certain embodiments, the carrier is a peptide comprising a T cell epitope.

[00201] Examples of carrier proteins which may be used in the present invention are tetanus toxoid (TT), diphtheria toxoid (DT), diphtheria toxin cross-reacting material 197 (CRM197), fragment C of TT, Keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), protein D, outer-membrane protein (OMP) and pneumolysin, diphtheria toxin cross-reacting material 197 (CRM197) or other DT point mutants, such as CRM176, CRM228, CRM 45 (Uchida et al J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103 and CRM107 and other mutations described in the art.

[00202] In certain embodiments, the glycan conjugate is of the formula (IV-a) or (IV-b): [Image disponible dans le document PDF, Image available in the PDF document] (IV-a); [Image disponible dans le document PDF, Image available in the PDF document] b) wherein m is an integer of 1 to 40, inclusive.

[00203] In certain embodiments, m is an integer of 1 to 30, inclusive. As generally defined herein, m is an integer of 1 to 20 inclusive. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, m is 4. In certain embodiments, m is 6. In certain embodiments, m is 8. In certain embodiments, m is 10. In certain embodiments, m is 15. In certain embodiments, m is 20. In certain embodiments, m is 30. In certain embodiments, m is 40.

[00204] In another aspect, the present invention provides a glycan conjugate mixture comprising at least two of the glycan conjugates as described herein. In certain embodiments, the average value of w in the glycan mixture is from about 1.0 to about 40.0. In certain embodiments, the average value of w in the glycan mixture is from about 1.0 to 10.0. In certain embodiments, the average value of w in the glycan mixture is about 5.7, 4.9, 2.9, 2.8, or 3.1. In certain embodiments, the average value of w in the glycan mixture is about 4.9, 2.9, 2.8, or 3.1.

[00205] In certain embodiments, the immunogenic compositions described herein include an immunogenically effective amount of a glycan conjugate of the invention.

[00206] The compounds of the invention can be synthesized using procedures known in the art or described herein. Also see US20140051127.

[00207] The immunogenic conjugate of the invention may include one or more molecules (e.g., 1-40, 1-20, 1-25, 1-30, 5-20, 5-25, 5-30, or 5-35) of the same or different SSEA-33 and / or SSEA-4 derivatives. Procedures for generating glycan conjugates are known in the art and described below. Also see US Patent No. 8,268,969.

[00208] In certain embodiments, the immunogenic composition of the invention may include one or more adjuvants. Suitable adjuvants are known in the art (e.g., C34, 7DW8-5, C17, C23, Gluco-C34, Aluminum salt, Squalene, MF59, and QS-21).

[00209] As used herein, the term "alum adjuvant" refers to an aluminum salt with immune adjuvant activity. This agent adsorbs and precipitates protein antigens in solution; the resulting precipitate improves vaccine immunogenicity by facilitating the slow release of antigen from the vaccine depot formed at the site of inoculation.

[00210] As used herein, the term "immunologic adjuvant" refers to a substance used in conjunction with an immunogen which enhances or modifies the immune response to the immunogen. The α-GalCer analogs of the present disclosure are used as immunologic adjuvants to modify or augment the effects of a vaccine by stimulating the immune system of a patient who is administered the vaccine to respond to the vaccine more vigorously. In an exemplary implementation, the analog C34 is used as an adjuvant. The structures of C34 and other alpha-galactosyl ceramide analogs and their use as adjuvants are disclosed in detail in US patent No. 7,928,077.

[00211] As used herein, the term "glycolipid" refers to a carbohydrate-attached lipid that serves as a marker for cellular recognition.

[00212] The glycolipids C34, Gluco-C34, C23 and 7DW8-5 have the following structures: [Image disponible dans le document PDF, Image available in the PDF document] C1, <com:Math><semantics>R=(CH2)24CH3<annotation encoding="application / x-tex">R = (CH_2)_{24}CH_3< / annotation>< / semantics>< / com:Math><com:Br / > C23, <com:Math><semantics>R=(CH2)7PhF<annotation encoding="application / x-tex">R = (CH_2)_7PhF< / annotation>< / semantics>< / com:Math><com:Br / > C34, <com:Math><semantics>R=(CH2)10PhOPhF<annotation encoding="application / x-tex">R = (CH_2)_{10}PhOPhF< / annotation>< / semantics>< / com:Math><com:Br / > 7DW8-5, <com:Math><semantics>R=(CH2)10PhF<annotation encoding="application / x-tex">R = (CH_2)_{10}PhF< / annotation>< / semantics>< / com:Math> [Image disponible dans le document PDF, Image available in the PDF document] [Image disponible dans le document PDF, Image available in the PDF document] [Image disponible dans le document PDF, Image available in the PDF document] [Image disponible dans le document PDF, Image available in the PDF document]

[00213] The immunogenic composition can further include a pharmaceutically acceptable excipient. In certain embodiments, the immunogenic compositions described herein include an pharmaceutically effective amount of a glycan conjugate of the invention.

[00214] In another aspect, the present invention provides a cancer vaccine comprising an immmunogenic composition described herein and a pharmaceutically acceptable excipient.

[00215] The cancer vaccines of the invention may include a single dose or multiple doses of the inventive glycan conjugates, a glycan conjugate mixture thereof, or immmunogenic compositions thereof. The provided cancer vaccines may be useful for treating or reducing the risk of cancers. The cancer vaccines may also include packaging information describing the use or prescribing information for the subject or a health care professional. Such information may be required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). The cancer vaccine may also optionally include a device for administration of the compound or composition, for example, a syringe for parenteral administration. Pharmaceutical Formulations

[00216] The immune composition is administered in a manner compatible with the dosage formulation, and in an amount that is therapeutically effective, protective and immunogenic. The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize antibodies, and if needed, to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dosage of the vaccine may also depend on the route of administration and varies according to the size of the host.

[0100] The immune composition of this invention can also be used to generate antibodies in animals for production of antibodies, which can be used in both cancer treatment and diagnosis. Methods of making 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" includes intact immunoglobulin molecules as well as fragments thereof, such as Fab, F(ab')2, Fv, scFv (single chain antibody), and dAb (domain antibody; Ward, et. al. (1989) Nature, 341, 544).

[0101] The compositions disclosed herein can be included in a pharmaceutical composition together with additional active agents, carriers, vehicles, excipients, or auxiliary agents identifiable by a person skilled in the art upon reading of the present disclosure.

[0102] The pharmaceutical compositions preferably comprise at least one pharmaceutically acceptable carrier. In such pharmaceutical compositions, the compositions disclosed herein form the "active compound," also referred to as the "active agent." As used herein the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Clinical Applications

[0103] The present invention provides glycan conjugates, immunogenic compositions or vaccines useful for the treatment of a proliferative disease such as cancer (e.g. lung cancer, large bowel cancer, pancreas cancer, biliary tract cancer, or endometrial cancer), benign neoplasm, or angiogenesis in a subject.

[0104] The immunogenic compositions or vaccines described herein can also be used to generate antibodies in human or animals for production of antibodies, which can be used in both cancer treatment and diagnosis. In some embodiments, the immunogenic compositions or vaccines described herein can also be used to generate antibodies for production of Globo H, SSEA-3 and / or SSEA-4 antibodies. Methods of making monoclonal and polyclonal antibodies and fragments thereof in human and / or 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" includes intact immunoglobulin molecules as well as fragments thereof, such as Fab, F(ab').sub.2, Fv, scFv (single chain antibody), and dAb (domain antibody; Ward, et. al. (1989) Nature, 341, 544).

[0105] Compositions comprising at least one anti-SSEA-3 / SSEA-4 / GLOBO H antibody or at least one polynucleotide comprising sequences encoding an anti-SSEA-3 / SSEA-4 / GLOBO H antibody are provided. In certain embodiments, a composition may be a pharmaceutical composition. As used herein, compositions comprise one or more antibodies that bind to one or more SSEA-3 / SSEA-4 / GLOBO H and / or one or more polynucleotides comprising sequences encoding one or more antibodies that bind to one or more SSEA-3 / SSEA- 4 / GLOBO H. These compositions may further comprise suitable carriers, such as pharmaceutically acceptable excipients including buffers, which are well known in the art.

[0106] Isolated antibodies and polynucleotides are also provided. In certain embodiments, the isolated antibodies and polynucleotides are substantially pure.

[0107] In one embodiment, anti-SSEA-3 / SSEA-4 / GLOBO H antibodies are monoclonal. In another embodiment, fragments of the anti-SSEA-3 / SSEA-4 / GLOBO H antibodies (e.g., Fab, Fab -SH and F(ab )2 fragments) are provided. These antibody fragments can be created by traditional means, such as enzymatic digestion, or may be generated by recombinant techniques. Such antibody fragments may be chimeric, humanized, or human. These fragments are useful for the diagnostic and therapeutic purposes set forth below.

[0108] Example of Generation of Anti-SSEA-3 / SSEA-4 / GLOBO H Antibodies Using a Phage Display Library

[0109] A variety of methods are known in the art for generating phage display libraries from which an antibody of interest can be obtained. One method of generating antibodies of interest is through the use of a phage antibody library as described in Lee et al., J. Mol. Biol. (2004), 340(5): 1073-93.

[0110] The anti-SSEA-3 / SSEA-4 / GLOBO H antibodies of the invention can be made by using combinatorial libraries to screen for synthetic antibody clones with the desired activity or activities. In principle, synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption / elution. Any of the anti-SSEA-3 / SSEA-4 / GLOBO H antibodies of the invention can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length anti-SSEA-3 / SSEA- 4 / GLOBO H antibody clone using the Fv sequences from the phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.

[0111] The antigen-binding domain of an antibody is formed from two variable (V) regions of about 110 amino acids, one each from the light (VL) and heavy (VH) chains, that both present three hypervariable loops or complementarity-determining regions (CDRs). Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, scFv encoding phage clones and Fab encoding phage clones are collectively referred to as "Fv phage clones" or "Fv clones".

[0112] Repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning the unrearranged V- gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

[0113] Filamentous phage is used to display antibody fragments by fusion to the minor coat protein pIII. The antibody fragments can be displayed as single chain Fv fragments, in which VH and VL domains are connected on the same polypeptide chain by a flexible polypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fab fragments, in which one chain is fused to pIII and the other is secreted into the bacterial host cell periplasm where assembly of a Fab-coat protein structure which becomes displayed on the phage surface by displacing some of the wild type coat proteins, e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

[0114] In general, nucleic acids encoding antibody gene fragments are obtained from immune cells harvested from humans or animals. If a library biased in favor of anti-SSEA-3 / SSEA- 4 / GLOBO H clones is desired, the subject is immunized with SSEA-3 / SSEA-4 / GLOBO H to generate an antibody response, and spleen cells and / or circulating B cells or other peripheral blood lymphocytes (PBLs) are recovered for library construction. In one embodiment, a human antibody gene fragment library biased in favor of anti-human SSEA-3 / SSEA- 4 / GLOBO H clones is obtained by generating an anti-human SSEA-3 / SSEA-4 / GLOBO H antibody response in transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) such that SSEA-3 / SSEA- 4 / GLOBO H immunization gives rise to B cells producing human antibodies against SSEA- 3 / SSEA-4 / GLOBO H. The generation of human antibody-producing transgenic mice is described below.

[0115] Additional enrichment for anti-SSEA-3 / SSEA-4 / GLOBO H reactive cell populations can be obtained by using a suitable screening procedure to isolate B cells expressing SSEA- 3 / SSEA-4 / GLOBO H-specific antibody, e.g., by cell separation with SSEA-3 / SSEA- 4 / GLOBO H affinity chromatography or adsorption of cells to fluorochrome-labeled SSEA- 3 / SSEA-4 / GLOBO H followed by flow-activated cell sorting (FACS).

[0116] Alternatively, the use of spleen cells and / or B cells or other PBLs from an unimmunized donor provides a better representation of the possible antibody repertoire, and also permits the construction of an antibody library using any animal (human or non-human) species in which SSEA-3 / SSEA-4 / GLOBO H is not antigenic. For libraries incorporating in vitro antibody gene construction, stem cells are harvested from the subject to provide nucleic acids encoding unrearranged antibody gene segments. The immune cells of interest can be obtained from a variety of animal species, such as human, mouse, rat, lagomorpha, luprine, canine, feline, porcine, bovine, equine, and avian species, etc.

[0117] Nucleic acid encoding antibody variable gene segments (including VH and VL) segments) are recovered from the cells of interest and amplified. In the case of rearranged VH and VL gene libraries, the desired DNA can be obtained by isolating genomic DNA or mRNA from lymphocytes followed by polymerase chain reaction (PCR) with primers matching the 5 and 3 ends of rearranged VH and VL genes as described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse V gene repertoires for expression. The V genes can be amplified from cDNA and genomic DNA, with back primers at the 5 end of the exon encoding the mature V-domain and forward primers based within the J-segment as described in Orlandi et al. (1989) and in Ward et al., Nature, 341: 544-546 (1989). However, for amplifying from cDNA, back primers can also be based in the leader exon as described in Jones et al., Biotechnol., 9: 88-89 (1991), and forward primers within the constant region as described in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732 (1989). To maximize complementarity, degeneracy can be incorporated in the primers as described in Orlandi et al. (1989) or Sastry et al. (1989). In certain embodiments, the library diversity is maximized by using PCR primers targeted to each V-gene family in order to amplify all available VH and VL arrangements present in the immune cell nucleic acid sample, e.g. as described in the method of Marks et al., J. Mol. Biol., 222: 581-597 (1991) or as described in the method of Orum et al., Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the amplified DNA into expression vectors, rare restriction sites can be introduced within the PCR primer as a tag at one end as described in Orlandi et al. (1989), or by further PCR amplification with a tagged primer as described in Clackson et al., Nature, 352: 624-628 (1991).

[0118] Repertoires of synthetically rearranged V genes can be derived in vitro from V gene segments. Most of the human VH-gene segments have been cloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227: 776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned segments (including all the major conformations of the H1 and H2 loop) can be used to generate diverse VH gene repertoires with PCR primers encoding H3 loops of diverse sequence and length as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). VH repertoires can also be made with all the sequence diversity focused in a long H3 loop of a single length as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). Human Vκ and Vλ segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used to make synthetic light chain repertoires. Synthetic V gene repertoires, based on a range of VH and VL folds, and L3 and H3 lengths, will encode antibodies of considerable structural diversity. Following amplification of V-gene encoding DNAs, germline V-gene segments can be rearranged in vitro according to the methods of Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

[0119] Repertoires of antibody fragments can be constructed by combining VH and VL gene repertoires together in several ways. Each repertoire can be created in different vectors, and the vectors recombined in vitro, e.g., as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo by combinatorial infection, e.g., the loxP system described in Waterhouse et al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivo recombination approach exploits the two-chain nature of Fab fragments to overcome the limit on library size imposed by E. coli transformation efficiency. Naive VH and VL repertoires are cloned separately, one into a phagemid and the other into a phage vector. The two libraries are then combined by phage infection of phagemid-containing bacteria so that each cell contains a different combination and the library size is limited only by the number of cells present (about 1012 clones). Both vectors contain in vivo recombination signals so that the VH and VL genes are recombined onto a single replicon and are co-packaged into phage virions. These huge libraries provide large numbers of diverse antibodies of good affinity (Kd <com:Math><semantics>−1<annotation encoding="application / x-tex">-1< / annotation>< / semantics>< / com:Math> of about <com:Math><semantics>10−8<annotation encoding="application / x-tex">10-8< / annotation>< / semantics>< / com:Math> M).

[0120] Alternatively, the repertoires may be cloned sequentially into the same vector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g. as described in Clackson et al., Nature, 352: 624-628 (1991). PCR assembly can also be used to join VH and VL DNAs with DNA encoding a flexible peptide spacer to form single chain Fv (scFv) repertoires. In yet another technique, "in cell PCR assembly" is used to combine VH and VL genes within lymphocytes by PCR and then clone repertoires of linked genes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837 (1992).

[0121] Screening of the libraries can be accomplished by any art-known technique. For example, SSEA-3 / SSEA-4 / GLOBO H targets can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other art- known method for panning phage display libraries.

[0122] The phage library samples are contacted with immobilized SSEA-3 / SSEA-4 / GLOBO H under conditions suitable for binding of at least a portion of the phage particles with the adsorbent. Normally, the conditions, including pH, ionic strength, temperature and the like are selected to mimic physiological conditions. The phages bound to the solid phase are washed and then eluted by acid, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or by alkali, e.g. as described in Marks et al., J. Mol. Biol., 222: 581-597 (1991), or by SSEA-3 / SSEA-4 / GLOBO H antigen competition, e.g. in a procedure similar to the antigen competition method of Clackson et al., Nature, 352: 624-628 (1991). Phages can be enriched 20-1,000-fold in a single round of selection. Moreover, the enriched phages can be grown in bacterial culture and subjected to further rounds of selection.

[0123] The efficiency of selection depends on many factors, including the kinetics of dissociation during washing, and whether multiple antibody fragments on a single phage can simultaneously engage with antigen. Antibodies with fast dissociation kinetics (and weak binding affinities) can be retained by use of short washes, multivalent phage display and high coating density of antigen in solid phase. The high density not only stabilizes the phage through multivalent interactions, but favors rebinding of phage that has dissociated. The selection of antibodies with slow dissociation kinetics (and good binding affinities) can be promoted by use of long washes and monovalent phage display as described in Bass et al., Proteins, 8: 309-314 (1990) and in WO 92 / 09690, and a low coating density of antigen as described in Marks et al., Biotechnol., 10: 779-783 (1992).

[0124] It is possible to select between phage antibodies of different affinities, even with affinities that differ slightly, for SSEA-3 / SSEA-4 / GLOBO H. However, random mutation of a selected antibody (e.g. as performed in some of the affinity maturation techniques described above) is likely to give rise to many mutants, most binding to antigen, and a few with higher affinity. With limiting SSEA-3 / SSEA-4 / GLOBO H, rare high affinity phage could be competed out. To retain all the higher affinity mutants, phages can be incubated with excess biotinylated SSEA-3 / SSEA-4 / GLOBO H, but with the biotinylated SSEA-3 / SSEA-4 / GLOBO H at a concentration of lower molarity than the target molar affinity constant for SSEA- 3 / SSEA-4 / GLOBO H. The high affinity-binding phages can then be captured by streptavidin- coated paramagnetic beads. Such "equilibrium capture" allows the antibodies to be selected according to their affinities of binding, with sensitivity that permits isolation of mutant clones with as little as two-fold higher affinity from a great excess of phages with lower affinity. Conditions used in washing phages bound to a solid phase can also be manipulated to discriminate on the basis of dissociation kinetics.

[0125] Anti-SSEA-3 / SSEA-4 / GLOBO H clones may be activity selected. In one embodiment, the invention provides anti-SSEA-3 / SSEA-4 / GLOBO H antibodies that block the binding between a SSEA-3 / SSEA-4 / GLOBO H ligand and SSEA-3 / SSEA-4 / GLOBO H, but do not block the binding between a SSEA-3 / SSEA-4 / GLOBO H ligand and a second protein. Fy clones corresponding to such anti-SSEA-3 / SSEA-4 / GLOBO H antibodies can be selected by (1) isolating anti-SSEA-3 / SSEA-4 / GLOBO H clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) selecting SSEA-3 / SSEA- 4 / GLOBO H and a second protein against which blocking and non-blocking activity, respectively, is desired; (3) adsorbing the anti-SSEA-3 / SSEA-4 / GLOBO H phage clones to immobilized SSEA-3 / SSEA-4 / GLOBO H; (4) using an excess of the second protein to elute any undesired clones that recognize SSEA-3 / SSEA-4 / GLOBO H-binding determinants which overlap or are shared with the binding determinants of the second protein; and (5) eluting the clones which remain adsorbed following step (4). Optionally, clones with the desired blocking / non-blocking properties can be further enriched by repeating the selection procedures described herein one or more times.

[0126] DNA encoding the Fv clones of the invention is readily isolated and sequenced using conventional procedures (e.g. by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from hybridoma or phage DNA template). Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the desired monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of antibody-encoding DNA include Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs, 130: 151 (1992).

[0127] DNA encoding the Fv clones of the invention can be combined with known DNA sequences encoding heavy chain and / or light chain constant regions (e.g. the appropriate DNA sequences can be obtained from Kabat et al., supra) to form clones encoding full or partial length heavy and / or light chains. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species. A Fv clone derived from the variable domain DNA of one animal (such as human) species and then fused to constant region DNA of another animal species to form coding sequence(s) for "hybrid", full length heavy chain and / or light chain is included in the definition of "chimeric" and "hybrid" antibody as used herein. In one embodiment, a Fv clone derived from human variable DNA is fused to human constant region DNA to form coding sequence(s) for all human, full or partial length heavy and / or light chains.

[0128] The antibodies produced by naive libraries (either natural or synthetic) can be of moderate affinity (Kd -1 of about 106 to 107 M-1), but affinity maturation can also be mimicked in vitro by constructing and reselecting from secondary libraries as described in Winter et al. (1994), supra. For example, mutation can be introduced at random in vitro by using error-prone polymerase (reported in Leung et al., Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol. Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl. Acad. Sci. USA, 89: 3576-3580 (1992). Additionally, affinity maturation can be performed by randomly mutating one or more CDRs, e.g. using PCR with primers carrying random sequence spanning the CDR of interest, in selected individual Fv clones and screening for higher affinity clones. WO 9607754 (published 14 Mar. 1996) described a method for inducing mutagenesis in a complementarity determining region of an immunoglobulin light chain to create a library of light chain genes. Another effective approach is to recombine the VH or VL domains selected by phage display with repertoires of naturally occurring V domain variants obtained from unimmunized donors and screen for higher affinity in several rounds of chain reshuffling as described in Marks et al., Biotechnol., 10: 779-783 (1992). This technique allows the production of antibodies and antibody fragments with affinities in the 10–9 M range.

[0129] Other Methods of Generating Anti-SSEA-3 / SSEA-4 / GLOBO H Antibodies

[0130] Other methods of generating and assessing the affinity of antibodies are well known in the art and are described, e.g., in Kohler et al., Nature 256: 495 (1975); U.S. Pat. No. 4,816,567; Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986; Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987; Munson et al., Anal. Biochem., 107:220 (1980); Engels et al., Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989); Abrahmsen et al., EMBO J., 4: 3901 (1985); Methods in Enzymology, vol. 44 (1976); Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984).

[0131] General Methods

[0132] In general, the invention provides affinity-matured SSEA-3 / SSEA-4 / GLOBO H antibodies. These antibodies have increased affinity and specificity for SSEA-3 / SSEA- 4 / GLOBO H. This increase in affinity and sensitivity permits the molecules of the invention to be used for applications and methods that are benefited by (a) the increased sensitivity of the molecules of the invention and / or (b) the tight binding of SSEA-3 / SSEA-4 / GLOBO H by the molecules of the invention.

[0133] In one embodiment, SSEA-3 / SSEA-4 / GLOBO H antibodies that are useful for treatment of SSEA-3 / SSEA-4 / GLOBO H-mediated disorders in which a partial or total blockade of one or more SSEA-3 / SSEA-4 / GLOBO H activities is desired. In one embodiment, the anti SSEA-3 / SSEA-4 / GLOBO H antibodies of the invention are used to treat cancer.

[0134] The anti- SSEA-3 / SSEA-4 / GLOBO H antibodies of the invention permit the sensitive and specific detection of the epitopes in straightforward and routine biomolecular assays such as immunoprecipitations, ELISAs, or immunomicroscopy without the need for mass spectrometry or genetic manipulation. In turn, this provides a significant advantage in both observing and elucidating the normal functioning of these pathways and in detecting when the pathways are functioning aberrantly.

[0135] The SSEA-3 / SSEA-4 / GLOBO H antibodies of the invention can also be used to determine the role in the development and pathogenesis of disease. For example, as described above, the SSEA-3 / SSEA-4 / GLOBO H antibodies of the invention can be used to determine whether the TACAs are normally temporally expressed which can be correlated with one or more disease states.

[0136] The SSEA-3 / SSEA-4 / GLOBO H antibodies of the invention can further be used to treat diseases in which one or more SSEA-3 / SSEA-4 / GLOBO Hs are aberrantly regulated or aberrantly functioning without interfering with the normal activity of SSEA-3 / SSEA- 4 / GLOBO Hs for which the anti-SSEA-3 / SSEA-4 / GLOBO H antibodies of the invention are not specific.

[0137] In another aspect, the anti- SSEA-3 / SSEA-4 / GLOBO H antibodies of the invention find utility as reagents for detection of cancer states in various cell types and tissues.

[0138] In yet another aspect, the present anti-SSEA-3 / SSEA-4 / GLOBO H antibodies are useful for the development of SSEA-3 / SSEA-4 / GLOBO H antagonists with blocking activity patterns similar to those of the subject antibodies of the invention. For example, anti-SSEA- 3 / SSEA-4 / GLOBO H antibodies of the invention can be used to determine and identify other antibodies that have the same SSEA-3 / SSEA-4 / GLOBO H binding characteristics and / or capabilities of blocking SSEA-3 / SSEA-4 / GLOBO H- pathways.

[0139] As a further example, anti-SSEA-3 / SSEA-4 / GLOBO H antibodies of the invention can be used to identify other anti-SSEA-3 / SSEA-4 / GLOBO H antibodies that bind substantially the same antigenic determinant(s) of SSEA-3 / SSEA-4 / GLOBO H as the antibodies exemplified herein, including linear and conformational epitopes.

[0140] The anti-SSEA-3 / SSEA-4 / GLOBO H antibodies of the invention can be used in assays based on the physiological pathways in which SSEA-3 / SSEA-4 / GLOBO H is involved to screen for small molecule antagonists of SSEA-3 / SSEA-4 / GLOBO H which will exhibit similar pharmacological effects in blocking the binding of one or more binding partners to SSEA-3 / SSEA-4 / GLOBO H as the antibody does.

[0141] Generation of antibodies can be achieved using routine skills in the art, including those described herein, such as the hybridoma technique and screening of phage displayed libraries of binder molecules. These methods are well-established in the art.

[0142] Briefly, the anti-SSEA-3 / SSEA-4 / GLOBO H antibodies of the invention can be made by using combinatorial libraries to screen for synthetic antibody clones with the desired activity or activities. In principle, synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption / elution. Any of the anti-SSEA-3 / SSEA-4 / GLOBO H antibodies of the invention can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length anti-SSEA-3 / SSEA-4 / GLOBO H antibody clone using the Fv sequences from the phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.

[0143] In one embodiment, anti-SSEA-3 / SSEA-4 / GLOBO H antibodies of the invention are monoclonal. Also encompassed within the scope of the invention are antibody fragments such as Fab, Fab, Fab, SH and F(ab, 2) fragments, and variations thereof, of the anti-SSEA- 3 / SSEA-4 / GLOBO H antibodies provided herein. These antibody fragments can be created by traditional means, such as enzymatic digestion, or may be generated by recombinant techniques. Such antibody fragments may be chimeric, human or humanized. These fragments are useful for the experimental, diagnostic, and therapeutic purposes set forth herein.

[0144] Monoclonal antibodies can be obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0145] The anti-SSEA-3 / SSEA-4 / GLOBO H monoclonal antibodies of the invention can be made using a variety of methods known in the art, including the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or alternatively they may be made by recombinant DNA methods (e.g., U.S. Pat. No. 4,816,567).

[0146] Vectors, Host Cells and Recombinant Methods

[0147] For recombinant production of an antibody of the invention, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. Host cells include, but are not limited to, cells of either prokaryotic or eukaryotic (generally mammalian) origin. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species.

[0148] Generating Antibodies Using Prokaryotic Host Cells

[0149] Vector Construction

[0150] Polynucleotide sequences encoding polypeptide components of the antibody of the invention can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present invention. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides. The vector components generally include, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert and a transcription termination sequence.

[0151] In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of particular antibodies are described in detail in Carter et al., U.S. Pat. No. 5,648,237.

[0152] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as λGEM<com:Sup>TM< / com:Sup>-11 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.

[0153] The expression vector of the invention may comprise two or more promoter-cistron pairs, encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence located upstream (5 ´) to a cistron that modulates its expression. Prokaryotic promoters typically fall into two classes, inducible and constitutive. Inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in the culture condition, e.g. the presence or absence of a nutrient or a change in temperature.

[0154] A large number of promoters recognized by a variety of potential host cells are well known. The selected promoter can be operably linked to cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the invention. Both the native promoter sequence and many heterologous promoters may be used to direct amplification and / or expression of the target genes. In some embodiments, heterologous promoters are utilized, as they generally permit greater transcription and higher yields of expressed target gene as compared to the native target polypeptide promoter.

[0155] Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the <com:Math><semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics>< / com:Math>- galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac or the trc promoter. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are suitable as well. Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to cistrons encoding the target light and heavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors to supply any required restriction sites.

[0156] In one aspect of the invention, each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane. In general, the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected for the purpose of this invention should be one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequences native to the heterologous polypeptides, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, Lamb, PhoE, Pelb, OmpA and MBP. In one embodiment of the invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

[0157] In another aspect, the production of the immunoglobulins according to the invention can occur in the cytoplasm of the host cell, and therefore does not require the pre...

Claims

1. A method of detecting a ESA hi PROCR hi SSEA-3 + or CD44 + CD24 - / lo SSEA-3 + breast cancer stem cell in a patient, the method comprising: contacting a sample that has been isolated from the patient with antibodies that bind to cancer stem cell markers SSEA-3, ESA, and PROCR, or antibodies that bind to cancer stem cell markers SSEA-3, CD24, and CD44 on the breast cancer stem cell; and detecting a specific binding of the antibodies that bind to the cancer stem cell markers on the breast cancer stem cell, thereby detecting the ESA hi PROCR hi SSEA-3 + or CD44 + CD24 - / lo SSEA-3 + breast cancer stem cell.

2. A method of detecting the presence of ESA hi PROCR hi SSEA-3 + or CD44 + CD24 - / lo SSEA-3 + breast cancer stem cells in a patient, the method comprising: analyzing an expression of cancer stem cell markers on cells in a sample isolated from the patient, wherein the cancer stem cell markers are i) SSEA-3, CD24, and CD44; or ii) SSEA-3, PROCR, and ESA, wherein the presence of cells expressing ESA hi PROCR hi SSEA-3 + or CD44 + CD24 - / lo SSEA-3 + indicates the presence of the ESA hi PROCR hi SSEA-3 + or CD44 + CD24 - / lo SSEA-3 + breast cancer stem cells.

3. The method according to claim 1 or 2, wherein the sample is stained with a binding agent that specifically binds to the cancer stem cell marker.

4. The method according to claim 1 or 2, further comprising testing the co-expression of a cancer stem cell marker selected from the group consisting of SSEA-4 and GloboH.

5. The method according to claim 1 or 2, further comprising analysis of a staining pattern, wherein the analysis of the staining pattern provides the relative distribution of breast cancer stem cells.

6. A method for identifying whether a breast cancer is present in a patient, comprising: determining whether a sample obtained from the patient comprises stem cells that overexpress β \beta -1,3-galactosyltransferase 5 ( β \beta 3GalT5); and assaying for the presence of cancer stem cell markers in the sample obtained from the patient, wherein the cancer stem cell markers are i) SSEA-3, CD24, and CD44; or ii) SSEA-3, PROCR, and ESA, wherein the presence of stem cells expressing ESA hi PROCR hi SSEA-3 + or CD44 + CD24 - / lo SSEA-3 + , and overexpressing β-1,3-galactosyltransferase 5 (β3GalT5) indicates that the breast cancer is present.

7. A method for identifying a population of breast cancer stem cells, the method comprising: a) providing a starting population of cancer cells, b) determining the expression level of cancer stem cell markers i) SSEA-3, CD24, and CD44; or ii) SSEA-3, PROCR, and ESA, in the starting population of cancer cells, c) selecting a population of cells expressing ESA hi PROCR hi SSEA-3 + or CD44 + CD24 - / lo SSEA-3 + , wherein said selected population of cells are breast cancer stem cells, and d) isolating and / or enriching for said population of cells selected in step c).

8. The method of any one of claims 1-6, wherein the sample is a bodily fluid sample.

9. The method of claim 8, wherein the bodily fluid sample is a blood sample.

10. The method of claim 1 or 2, wherein the detecting is done by using flow cytometry.

11. The method of claim 2, wherein the analyzing is done by using flow cytometry.

12. The method of claim 6 or 7, wherein the determining is done by using flow cytometry.