BR112015016381B1 - VACCINE, AND, PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

VACCINE, AND, PHARMACEUTICAL COMPOSITION. The present invention provides vaccines that comprise a carbohydrate antigen conjugated with a diphtheria toxin (DT) as a carrier protein, in which the ratio of the number of molecules of the carbohydrate antigen to that of the carrier protein is greater than 5:1. Also disclosed herein is a novel saponin adjuvant and methods for inhibiting cancer cells by administering an effective amount of the vaccine disclosed herein.

Description

CROSS REFERENCE TO RELATED ORDERS

[0001] This application claims the benefit of U.S. patent application number 61/748,880, filed January 4, 2013, the full disclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Cancer vaccines are designed to treat cancers by increasing the body's natural ability to protect itself through the immune system. They have always represented a very attractive therapeutic approach, especially given the several disadvantages of conventional surgery, radiation and chemotherapy in cancer management. However, due to the low immunogenicity of the carbohydrate cancer antigen and the fact that many synthetic vaccines induce mainly IgM and to some extent an IgG antibody, the effectiveness of such a cancer vaccine is still low. Various approaches have been explored, such as the use of an adjuvant, to aid immune recognition and activation.

[0003] There is an unmet need to develop a cancer vaccine and an effective adjuvant with an improved immune response, especially an IgG response. The present invention provides vaccines against carbohydrate antigens and an adjuvant to meet these and other needs.

BRIEF SUMMARY OF THE INVENTION

[0004] In one embodiment, the present invention discloses a vaccine comprising a carbohydrate antigen, or its immunogenic fragment; and a toxoid protein, wherein the ratio of carbohydrate antigen to toxoid protein ranges from 5:1 to 39:1, wherein the ratio represents the number of carbohydrate antigen molecules relative to the toxoid protein. IgG production from the vaccine with a carbohydrate antigen to toxoid protein ratio ranging from 5:1 up to 39:1 was found to be higher when compared to that of a vaccine with an equal carbohydrate antigen to toxoid protein ratio. or below 4:1.

[0005] An embodiment of this invention offers the isolated compounds of formula (I) or pharmaceutically acceptable salts thereof; where R1 is selected from β-D-Apiose or β-D-Xylose; R2 and R3 are selected from H, alkyl or

[0006] Another embodiment of this invention offers pharmaceutical compositions comprising a compound of formula (I) or pharmaceutically acceptable salts thereof, wherein R1 is selected from β-D-apiose or β-D-Xylose R2 and R3 are selected from H, alkyl or and a pharmaceutically acceptable carrier.

[0007] A third embodiment of this invention offers a new saponin adjuvant, OBI-821, which comprises compound 1857 V1A, compound 1857 V1B, compound 1857 V2A and compound 1857 V2B.

[0008] A fourth embodiment of the present invention provides vaccines that comprise a carbohydrate antigen, or its immunogenic fragment; and OBI-821 saponin adjuvant. In one embodiment, the vaccine also comprises a carrier protein. The IgG production, antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) activities of the vaccine with the OBI-821 saponin adjuvant were found to be higher compared with those of a vaccine without the OBI-821 saponin adjuvant.

[0009] The present invention is also directed to methods for (i) inhibiting cancer cells, comprising administering an effective amount of the vaccine described herein, wherein cancer cells are inhibited; and, (ii) inducing an immune response, comprising administering an effective amount of the vaccine described herein to a subject in need thereof.

[0010] The present invention also describes a pharmaceutical composition comprising the vaccine described herein and a pharmaceutically acceptable excipient or carrier.

[0011] It should be understood that statements containing these terms do not limit the subject matter described herein or the meaning and scope of the patent claims below. The embodiments of the invention covered by this patent are defined by the appended claims, and not by this summary. This summary is a high-level overview of various aspects of the invention and introduces some of the concepts that are further described in the Detailed Description section below. This summary is not intended to identify the key or essential characteristics of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of application of the claimed subject matter. The subject matter is to be understood by reference to the appropriate portions of the entire specification, any or all drawings and each claim. The invention will become more apparent when reading the accompanying figures and detailed description that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Illustrative embodiments of the present invention are described in detail below with reference to the following figures:

[0013] Fig. 1A is a bar graph illustrating the anti-globo H IgG titer on day 24 for the following compositions: Globo saponin H/KLH/OBI-821, Globo saponin H/DT/OBI-821, Globo H/DT/C34 and Globo H/KLH/C34.

[0014] Fig. 1B is a line graph illustrating the anti-Globo H IgG titer over a 24-day period of the compositions in Fig. 1A.

[0015] Fig. 2 is a set of bar graphs showing in vivo ADCC and CDC activities of the G2 vaccine (Globo H/DT (8:1)); G3 vaccine (Globo H/DT (8:1)/OBI-821); and G4 vaccine (Globo H/DT (24:1)/OBI-821) in mice over a 24-day period. Fig. 2A illustrates the ADCD raw data, Fig. 2B illustrates the ADCD normalized data, Fig. 2C illustrates the CDC raw data, and Fig. 2D illustrates the CDC normalized data.

[0016] Fig. 3A and Fig. 3B are line graphs illustrating the total IgM and IgG titers of the following compositions over a 24-day period: G1 (Globo H/KLH/OBI-821), G2 (Globo H/DT (3:1)/OBI-821), G3 and G4 (Globo H/DT (8:1)/OBI-821), G5 (Globo H/DT (8:1)/C34) , G6 (Globo H/KLH/C34), G7 (Globo H/DT(16:1)/ OBI-821) and G8 (PBS).

[0017] Fig. 4 is a set of bar graphs showing the IgM and IgG response on Day 10, Day 17 and Day 24 of the compositions enumerated in Fig. 3: Panel (A) - (C) illustrates the IgM response of the compositions listed in Figure 3. on day 10, 17 and 24, respectively. Panel (D) - (F) illustrates the IgG response of the compositions listed in Figure 3 on day 10, 17 and 24, respectively.

[0018] Figures 5A-5C are mass spectrum images of OBI-821 (comprising compounds 1989 and 1857).

[0019] Fig. 6 is an LC-UV chromatographic image of OBI-821.

[0020] Fig. 7 is a set of LC-MS chromatographic images of OBI-821.

DETAILED DESCRIPTION OF THE INVENTION

[0021] In order to enable a clear and immediate understanding of the present invention, some terms are defined here. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as are normally understood by one skilled in the field to which this invention belongs.

[0022] An "effective amount", as used herein, refers to a dose of the pharmaceutical composition or vaccine that is sufficient to reduce the symptoms and signs of cancer, which include, but are not limited to, weight loss , pain and tumor mass, which is detectable clinically as a palpable mass or radiologically through various imaging methods.

[0023] The term “subject” may refer to a vertebrate with cancer or to a vertebrate for which cancer treatment is considered necessary. Subjects include warm-blooded animals, such as mammals, such as a primate, and, more preferably, a human. Non-human primates are subjects too. The term subject includes domestic animals such as dogs, cats, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, gerbil, guinea pig). da-India, etc.). Thus, veterinary uses and medical formulations are contemplated here.

[0024] As used herein, the term "alkyl" refers to a monovalent linear or branched hydrocarbon containing, unless otherwise indicated, 1-20 carbon atoms, for example, C1-C8 or C1- C4, which can be substituted or unsubstituted (other chain lengths, e.g. 21-30, can be encompassed by the invention). Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl and t-butyl.

[0025] The term "substantially pure" means substantially free of compounds normally associated with saponin in its natural state and exhibiting a constant and reproducible chromatographic response, elution profiles and biological activity. The term "substantially pure" is not intended to exclude artificial or synthetic mixtures of saponin with other compounds. All numbers here can be understood as modified by "about".

Vaccines with a higher carbohydrate ratio

[0026] Tumor-associated carbohydrate antigens generally exhibit low immunogenicity. A carbohydrate antigen conjugated with a carrier protein was adopted to increase the immunogenicity of said carbohydrate antigen. For example, about 700 Globo H molecules are conjugated to a non-toxic keyhole limpet hemocyanin (KLH) protein, an average of about 2 to 4 Globo H molecules are conjugated to diphtheria toxin (DT), about 8 Globo H molecules H are conjugated with bovine serum albumin (BSA) and about 6 Globo H molecules are conjugated with tetanus toxoid (Table 1 of US Patent No. 8,268,969).

[0027] The present invention offers a vaccine comprising a carbohydrate antigen, or immunogenic fragment thereof; and a toxoid protein, in which the ratio of carbohydrate antigen to toxoid protein varies from 5:1 to 24:1, in which the ratio reflects the number of molecules of the carbohydrate antigen or its immunogenic fragment in relation to the molecules of the toxoid protein. Such a vaccine exhibits improved immunogenicity compared to a vaccine with a ratio of carbohydrate antigen molecules to toxoid protein molecules equal to or less than 4:1. Other ranges are also encompassed by the invention, including ratios of the number of antigen molecules. carbohydrate, or its immunogenic fragment, relative to toxoid protein molecules of 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1 , 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25 :1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1 , 38:1 or 39:1.

[0028] In one embodiment, the toxoid protein is tetanus toxoid (TT) and the ratio of carbohydrate antigen to TT in the carbohydrate-TT vaccine ranges from 7:1 to 12:1.

[0029] The present invention offers a vaccine that comprises a carbohydrate antigen, or immunogenic fragment thereof; and a diphtheria toxin (DT), in which the ratio of carbohydrate antigen to DT varies from 5:1 to 39:1, where the ratio reflects the number of molecules of the carbohydrate antigen or its immunogenic fragment in relation to the DT molecules . In another embodiment, the ratio of carbohydrate antigen to DT in the carbohydrate-DT vaccine ranges from 8:1 to 24:1.

[0030] Examples of carbohydrate antigens include, but are not limited to Globo H, stage-specific embryonic antigen 3 (SSEA 3) (also called Gb5), stage-specific embryonic antigen 4 (SSEA-4), Gb-4 , Gb -3, Lewis antigens such as sLex, Lex, sLea, Lea, Ley, polysaccharide antigens such as polysialic acid (PSA), sTn(c), Tn(c), Thomsen-Friedenreich antigens (TF ( c)), gangliosides, such as GD1, GD2, GD3, fucosyl, GM1, GM2, GM3, GD1α and GM2. Other carbohydrate antigens include, but are not limited to: α-galactose, α-Man-6-phosphate, α-L-rhamnose, α-GalNAc (Tn), α-NeuAc-OCH2C6H4- pNHCOOCH2, Fucα1-2Galβ1-4GalNAcβ ( H types3), NeuAcα2-8NeuAcα, (NeuAcα2- 8) 2 polysialic acid, NeuAca2-6Galb, NeuAcb2-6Gala (STN), Gala1-3Galb1- 4GlaNAcb (NeuAca2-8) 3, GalNAcαa-3 (Fucα1-2) Galβ ( Blood Group A), Galα1-3 (Fucα1-2) Galβ (Blood Group B), 6Gal-HSO3-SiaLex, 6GluNAc-HSO3-SiaLex and α2-6 biantennary sialylated N-glycans. In one embodiment, the carbohydrate antigen is Globe H. "Globe H" is a hexasaccharide (Fucαl^2Galβ1^3GalNAcβ1^3Galα1^4Galβ1^4Glcβ1), which was initially isolated from the human breast cancer cell line MCF- 7 (Menard S, Tagliabue E, Canevari S, Fossati G, Colnaghi MI. (1983). Generation of monoclonal antibodies that react with normal and human breast cancer cells. Cancer Res, 43, 1295-300; and Bremer EG , Levery SB, Sonnino S, Ghidoni R, Canevari S, Kannagi R, Hakomori S. (1984) Characterization of a glycosphingolipid antigen defined by the MBr1 monoclonal antibody expressed on normal and neoplastic epithelial cells of the human mammary gland. J Biol Chem, 259, 14773-7). Globo H is expressed in a variety of epithelial cell tumors, such as colon, ovarian, gastric, pancreatic, endometrial, lung, prostate and breast cancer (Menard S et al. supra; Bremer EG et al ., supra; Canevari S, Fossati G, Balsari A, Sonnino S, Colnaghi MI. (1983). Globo H is commercially available (e.g., Carbosynth, UK) and can be synthesized by attaching glycoside to ceramide using methods known in the area.

[0031] The vaccine with a ratio of carbon antigen to toxoid protein greater than or equal to 5: 1 is manufactured in a basic condition, that is, at a pH greater than or equal to 8, greater than or equal to 9, greater than or equal to 10 , greater than or equal to 11 or greater than or equal to 12. The ratio of carbohydrate antigen to toxoid protein can be determined by methods known in the art, for example, MALDI-TOF Mass Spectrometry. US Patent No. 8,268,969; see also, Morelle W, Faid V, Chirat F, Michalski JC. Methods Mol Biol. 2009;534:5-21. doi: 10.1007/978-1-59745-022-5_1.Analysis of N- and O-linked glycans from glycoproteins using MALDI-TOF mass spectrometry.

[0032] The vaccine may further comprise an adjuvant, where the adjuvant is a saponin, such as OBI-821, which is described here, or synthetic analogues of α-galactosyl-ceramide (GalCer or C1).

[0033] The terms "α-galactosyl-ceramide" and "α-GalCer" refer to a glycolipid that stimulates natural killer T cells to produce both TH1 and TH2 cytokines, as described in Patent No. 8,268,969, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the α-GalCer adjuvant has the following structure: where R is (CH2)24CH3, (CH2)7PhF, (CH2)10PhOPhF or (CH2)10PhF.

[0034] In one embodiment, R is (CH2)10PhOPhF, known as adjuvant C34 with the following structure:

New Saponrna Adjuvant

[0035] The present invention provides OBI-821 saponins that can be substantially pure. The invention encompasses both OBI-821 saponins which are substantially pure and biologically active fragments. The invention may also encompass impure forms of OBI-821 saponins. Purified OBI-821 saponins exhibit an enhanced adjuvant effect when administered with the vaccine described herein or mixed with other substantially pure or saponin-free saponin adjuvants. OBI-821 saponins are naturally occurring glycosides, extracted with high purity from the bark of the Quillaja saponaria tree, (quilaia), by high pressure liquid chromatography (HPLC), low pressure liquid chromatography with silica and hydrophilic interactive chromatography ( HILIC) as described, for example, in U.S. Patent No. 5,057,540 and U.S. Patent No. 6,524,584, the contents of which are incorporated by reference in their entirety. High pressure liquid chromatography analysis shows that OBI-821 is a mixture of structurally related isomeric compounds. Different isomeric compounds purified from the OBI-821 saponins were identified and disclosed here.

[0036] OBI-821 saponin comprises at least one isolated compound of formula I as follows: where R1 is β-D-Apiose or β-D-Xylose; and R2 and R3 are independently H, alkyl,

[0037] OBI-821 saponin may also comprise an isolated compound of formula I in which (i) R1 is β-D-apiose, R2 is the fatty acyl portion of compound 1989 described above, and R3 is H (compound 1989 V1A); (ii) R1 is β-D-apiose, R2 is H, and R3 is the fatty acyl portion of compound 1989 described above (compound 1989 V1B); (iii) R1 is β-D-xylose, R2 represents the fatty acyl moiety of compound 1989 described above, and R3 is H (compound 1989 V2A); or (iv) R1 is β-D-xylose, R2 is H, and R3 is the fatty acyl moiety of compound 1989 described above (compound 1989 V2B). Collectively, compound 1989 V1A, compound 1989 V1B, compound 1989 V2A, and compound 1989 V2B are referred to as the "1989 compound mixture."

[0038] Table 1 summarizes the functional groups of compounds 1989 and the mole % of each compound 1989 in the mixture of compounds 1989. TABLE 1

[0039] OBI-821 saponin may comprise an isolated compound of formula I wherein: (i) R1 is β-D-apiose, R2 is the fatty acyl portion of compound 1957 described above, and R3 is H (compound 1857 V1A ); (ii) R1 is β-D-apiose, R2 is H, and R3 is the fatty acyl portion of compound 1857 described above (compound 1857 V1B); (iii) R1 is β-D-xylose, R2 represents the fatty acyl moiety of compound 1857 described above, and R3 is H (compound 1857 V2A); or (iv) R1 is β-D-xylose, R2 is H, and R3 is the fatty acyl moiety of compound 1857 described above (compound 1857 V2B). Collectively, compound 1857 V1A, compound 1857 V1B, compound 1857 V2A, and compound 1857 V2B are referred to as a "mixture of compounds 1857."

[0040] Table 2 summarizes the functional groups of compounds 1857 and the mol % of each compound 1857 in the mixture of compounds 1857. TABLE 2

[0041] OBI-821 saponin comprises one or more of the following compounds: (i) compound 1857 V1A; (ii) compound 1857 V1B; (iii) compound 1857 V2A; (iii) compound 1857 V2B; (iv) compound 1989 V1A; (v) compound 1989 V1B; (vi) compound 1989 V2A; or (vii) compound 1989 V2B. The percentages of the mixture of compounds 1857 and the mixture of compounds 1989 in the OBI-821 saponin can vary as follows: (i) about 1 mol % to about 15 mol % of OBI-821 comprising a mixture of compounds 1857; and (ii) about 85 mol% to about 99 mol% OBI-821 comprising a mixture of compounds 1989.

[0042] The entire mole percentage can vary in increments of 0.1% (e.g., about 87% to about 90%, about 90.5% to about 97%, about 3.5% to about from 11%, about 10% to about 14%).

[0043] The mixture of compounds 1989 may comprise about 6070 mol% of compound 1989 V1A; about 1-5 mol% compound 1989 V1B; about 30-40 mol% of compound 1989 V2A; and, about 0.13 mol% of compound 1989 V2B. The entire mole percentage can vary by an increment of 0.1 (e.g., 65%, 2.5%, 35.6%).

[0044] The mixture of compounds 1857 may comprise about 6070 mol% of compound 1857 V1A; about 1-5 mol% compound 1857 V1B; about 30-40 mol% of compound 1857 V2A; and, about 0.13 mol% of compound 1857 V2B. The entire mole percentage can vary by an increment of 0.1 (e.g., 67%, 1.5%, 33.9%).

[0045] In another embodiment, substantially pure OBI-821 is purified from a crude extract of Quillaja saponaria, wherein said OBI-821 is characterized by a predominant single peak comprising 90% or more of the total area of all peaks in a chromatogram, excluding the solvent peak, when analyzed on reversed-phase HPLC on a Symmetry C18 column, having a particle size of 5 μm, pore size 100 Å, 4.6 mm internal diameter x 25 cm long with an elution program comprising the mobile phase of A: B 95%: 5% to 75%: 25% in 11 minutes, the mobile phase being distilled water with 0.1% trifluoroacetic acid, and the mobile phase B being acetonitrile with 0.1% trifluoroacetic acid at a flow rate of 1 ml/min.

[0046] In one embodiment, the pharmaceutical composition comprises the compound of formula (I) where, R1 is β-D-apiose or β-D-xylose; and R2 and R3 are independently H, alkyl, or and a pharmaceutically acceptable carrier.

[0047] The vaccine may comprise a carbohydrate antigen or its immunogenic fragment and an OBI-821 saponin. In another embodiment, the vaccine comprises a carbohydrate antigen selected from Globo H, SSEA-3, SSEA-4, Gb-4 or a mixture thereof, a DT and an OBI-821 saponin. In yet another embodiment, the vaccine may comprise a carbohydrate antigen or immunogenic fragment thereof, a carrier protein and an OBI-821 saponin. Non-limiting examples of the carrier protein include toxoid proteins and non-toxoid proteins such as KLH.

toxoid protein

[0048] The toxoid protein conjugated to the carbohydrate antigen can be a diphtheria toxin (DT) or tetanus toxoid (TT).

[0049] Toxins can be inactivated, for example, by treatment with formaldehyde, glutaraldehyde, UDP-aldehyde, peroxide, oxygen or by mutation (for example, using recombinant methods). Relyveld et al., Methods in Enzymology, 93:24, 1983. Woodrow and Levine, eds., New Generation Vaccines, Marcel Dekker, Inc., New York, 1989. Genth et al., Inf. and Immun., 68 (3):1094-1101, 2000. Mutant diphtheria toxins with reduced toxicity can also be produced using recombinant methods. US Patent Numbers 5,085,862; 5,221,618; 5,244,657; 5,332,583; 5,358,868; and 5,433,945.

[0050] DT consists of materials that cross-react with diphtheria toxin (DT-CRM) or diphtheria toxoids. A DT-CRM refers to a diphtheria toxin that has been mutated, for example by mutation or chemical modification, so that it no longer has ADP-ribosyl in sufficient quantity. Non-limiting examples of DT-CRM include DT-CRM 30, DT-CRM 45, DT-CRM 176, DT-CRM 197 and DT-CRM 228. A diphtheria toxoid is a diphtheria toxin inactivated by formaldehyde. DT is commercially available from or can be prepared by methods known in the art, such as recombinant DNA technology as described in U.S. Patent No. 5,614,382, the contents of which are incorporated herein by reference in their entirety.

[0051] The carbohydrate antigen of the vaccine described herein can be covalently linked to a carrier protein through a p-nitrophenyl linker by a synthetic process described in US Patent No. 8,268,969, the contents of which are incorporated by reference in its entirety.

[0052] The vaccines of the present invention can induce one or more of the following activities: a higher IgG titer compared to the IgM titer, a greater complement-dependent cytotoxicity (CDC) activity, and/or a greater complement-mediated cytotoxicity activity. antibody-dependent cells (ADCC). In another embodiment, the vaccines induce one or more of the following cells: natural killer cells, CD4 + T lymphocytes or CD8 + T lymphocytes. Other immunological parameters can be measured, including, but not limited to, T helper cell activation.

[0053] The invention also provides a pharmaceutical composition comprising the vaccine described herein and a pharmaceutically acceptable vehicle, excipient or carrier. Acceptable carriers are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. Furthermore, the carrier may contain other excipients, such as wetting or emulsifying agents, pH buffering agents or adjuvants. Pharmaceutically acceptable carriers may contain a physiologically acceptable compound that acts to, for example, stabilize or increase or decrease the absorption or clearance rates of the pharmaceutical compositions of the invention. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose or dextrans, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, detergents, liposomal carriers, or other stabilizers and/or buffers. . Excipients may be non-ionic surfactants, polyvinylpyrrolidone, human serum albumin, aluminum hydroxide, agents with anesthetic action and various unmodified and derivatized cyclodextrins. More preferably, the nonionic surfactants may include polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80. The polyvinylpyrrolidone may preferably be Plasdone C15, a pharmaceutical grade of polyvinylpyrrolidone. The agent with anesthetic action is preferably benzyl alcohol. Other physiologically acceptable compounds include wetting agents, emulsifiers, dispersing agents or preservatives. See, for example, the twenty-first edition of Remington’s Pharmaceutical Science, Mack Publishing Company, Easton, Pa. (“Remington’s”). The pharmaceutical compositions of the present invention may also include auxiliary substances, such as pharmacological agents, cytokines or other biological response modifiers. The pharmaceutical composition comprising such an excipient or carrier is formulated by well-known conventional methods.

[0054] The vaccine can be formulated for the following route of administration: intramuscular, intradermal, oral, dermal, nasal, buccal, rectal, vaginal, inhalation or subcutaneous administration. Other modes of administration may be applicable as long as satisfactory immunogenicity can be induced.

[0055] The pharmaceutical compositions of the present invention can be prepared in injectable form, either as liquid solutions or suspensions, or as solid forms that are suitable for solution or suspension in liquid vehicles prior to injection. The pharmaceutical composition can also be prepared in solid, emulsified form, or the active ingredient can be encapsulated in liposomal vehicles or other particulate carriers used for sustained administration. For example, the pharmaceutical composition may be in the form of an oil emulsion, water-in-oil emulsion, water-in-oil-in-water emulsion, site-specific emulsion, long-stay emulsions, sticky emulsion, microemulsion, nanoemulsion, liposomes, microparticles , microspheres, nanospheres, nanoparticles and various natural or synthetic polymers, such as non-resorbable impermeable polymers such as ethylene vinyl acetate copolymers and Hytrel® copolymers, swellable polymers such as hydrogels, or reabsorbable polymers such as collagen and certain polyacids or polyesters, such such as those used to make resorbable sutures, which allow the sustained release of the vaccine.

[0056] Pharmaceutically acceptable salts of the compounds of the invention and physiologically functional derivatives thereof include salts derived from an appropriate base, such as an alkali metal (e.g., sodium, potassium), an alkaline earth metal (e.g., calcium, magnesium), ammonium and NX4+ (where X is C1-C4 alkyl). Pharmaceutically acceptable salts of an amino group include salts of organic carboxylic acids, such as the tartaric, aliphatic, cycloaliphatic, aromatic, heterocyclic, carboxylic and sulfonic classes of organic acids, such as, for example, formic, glucuronic, malic, maleic acid. , fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic, hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, stearic, alginic, hydroxybutyric, cyclohexyl-aminosulfonic, galactaric and galacturonic and the like, lactobionic, fumaric and succinic acids; organic sulfonic acids, such as methanesulfonic, ethanesulfonic, isothionic, benzenesulfonic and p-toluenesulfonic acids; and inorganic acids such as hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, sulfamic, phosphoric acid and the like. Pharmaceutically acceptable salts of a compound having a hydroxy group consist of the anion of said compound in combination with a suitable cation such as Na+, NH4+ or NX4+ (wherein X is, for example, a C1 - C4 alkyl group), Ca++, Li+ , Mg++, or K+ and zinc or organic salts made from primary, secondary and tertiary amines, cyclic amines, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), procaine and the like. All such salts can be prepared by conventional means from the corresponding compound by reacting, for example, the appropriate acid or base with the compound in free form.

Methods for inducing immune response/inhibiting cancer cells

[0057] Another aspect of the present invention is directed to methods for inducing an immune response comprising administering an effective amount of the vaccine described herein to a subject in need thereof. The immune response includes, but is not limited to, the NK cell response, ADCC and CDC activity, and IgM and IgG production.

[0058] In yet another aspect, the present invention provides methods for inhibiting cancer cells, including administering an effective amount of the vaccine described herein to a subject in need thereof. In one embodiment, the cancer is selected from breast cancer, lung cancer, esophageal cancer, rectal cancer, biliary cancer, liver cancer, mouth cancer, gastric cancer, colon cancer, nasopharyngeal cancer, kidney, brain tumor, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, pancreatic cancer, testicular cancer, bladder cancer, head and neck cancer, oral cancer, neuroendocrine cancer, adrenal cancer, breast cancer thyroid, bone cancer, skin cancer (e.g. basal cell carcinoma, squamous cell carcinoma or melanoma). In another embodiment, the cancer is a Globo H-expressing cancer. Non-limiting examples of Globo H-expressing cancers include breast cancer, gastric cancer, lung cancer, colon cancer, pancreatic cancer, prostate cancer, ovarian and endometrial cancer. The antibody generated by the vaccine, such as the anti-Globo H antibody, inherently inhibits Globo H-expressing cancer.

[0059] In certain embodiments, the effective amount of a vaccine serves to induce desired immunological effects, such as stimulating the production of IgG against a specific carbohydrate antigen (e.g. Globo H) in a subject. The effective amount or dose of a vaccine or pharmaceutical composition may vary according to the amount of carbohydrate antigen, the type of adjuvant used, the mode of administration and the age, size and condition of the subject to be treated. The precise amount of vaccine or pharmaceutical composition required to induce immunogenicity will be determined by the physician.

[0060] The vaccine can be administered as an initial dose with or without one or more booster doses at specific time intervals, to achieve a long-term immunological protection effect between several months and several years. The frequency of administration may vary depending on any of several factors, for example, the severity of symptoms, degree of immunoprotection desired, whether the pharmaceutical composition is used for prophylactic or curative purposes, etc. For example, in one embodiment, the pharmaceutical composition according to the invention is administered once a month, twice a month, three times a month, every other week, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, every other day, daily, twice a day, or three times a day. The vaccine may also be administered with other conventional therapy, such as chemotherapy, targeted therapy or antibody therapy directed against tumor-associated carbohydrate antigen for treating cancer, either simultaneously or sequentially.

[0061] The present invention is further illustrated by the following examples, which are provided for demonstration purposes, not limitation. Those skilled in the art should, in light of the present description, take into account that many changes can be made to the specific embodiments that are disclosed and still obtain the same or similar result without departing from the spirit and scope of the invention.

Example 1: Preparation of a vaccine with a higher carbohydrate to toxin protein ratio and extraction of OBI-821

[0062] Globe H was conjugated with KLH or DT, according to methods known in the art, for example, as described in American Patent No. 6,544,952 or 8,268,969, the contents of which are incorporated herein by reference in their entirety. The resulting vaccine comprised Globo H:DT (ratio of Globo H to DT molecules=2-4:1)

General Glycoconjugate Generation Procedure

[0063] The glycoconjugates were manufactured as follows:

[0064] BSA, DT-CRM197 and Tetanus Toxoid (Adimmune, Taiwan) were dissolved in 100 mM phosphate buffer pH 7.2 (~5 mg/ml) and 30 to 40 equivalents of Globo H monoester 35 were added to the solution. The mixture was gently stirred for 24 h at room temperature. The mixture was then diluted with deionized water and dialyzed against 5 changes of deionized water. The solution was then freeze-dried until a white powder was produced. The Globo H-protein conjugates obtained can be characterized through MALDI-TOF analysis to determine the rate of carbohydrate incorporation. 41 (GH-BSA), MALDI-TOF found 76029, 42 (GH-DT-CRM197) found 62138, 43 (GH-TT) found 162902, 44 (GH-BaMV) was not determined. MALDI-TOF MS Analysis for Glycoconjugates Glycoconjugates and primary carrier proteins can be reconstituted with ddH2O (~1 μg/μL). The matrix, sinapinic acid, was freshly prepared with acetonitrile and deionized water 1:1, resulting in a final matrix concentration of 10 mg/ml, including 0.1% TFA. The matrix solution and glycoconjugates were gently loaded and mixed and then the plate was air dried. Calibration with bovine serum albumin was mandatory before measurement. Each primary protein and glycoconjugate sample was detected in linear positive mode. The average molecular weight allows the calculation of the average number of carbohydrate molecules incorporated into the carrier protein.

[0065] A vaccine with a carbohydrate antigen molecule:toxin protein molecule ratio above 5:1 was manufactured according to the following steps: (a) 10 ml-25 ml of Globo H (available from OBI Pharma, Taiwan) and p-nitrophenyl ester ligand (available from OBI Pharma, Taiwan) were dissolved in 25 μl of DMF (commercially available from Sigma-Aldrich, USA). (b) 25 mg of TD was dissolved with 2.5 ml of phosphate buffer (i.e., a basic buffer with pH > 8). (c) The mixture in step (a) was added to the mixture in step (g) at room temperature overnight. The resulting mixture had a pH between 8-9.2

[0066] Results: 10 ml of Globo H resulted in vaccines comprising Globo H: DT (8: 1) and 25 ml of Globo H resulted in vaccines comprising Globo H: DT (24: 1), as determined by MALDI-TOF MS.

OBI-821 saponin preparation

[0067] OBI-821 saponin was extracted from Quillaja saponaria extract in accordance with the following steps: (a) Quillaja saponaria extract was pre-filtered by large particle C18 reverse phase chromatography, and then purified by silica-based normal preparative phase chromatography. This resulted in the raw OBI-821. (b) The crude OBI-821 in Step (a) was again prefiltered by large particle C18 reversed-phase chromatography, followed by preparative reversed-phase HPLC. The OBI-821 substance was sequentially finished through a desalination and freeze-drying process.

[0068] The OBI-821 saponin extracted from the bark of the Quillaja saponaria Molina tree was analyzed by mass spectrometry.

[0069] The mass peak at 1989.01 in Fig. 5A, the mass peak at 1989.12 in Fig. 5B, and the mass peak at 1989.13 illustrate the presence of compounds with a molecular weight of about 1989 .

[0070] The molar ratio of compounds with a molecular weight of about 1989 are: 89.8% in Fig. 5A, 96.8% in Fig. 5B and 87.0% in Fig. 5C. Similarly, the mass peak at 1856.97 in Fig. 5A, the mass peak at 1856.02 in Fig. 5B, and the mass peak at 1857.09 illustrate the presence of compounds with a molecular weight of ca. 1857. The molar ratio of compounds with a molecular weight of about 1857 are: 10.2% in Fig. 5A, 3.2% in Fig. 5B, and 13% in Fig. 5C.

[0071] The purified OBI-821 saponin was also analyzed by chromatography. Fig. 6 is an LC-UV chromatographic image (Column: PolyLC PolyHYDROXYETHYL A 200* 4.6mm 5μm, 300A). The first peak illustrates the presence of compounds 1989 V1 (A & B) and compounds 1857 V1 (A & B) (about 65.94%) and the second peak illustrates the presence of compounds 1989 V2 (A & B) and compounds 1857 V2 (A & B) (about 34.06%). Fig. 7 is a LC-MS chromatographic image (Column: Waters Symmetry ODS 150 * 2.1 mm). Peak 1 in the upper panel illustrates the presence of compound 1989 V1B and V2B (about 2.2%), while Peak 4 illustrates the presence of compound 1989 V1A and V2A (about 97.8%). Peak 2 in the lower panel illustrates the presence of compound 1857 V1B and compound 1857 V2B (about 1.9%) and Peak 3 illustrates the presence of compound 1857 V1A and compound 1857 V2A.

Example 2: Immunogenicity of vaccines with a higher carbohydrate/protein toxin ratio and efficacy of the OBI-281 saponin adjuvant

[0072] An in vivo assessment of the immunogenicity of the Globo H/DT (8:1) vaccine in Example 1 and the efficacy of the OBI-821 saponin adjuvant was carried out using CL57B/6 mice.

[0073] CL57B/6 mice approximately 8 weeks old were randomized into the following 4 study groups:

[0074] Blood samples were collected via the retro-orbital route or facial vein without anticoagulant, before the first injection or Day 0, and three days after each injection (i.e., on Days 10, 17 and 24). Blood samples were centrifuged to separate serum and blood cells. Sera were collected and stored at -20°C, and were subsequently analyzed by ELISA. Serum from each mouse was serially diluted for anti-Globo H IgG analysis. Globo H-ceramide was coated on the assay plate overnight before being blocked for 30 minutes with 1X blocking buffer (Sigma) and washed. with PBST. The diluted serum samples were added to the assay plate, incubated for 1 hour at room temperature (RT), and washed. Goat anti-mouse IgG-AP secondary antibody (Southern Biotech) was added to the sample and incubated for 45 minutes at room temperature. The plate was washed again, followed by addition of chromogen substrate and incubation at 37°C for 20 minutes. The reaction was terminated by the addition of a stop solution. Optical density was quantified by a plate reader (Molecular Device) at 405 nm wavelength. The Mann-Whitney t test was used for statistical analysis. Fig. 1A and Fig. 1B show the quantitative anti-Globo H IgG titer of the tested vaccines.

[0075] Results: The IgG titer of mice immunized with Globo H/DT (8:1 ratio) was significantly higher than that of Globo H/KLH with a C34 adjuvant (P < 0.01). The IgG titer of mice immunized with Globo H/DT (ratio 8:1) was higher than that of Globo H/KLH with an OBI-821 saponin adjuvant. (See Fig. 1(A). Regardless of type of carrier protein used, the OBI-821 saponin induced a higher and statistically significant IgG titer compared to the C34 adjuvant (P<0.05, see Fig. 1A and Fig 1B).

Example 3: Assessment of the immunogenicity of vaccines with a higher carbohydrate/protein toxin ratio and the efficacy of the OBI-281 saponin adjuvant using the ADCC and CDC assays.

[0076] Four groups of Lewis rats were immunized with the vaccines in Table 3 Table 3: Vaccine Composition

[0077] Mice were immunized subcutaneously with the vaccines listed in Table 3, on day 0, 7, 14 and 21. Peripheral blood mononuclear cells (PBMC) and plasma were collected before the first injection (i.e. Day 0) and on Day 10, Day 17 and Day 24.

[0078] The ADCC and CDC assays were carried out using a calcein AM release method known in the art. The process is described as follows:

Labeling target cells with Calcein AM

[0079] MCF-7 breast cancer cells (target cells) were cultured in Minimum Essential Medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate and 0.01 mg/ml insulin, 10% serum bovine fetal. Target cells were added to 96-well plates (5x103 cells per well) and incubated at 37°C in a humidified 5% CO2 atmosphere overnight. The medium was discarded and each well was washed once with PBS. 100 L of 20 M calcein-AM solution was added to each well (2 mmol per well) and incubated at 37C in a humidified 5% CO2 atmosphere for 2 hours. The supernatant was dried and each well was washed three times with PBS. Target cell incubated with plasma sample

[0080] The plasma sample was heat inactivated and 50 L of 1/5X heat inactivated plasma sample was added into each well except the "full release" control and "background" control. The total final dilution would be 1/10X after adding 50 L of PBMC or serum. The plates were incubated at 37°C (in the dark) for 30 min.

Target cell incubated with PBMC or complement

[0081] After incubation, 50 microliters of PBMC (2 x 106 cells/mL) (for E:T ratio: 20:1) was added to each well of the ADCC assay and 50 microliters of serum diluted 1/10X was added to each well in the CDC assay, with the exception of the "full release" control and "background" control. The reaction mixtures were incubated at 37°C in a humidified atmosphere of 5% CO2 for 4 hours. MEM without phenol red containing 2% Triton solution (50 microliters) was added to the "full release" control for the last 15 min of incubation time and MEM without phenol red (50 microliters) was added to the "background" control. ". The plates were centrifuged at 100 g for 5 min, and then 80 microliters of the supernatant was transferred to black 96-well plates. Fluorescence was measured at wavelengths of 485 nm excitation and 538 nm emission.

[0082] Figs. 2A to 2D show the in vivo ADCC and CDC activities of the G2, G3 and G4 vaccines.

[0083] Results: As illustrated in Fig. 2B and 2D, the ADCC and CDC activities of the G3 vaccine (with an OBI-821 saponin adjuvant) on Day 24 were higher than those of the G2 vaccine (without saponin adjuvant). As illustrated in Fig. 2B and 2D, the ADCC and CDC activities of the G4 vaccine (Globo H/DT ratio of 24:1) on Day 24 were higher than those of the G3 vaccine (Globo H/DT ratio of 8:1). These results show that the OBI-821 saponin adjuvant and a vaccine with a carbohydrate antigen/toxin protein ratio above 5:1 amplify and induce a longer lasting ADCC and CDC response.

Example 4: Immune response of vaccines with a higher carbohydrate/protein toxin ratio and efficacy of the OBI-821 saponin adjuvant

[0084] An in vivo evaluation of the Globo H/DT (8:1) and Globo H/DT (16:1) vaccines in Example 1 and the OBI-821 saponin adjuvant was performed using CL57B/6 or Balb/ mice. w.

[0085] CL57B/6 mice approximately 8 weeks old were randomized into the following 8 study groups:

[0086] Blood samples were collected via the retro-orbital route or facial vein without anticoagulant, before the first injection or Day 0, Day 10, 17 and 24. The blood samples were centrifuged to separate the serum and blood cells. Sera were collected and stored at -20°C, and were subsequently analyzed by ELISA. Serum from each mouse was serially diluted for analysis of IgG and IgM anti-Globo H. Fig. 3A, Fig. 3B and Fig. 4 show the quantitative titer of IgG and IgM anti-Globo H of the vaccines tested.

[0087] Results: Vaccines with OBI-821 saponin adjuvant induce more IgM anti-H Globo and IgG anti-Globo H in a statistically significant way than vaccines with C34 adjuvant (see Figures 3A, 3B and 4). The following statistically significant differences were noted: - The IgM titer of the G3 vaccine (OBI-821 saponin) was significantly higher than that of the G5 vaccine (C34) on Day 17 (p=0.02); - The IgM titer of the G1 vaccine (OBI-821 saponin) was significantly higher than that of the G6 vaccine (C34) on Day 17 (p=0.03); - The IgM titer of the G3 vaccine (OBI-821 saponin) was significantly higher than that of the G5 vaccine (C34) on Day 24 (p=0.03); - The IgG titer of the G3 vaccine (OBI-821 saponin) was significantly higher than that of the G5 vaccine (C34) on Day 17 (p=0.001); - The IgG titer of the G1 vaccine (OBI-821 saponin) was significantly higher than that of the G6 vaccine (C34) on Day 17 (p=0.003); - The IgG titer of the G3 vaccine (OBI-821 saponin) was significantly higher than that of the G5 vaccine (C34) on Day 24 (p=0.03) and - The IgG titer of the G1 vaccine (OBI-821 saponin ) was significantly higher than that of the G6 (C34) vaccine on Day 24 (p=0.004);

[0088] These results show that the OBI-821 saponin adjuvant significantly amplifies the IgM and IgG response compared to the C34 adjuvant.

[0089] The Globo H/KLH/OBI-821 saponin (G1) vaccine induces significantly higher IgM and IgG titers than the Globo H/DT(3:1)/OBI-821 saponin (G2) on Day 17 and Day 24. Without being tied to a particular theory, it is believed that G1 has a higher carbohydrate density (around 700 Globo H units per KLH carrier protein) and that it provoked a stronger immune response, while G2 would have a lower carbohydrate density (3 Globo H units per DT carrier protein) and would have provoked a weaker immune response. The following statistically significant differences were noted: - The IgM titer of the G1 vaccine (KLH) was significantly higher than that of the G2 vaccine (DT) on Day17 (p=0.003); - The IgM titer of the G1 vaccine (KLH) was significantly higher than that of the G2 vaccine (DT) on Day 24 (p=0.03); - The IgG titer of the G1 vaccine (KLH) was significantly higher than that of the G2 vaccine (DT) on Day 24 (p=0.004);

[0090] The IgM and IgG titers of Globo H/DT (8: 1 - ratio of Globo H to DT molecules) / OBI-821 saponin (G3 and G4) and Globo H/ DT (16: 1 - ratio of molecules of Globo H for DT)/saponin OBI-821 (G7) are comparable to those of Globo H/KLH/saponin OBI-821 (G1) at Day 17 and Day 24. (See Fig. 4). Despite lower carbohydrate density than that of GH-KLH (700:1 ratio of Globo H to DT molecules), GH-DT (8:1 ratio of Globe H to DT molecules) exhibited immunogenicity. comparable to that of GH-KLH.

[0091] Vaccines with a higher Globo H/DT ratio (8:1 or 161- ratio of Globo H to DT molecules) induce higher and longer-lasting IgM and IgG titers compared to vaccine titers with a ratio smaller than Globo H/DT (3:1). The following statistically significant differences were noted: - The IgM titer of the G3 vaccine (8:1 ratio of Globo H molecules to DT) was significantly higher than that of the G2 vaccine (3:1 ratio) on Day 17 ( p = 0.02), - The IgM titer of the G7 vaccine (16:1 ratio of Globo H molecules to DT) was significantly higher than that of the G2 vaccine (3:1 ratio) on Day 17 (p = 0.006), - The IgG titer of the G3 vaccine (8:1 ratio of Globo H molecules to DT) was significantly higher than that of the G2 vaccine (3:1 ratio) on Day 17 (p = 0, 01), - The IgG titer of the G7 vaccine (16:1 ratio of Globo H molecules to DT) was significantly higher than that of the G2 vaccine (3:1 ratio) on Day 17 (p = 0.03 ), - The IgG titer of the G3 vaccine (8:1 ratio of Globo H molecules to DT) was significantly higher than that of the G2 vaccine (3:1 ratio) on Day 24 (p = 0.01) , - The IgG titer of the G7 vaccine (16:1 ratio of Globo H to DT molecules) was significantly higher than that of the G2 vaccine (3:1 ratio of Globo H to DT molecules) on Day 24 (p = 0.01), IgG titers of Globo H/DT (8: 1 - ratio of Globo H to DT molecules) / OBI-821 saponin (G3) and Globo H/DT (16:1) / saponin OBI-821 (G7) are significantly higher than those of Globo H/DT (3:1- ratio of Globo H to DT molecules) / saponin OBI- 821 (G2) on Day 17 and 25 (P < 0.05).

[0092] All publications, patents and patent applications cited in this specification are incorporated herein by reference as if each particular publication or patent application were specifically and individually indicated to be incorporated by reference.

[0093] The present invention should not be limited to the scope of the embodiments disclosed herein, which are intended to be exclusive illustrations of individual aspects of the invention, but not a limitation of the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and teachings and are likewise intended to fall within the scope of the invention. Such modifications or other embodiments may be practiced without departing from the true scope and spirit of the invention.

Claims (13)

1. Vaccine characterized by the fact that it comprises: (a) a carbohydrate antigen associated with a tumor or its immunogenic fragment; and (b) a diphtheria toxin (DT), in which the ratio of tumor-associated carbohydrate antigen or immunogenic fragment thereof to DT ranges from 5:1 to 39:1, and the tumor-associated carbohydrate antigen is selected from Globo H, stage-specific embryonic antigen-3 (SSEA-3) or stage-specific embryonic antigen-4 (SSEA-4). 2. Vaccine according to claim 1, characterized by the fact that the proportion of tumor-associated carbohydrate antigen or its immunogenic fragment and the DT varies between 8: 1 and 24: 1. 3. Vaccine according to claim 1, characterized by the fact that the tumor-associated carbohydrate antigen is Globo H. 4. Vaccine according to claim 1, characterized in that DT is a cross-reactive material with diphtheria toxin or diphtheria toxoid. 5. Vaccine according to claim 4, characterized in that the material cross-reactive with diphtheria toxin is selected from CRM30, CRM45, CRM 176, CRM 197 or CRM 228. 6. Vaccine according to claim 1, characterized by the fact that it further comprises a saponin adjuvant. 7. Vaccine according to claim 6, characterized by the fact that the saponin adjuvant is OBI-821 saponin. 8. Vaccine according to claim 1, characterized by the fact that it further comprises an α-galactosyl-ceramide (α-GalCer) adjuvant. 9. Vaccine according to claim 8, characterized by the fact that the α-GalCer adjuvant has the following structure: 10. Pharmaceutical composition, characterized by the fact that it comprises (a) the vaccine as defined in claim 1; and (b) a pharmaceutically acceptable carrier. 11. Vaccine according to any one of claims 1 to 9, characterized by the fact that it is for inhibiting cancerous cells, comprising administering the vaccine to a subject in need thereof, where the cancerous cells are inhibited. 12. Vaccine according to claim 11, characterized by the fact that the cancer is a cancer that expresses Globo H. 13. Vaccine according to claim 12, characterized by the fact that the cancer expressing Globo H is breast cancer, lung cancer, esophageal cancer, rectal cancer, biliary cancer, liver cancer, oral cancer, gastric cancer , colon cancer, nasopharyngeal cancer, kidney cancer, brain tumor, cervical cancer, pancreatic cancer, testicular cancer, bladder cancer, head and neck cancer, oral cancer, neuroendocrine cancer, adrenal cancer, thyroid cancer, bone cancer, skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, prostate cancer, ovarian cancer and endometrial cancer.