HK1221476B - Compositions of a carbohydrate vaccine for inducing immune responses and uses thereof in cancer treatment - Google Patents

Description

Sugar vaccine composition for inducing immune response and use thereof in treating cancer

Technical Field

The present invention generally encompasses compositions and methods for cancer immunotherapy and particularly encompasses immunogenic glycoconjugates capable of stimulating an anti-cancer immune response.

Background of the Invention

The use of synthetic glycoconjugates to elicit antibodies was first demonstrated by Goebel and Avery in 1929 (Goebel, W.F. and Avery, O.T., J. Exp. Med., 1929, 50, 521; Avery, O.T. and Goebel, W.F., J. Exp. Med., 1929, 50, 533). The saccharide was linked to a carrier protein via a diazophenyl glycoside. Polyclonal antibodies were generated by immunizing rabbits with the synthetic antigen. Other researchers (Allen, P.Z. and Goldstein, I.J., Biochemistry, 1967, 6, 3029; Rude, E. and Delius, M.M., Carbohydr. Res., 1968, 8, 219; Himmelspach, K. et al., Eur. J. Immunol., 1971, 1, 106; Fielder, R.J. et al., J. Immunol., 1970, 105, 265) developed similar techniques for conjugating sugars to protein carriers.

Glycoconjugates can be used in active immunotherapies generated from vaccination to specifically target known targets on tumor cells. Responses to saccharide antigens normally do not involve the use of T cells that would aid in tumor rejection in vivo. Although complete tumor rejection is considered unlikely due to conjugate vaccination, such treatments would enhance immune surveillance and potentially reduce the recurrence of new tumor colonies. (Dennis, J., Oxford Glycosystems Glyconews Second, 1992; Lloyd, K.O., cited in Specific Immunotherapy of Cancer with Vaccines, 1993, New York Academy of Sciences, 50-58). Toyokuni and Singhal have described a synthetic saccharide conjugate (Toyokuni, T. et al., J. Am. Chem. Soc., 1994, 116, 395) that stimulates measurable IgG titers, a significant result because IgG responses are typically associated with the recruitment of helper T cells.

The carbohydrate antigen Globo H (Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glc) was first isolated and identified as a ceramide-linked glycolipid from breast cancer MCF-7 cells by Hakomori et al. in 1984 (Bremer EG et al., (1984) J Biol Chem 259:14773-14777). Additional studies using anti-Globo H monoclonal antibodies have shown that Globo H is present in many other cancers, including prostate, gastric, pancreatic, lung, ovarian, and colon cancers, and is expressed only in low amounts on the luminal surface of normal secretory tissues, where it is not readily accessible to the immune system. (Ragupathi G et al., (1997) Angew Chem Int Ed 36:125-128 (1997). In addition, it has been established that the serum of breast cancer patients contains high levels of anti-Globo H antibodies. (Gilewski T et al., (2001) Proc Natl Acad Sci USA 98:3270-3275; Huang C-Y et al., (2006) Proc Natl Acad Sci USA 103:15-20; Wang C-C et al., (2008) Proc Natl Acad Sci USA105(33):11661-11666). Patients with Globo H-positive tumors show a shorter survival period than patients with Globo H-negative tumors. (Chang, Y-J et al., (2007) Proc Natl Acad Sci USA 104(25):10299-10304). These studies have led to the discovery of the possibility that Globo H may be an important target of breast cancer research. H, a hexasaccharide epitope, has become an attractive tumor marker and a viable target for the development of cancer vaccines.

The synthetic Globo H vaccine, combined with an immune adjuvant, has been shown to induce primarily IgM and, to a lesser extent, IgG antibodies in both prostate cancer patients and metastatic breast cancer patients. In Phase I clinical trials, the vaccine also demonstrated minimal toxicity, with transient local skin reactions at the vaccination site. (Gilewski T et al., (2001) Proc Natl Acad Sci USA 98:3270-3275; Ragupathi G et al., (1997) Angew Chem Int Ed 36:125-128; Slovin SF et al., (1997) Proc Natl Acad Sci USA 96:5710-5715). Mild flu-like symptoms have been observed in some of these patients, possibly related to the side effects of QS-21. A pentavalent vaccine containing five prostate and breast cancer-associated carbohydrate antigens—Globo-H, GM2, STn, TF, and Tn—conjugated to a maleimide-modified carrier protein, KLH, has been reported to produce anti-Globo H sera with higher IgG titers than IgM in ELISA assays (Zhu J. et al., (2009) J. Am. Chem. Soc. 131(26):9298-9303).

KLH is known to contain glycosylated polypeptide subunits that assemble to form decamer (10-mer) particles, 20-mer particles, and larger particles. These multimeric structures have been characterized using ultracentrifugation techniques, which yield sedimentation coefficients of 11-19 s for dissociated subunits and 92-10 s for 20-mer multimers. It is further known that a variety of factors can affect the size distribution of molluscan hemocyanins (including KLH). These factors include ionic strength, pH, temperature, pO ₂ , and the availability of certain divalent cations, particularly calcium and magnesium ions. The present inventors have developed compositions with increased effectiveness consisting primarily of KLH dimers and trimers linked to multiple Globo H moieties.

Although vaccines have been developed to elicit antibody responses against Globo H, their anticancer effects are unsatisfactory due to the low antigenicity of Globo H. New vaccines that can elicit high-level immune responses targeting Globo H are needed.

Summary of the Invention

The present invention generally encompasses therapeutic and/or prophylactic compositions comprising Globo H, as well as immunotherapeutics, vaccines, dosage forms, kits, and methods of making and treating the same.

In one embodiment, the invention encompasses an isolated therapeutic conjugate comprising a Globo H moiety linked to a keyhole limpet hemocyanin (KLH) moiety subunit. In certain embodiments, the linkage is a covalent bond.

In another embodiment, the present invention encompasses an isolated therapeutic conjugate comprising a Globo H moiety covalently linked to a keyhole limpet hemocyanin (KLH) moiety subunit, wherein the KLH is a derivatized KLH. As used herein, when referring to Globo-H and KLH, the term "covalently linked" means: Globo-H is covalently linked directly to KLH, or Globo-H is covalently linked to a derivatized KLH (as described herein), or Globo-H is covalently linked to KLH via a linker group (as described herein), or Globo-H is covalently linked to KLH via a linker group and a derivatized KLH.

In certain exemplary embodiments, the derivatized KLH of the present invention has the following structure:

In another embodiment, the present invention encompasses an isolated therapeutic conjugate comprising a Globo H moiety covalently linked to a keyhole limpet hemocyanin (KLH) moiety subunit via a linker molecule.

In a preferred embodiment, the Globo H moiety is bound to a lysine residue of a KLH moiety subunit.

In one embodiment, there are exactly or approximately 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 lysine residues per KLH portion subunit that are available for binding or actually bind directly or indirectly to the Globo H portion.

In another embodiment, the present invention encompasses an isolated therapeutic conjugate comprising a Globo H moiety covalently linked to a keyhole limpet hemocyanin (KLH) moiety subunit via a 4-(4-N-maleimidomethyl)cyclohexane-1-carboxyhydrazide (MMCCH) linker group. The MMCCH linker of the present invention has the following structure:

In another illustrative embodiment, the invention encompasses an isolated therapeutic conjugate having the following overall structure:

wherein n is an integer from about 1 to about 160. In certain embodiments, a monomeric KLH moiety may include from about 1 to about 160 Globo H moieties. One of ordinary skill in the art will recognize that these structures are shown as iminium hydrochloride salts, but may also exist or coexist as imines. Thus, the present invention encompasses imines and their salts, including iminium hydrochloride salts. In certain embodiments, a monomeric KLH moiety may include from about 1 to about 125 Globo H moieties. In certain embodiments, a monomeric KLH moiety may include from about 1 to about 100 Globo H moieties. In certain embodiments, a monomeric KLH moiety may include from about 1 to about 75 Globo H moieties. In certain embodiments, a monomeric KLH moiety may include from about 1 to about 50 Globo H moieties. In certain embodiments, a monomeric KLH moiety may include from about 1 to about 25 Globo H moieties. In certain embodiments, a monomeric KLH moiety may include from about 1 to about 10 Globo H moieties.

In certain embodiments, the Globo H moiety is covalently conjugated to the KLH moiety at a basic amino acid residue. In certain embodiments, the basic amino acid residue is arginine, lysine, histidine, or a combination thereof.

In another embodiment, the Globo H moiety is bound to a lysine conjugation site on a monomeric KLH moiety subunit.

In another embodiment, there are exactly or approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 lysine conjugation sites available for or actually bound to the Globo H moiety per monomeric KLH portion subunit. In another embodiment, there are 62, 66, 67, 68, 70, 72, 76, 86, 87, 88, 90, 92, 93, 100 such lysine conjugation sites per KLH portion subunit.

In certain therapeutic composition embodiments containing a mixture of partial subunits (e.g., KLH1 and KLH2 or variants thereof), the total number of available lysines (for both subunits) as counted collectively across the different subunit types can be exactly or approximately 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, or 310. In such embodiments, there are or may be exactly or approximately 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 lysine conjugation sites together across different subunits (e.g., KLH1 and KLH2 or variants thereof). In other such embodiments, there are 136, 137, 141, 140, 143, 147, or 155 lysine conjugation sites.

In another illustrative embodiment, the invention encompasses an isolated therapeutic conjugate having the following overall structure:

Wherein n is independently an integer from about 1 to about 3000, and m is independently an integer from about 1 to about 20. In certain embodiments, when m is greater than 1, the KLH moieties can aggregate to form a multimeric structure. In certain embodiments, the aggregation is a covalent bond. In certain other embodiments, the aggregation is not a covalent bond (e.g., the aggregation is formed by H-bond interactions or hydrophobic interactions). In certain embodiments, a monomeric KLH moiety (i.e., where m=1) can include from about 1 to about 160 Globo H moieties. In certain embodiments, a dimeric KLH moiety (i.e., where m=2) can include from about 1 to about 300 Globo H moieties. In certain embodiments, a trimeric KLH moiety (i.e., where m=3) can include from about 1 to about 450 Globo H moieties. In certain embodiments, a tetrameric KLH moiety (i.e., where m=4) can include from about 1 to about 600 Globo H moieties. In certain embodiments, a pentameric KLH moiety (ie, wherein m=5) can include from about 1 to about 750 Globo H moieties.

In another illustrative embodiment, the invention encompasses an isolated therapeutic conjugate having the following overall structure:

wherein n is independently an integer from about 1 to about 150, and m is independently an integer from about 1 to about 20.

In another embodiment, the invention encompasses an isolated therapeutic conjugate having the following overall structure:

wherein n is independently an integer from about 1 to about 160, and wherein m is independently an integer from about 1 to about 20. In certain embodiments, m is an integer from about 1 to about 5. In certain embodiments, m is an integer from about 1 to about 3. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, m is 3. In certain embodiments, m is 4. In certain embodiments, m is 5. In certain embodiments, m is 6. In certain embodiments, m is 7. In certain embodiments, m is 8. In certain embodiments, m is 9. In certain embodiments, m is 10. In certain embodiments, m is 11. In certain embodiments, m is 12. In certain embodiments, m is 13. In certain embodiments, m is 14. In certain embodiments, m is 15. In certain embodiments, m is 16. In certain embodiments, m is 17. In certain embodiments, m is 18. In certain embodiments, m is 19. In certain embodiments, m is 20. In certain embodiments, for any of the above embodiments, when m is 1 to 20, each n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 ,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125 5, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160.

In certain embodiments, there is more than one Globo H moiety attached to each monomeric KLH moiety. In certain exemplary embodiments, the more than one Globo H moiety attached to each monomeric KLH moiety is linked via a linker. In other exemplary embodiments, the more than one Globo H moiety attached to each KLH moiety is linked via a linker and is linked to a derivatized KLH moiety.

In another embodiment, the ratio of the Globo H moiety to the KLH moiety subunit is at least 1. In another embodiment, the ratio of the Globo H moiety to the KLH moiety subunit is at least 10. In another embodiment, the ratio of the Globo H moiety to the KLH moiety subunit is at least 25. In another embodiment, the ratio of the Globo H moiety to the KLH moiety subunit is at least 50. In yet another embodiment, the ratio of the Globo H moiety to the KLH moiety subunit is at least 100. In yet another embodiment, the ratio of the Globo H moiety to the KLH moiety subunit is at least 150. In yet another embodiment, the ratio of the Globo H moiety to the KLH moiety subunit is at least 500. In yet another embodiment, the ratio of the Globo H moiety to the KLH moiety subunit is at least 750. In yet another embodiment, the ratio of the Globo H moiety to the KLH moiety subunit is at least 1000. In yet another embodiment, the ratio of the Globo H moiety to the KLH moiety subunit is at least 1500. In yet another embodiment, the ratio of Globo H moiety to KLH moiety subunits is at least 2000.

In various embodiments, the invention encompasses a single KLH monomer to multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) KLH subunits, each having multiple Globo H moieties attached. In certain embodiments, the ratio of Globo H moieties to KLH moieties is the same. In other embodiments, the ratio of Globo H moieties to KLH moieties is different.

Another embodiment of the present invention encompasses a composition comprising at least two KLH moieties. For example, the derivatized KLH moieties are in the form of dimers. In another embodiment, the at least two KLH moieties are identical. In another embodiment, the at least two KLH moieties are different. In yet another embodiment, the at least two KLH moieties have the same Globo H moiety to KLH moiety subunit ratio. In yet another embodiment, the at least two KLH moieties have different Globo H moiety to KLH moiety subunit ratios.

Another embodiment of the present invention encompasses a therapeutic composition comprising at least three KLH moieties (e.g., derivatized KLH moieties in trimer form). In certain embodiments, the at least three KLH moieties are identical. In another embodiment, the at least three KLH moieties are not identical. In yet another embodiment, the at least three KLH moieties have the same Globo H moiety to KLH moiety subunit ratio. In yet another embodiment, the at least three KLH moieties have different Globo H moieties to KLH moiety subunit ratios.

Another embodiment of the present invention encompasses a therapeutic composition comprising at least four KLH moieties (e.g., derivatized KLH moieties in tetrameric form). In certain embodiments, the at least four KLH moieties are identical. In another embodiment, the at least four KLH moieties are not identical. In yet another embodiment, the at least four KLH moieties have the same Globo H moiety to KLH moiety subunit ratio. In yet another embodiment, the at least four KLH moieties have different Globo H moieties to KLH moiety subunit ratios.

Another embodiment of the present invention encompasses a therapeutic composition comprising at least five KLH moieties (e.g., derivatized KLH moieties in pentameric form). In certain embodiments, the at least five KLH moieties are identical. In another embodiment, the at least five KLH moieties are not identical. In yet another embodiment, the at least five KLH moieties have the same Globo H moiety to KLH moiety subunit ratio. In yet another embodiment, the at least five KLH moieties have different Globo H moieties to KLH moiety subunit ratios.

Another embodiment of the present invention encompasses a therapeutic composition comprising at least six KLH moieties (e.g., derivatized KLH moieties in hexameric form). In certain embodiments, the at least six KLH moieties are identical. In another embodiment, the at least six KLH moieties are not identical. In yet another embodiment, the at least six KLH moieties have the same Globo H moiety to KLH moiety subunit ratio. In yet another embodiment, the at least six KLH moieties have different Globo H moieties to KLH moiety subunit ratios.

In one embodiment, the Globo H moiety comprises (Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glc). In another embodiment, the KLH moiety subunit is a KLH-1 or KLH-2 moiety or a combination thereof. As used herein, the term "KLH" refers to KLH-1, KLH-2 and/or a combination thereof.

In another embodiment, the KLH partial subunit is at least 99% identical to the corresponding naturally occurring KLH partial subunit.

In another embodiment, the KLH partial subunit is at least 95% identical to the corresponding naturally occurring KLH partial subunit.

In another embodiment, the KLH partial subunit is at least 90% identical to the corresponding naturally occurring KLH partial subunit.

In another embodiment, the KLH partial subunit is at least 80% identical to the corresponding naturally occurring KLH partial subunit.

In another embodiment, the KLH partial subunit is at least 70% identical to the corresponding naturally occurring KLH partial subunit.

In another embodiment, the KLH partial subunit is at least 60% identical to the corresponding naturally occurring KLH partial subunit.

In another embodiment, the Globo H moiety is covalently linked to the keyhole limpet hemocyanin (KLH) moiety subunit via a linker. In yet another embodiment, the Globo H moiety is covalently linked to the keyhole limpet hemocyanin (KLH) moiety subunit via a 4-(4-N-maleimidomethyl)cyclohexane-1-carboxyhydrazide (MMCCH) bond. In yet another embodiment, the Globo H moiety is covalently linked to a derivatized keyhole limpet hemocyanin (KLH) moiety subunit and linked by a 4-(4-N-maleimidomethyl)cyclohexane-1-carboxyhydrazide (MMCCH) bond.

In another embodiment, the isolated therapeutic conjugate has an epitope ratio of at least or about 150 based on a KLH monomer having a molecular weight of about 350 KDa to about 400 KDa. In another embodiment, the isolated therapeutic conjugate has an epitope ratio of at least or about 100. In yet another embodiment, the isolated therapeutic conjugate has an epitope ratio of at least or about 75. In still yet another embodiment, the isolated therapeutic conjugate has an epitope ratio of at least or about 50. In yet another embodiment, the isolated therapeutic conjugate has an epitope ratio of at least or about 25. In yet another embodiment, the isolated therapeutic conjugate has an epitope ratio of at least or about 15. In yet another embodiment, the isolated therapeutic conjugate has an epitope ratio of at least or about 5. In yet another embodiment, the isolated therapeutic conjugate has an epitope ratio of at least or about 1.

Another embodiment of the present invention encompasses a pharmaceutical composition comprising KLH moiety subunits, wherein each KLH moiety subunit comprises one or more Globo H moieties covalently linked to a keyhole limpet hemocyanin (KLH) moiety subunit. In certain embodiments, the pharmaceutical composition comprises a dimer of at least two KLH moiety subunits, wherein each KLH moiety subunit comprises one or more Globo H moieties covalently linked to the KLH moiety subunit. In certain embodiments, the pharmaceutical composition comprises a trimer of at least three KLH moiety subunits, wherein each KLH moiety subunit comprises one or more Globo H moieties covalently linked to the KLH moiety subunit. In certain embodiments, the pharmaceutical composition comprises at least four KLH moiety subunits, wherein each KLH moiety subunit comprises one or more Globo H moieties covalently linked to the KLH moiety subunit. In certain embodiments, the pharmaceutical composition comprises a mixture of KLH moiety subunits (e.g., monomers, dimers, trimers, tetramers, pentamers, etc.), wherein each KLH moiety subunit comprises a plurality of Globo H moieties covalently linked to the KLH moiety subunit.

Another aspect of the present invention relates to a pharmaceutical composition comprising a monomer, dimer, trimer, tetramer or pentamer of a KLH moiety, or a combination thereof, wherein each KLH comprises one or more Globo H moieties covalently linked to a subunit of a keyhole limpet hemocyanin (KLH) moiety.

In one embodiment of the present invention, the epitope ratio of the therapeutic conjugates in the composition ranges from about 1 to 3000. In another embodiment, the epitope ratio of the therapeutic conjugates in the composition ranges from about 75 to 2000. In another embodiment, the epitope ratio of the therapeutic conjugates in the composition ranges from about 100 to 1000. In yet another embodiment, the average epitope ratio of the therapeutic conjugates in the composition ranges from about 150 to 500.

In another embodiment, about 1% to 99% of the therapeutic conjugate in the composition is a KLH monomer. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition is a KLH dimer. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition is a KLH trimer. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition is a KLH tetramer. In yet another embodiment, about 1% to 99% of the therapeutic conjugate in the composition is a KLH pentamer. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition comprises 6 KLH subunits. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition comprises 7 KLH subunits. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition comprises 8 KLH subunits. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition comprises 9 KLH subunits. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition comprises 10 KLH subunits. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition comprises 11 KLH subunits. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition comprises 12 KLH subunits. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition comprises 13 KLH subunits. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition comprises 14 KLH subunits. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition comprises 15 KLH subunits. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition comprises 16 KLH subunits. In yet another embodiment, about 0% to 99% of the therapeutic conjugate in the composition comprises 17 KLH subunits. In yet another embodiment, about 0% to 99% of the therapeutic conjugates in the composition comprise 18 KLH subunits. In yet another embodiment, about 0% to 99% of the therapeutic conjugates in the composition comprise 19 KLH subunits. In yet another embodiment, about 0% to 99% of the therapeutic conjugates in the composition comprise 20 KLH subunits. In yet another embodiment, about 1% to 99% of the therapeutic conjugates in the composition are monomers, dimers, trimers, tetramers, or combinations thereof. In yet another embodiment, about 99% of the therapeutic conjugates in the composition are monomers, dimers, trimers, tetramers, or combinations thereof.

In another embodiment, the pharmaceutical composition comprises an adjuvant including, but not limited to, Freund's adjuvant, a Toll-like receptor molecule, LPS, a lipoprotein, a lipopeptide, flagella, double-stranded RNA, viral DNA, unmethylated CpG islands, levamisole, bacillus calmette-guerin, isoprinosine, Zadaxin, a PD-1 antagonist, a PD-1 antibody, a CTLA antagonist, a CTLA antibody, an interleukin, a cytokine, GM-CSF, an aluminum salt-based glycolipid, aluminum phosphate, alum, aluminum hydroxide, liposomes, a TLR2 agonist, nanoparticles, monophosphate lipid A, OPT-821 saponin, QS-21 saponin, oil-in-water nanoemulsions, and bacteria-like particles.

In another embodiment, the pharmaceutical composition comprises a cytokine selected from the group consisting of IL-2, IL-12, IL-18, IFN-γ, TNF, IL-4, IL-10, IL-13, IL-21, GM-CSF, and TGF-β. In yet another embodiment, the pharmaceutical composition comprises a chemokine.

In yet another embodiment, the therapeutic agent is administered as a pharmaceutical composition.

In yet another embodiment, the pharmaceutical composition comprises a monoclonal antibody, a chemotherapeutic agent, a hormonal therapeutic agent, a retinoid receptor modulator, a cytotoxic/cytostatic drug, an anti-tumor agent, an antiproliferative agent, an anti-mTOR agent, an anti-Her2 agent, an anti-EGFR agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, a nitrogen mustard, a nitrosourea, an angiogenesis inhibitor, bevacizumab, a cell proliferation and survival signaling pathway inhibitor, an apoptosis inducer, an agent that interferes with a cell cycle checkpoint, an agent that interferes with a receptor tyrosine kinase (RTK), an integrin blocker, an NSAID, a PPAR agonist, an inhibitor of intrinsic multidrug resistance (MDR), an antiemetic agent, an agent useful in treating anemia, an agent useful in treating neutropenia, an immunopotentiating agent, a bisphosphonate, an aromatase inhibitor, an agent that induces terminal differentiation of tumor cells, a gamma-secretase inhibitor, a cancer vaccine (e.g., an active immunotherapy therapeutic agent), a monoclonal antibody therapeutic agent (e.g., a passive immunotherapy therapeutic agent), and any combination thereof.

In another embodiment, the therapeutic composition of the present invention may further comprise a PD-1/PD-L1 inhibitor (cytotoxic T lymphocyte (CTL) immunotherapy), a CTLA-4 immunotherapy, a CDK4/6 inhibitor (targeted therapy), a PI3K inhibitor (targeted therapy), an mTOR inhibitor (targeted therapy), an AKT inhibitor (targeted therapy), and a pan-Her inhibitor (targeted therapy). These inhibitors may also be modified to produce corresponding monoclonal antibodies. Such antibodies may be included in the therapeutic composition of the present invention.

In another embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. In yet another embodiment, the pharmaceutical composition is a cancer vaccine. In yet another embodiment, the pharmaceutical composition is formulated for subcutaneous administration. In yet another embodiment, the pharmaceutical composition is formulated for intramuscular administration. In yet another embodiment, the pharmaceutical composition is formulated for intra-arterial administration. In yet another embodiment, the pharmaceutical composition is formulated for intravenous administration.

Another embodiment of the present invention encompasses a method of treating cancer in a patient in need thereof, comprising administering an effective amount of a therapeutic composition comprising Globo H and KLH. In one embodiment, the patient has been diagnosed with or is suspected of having cancer. In another embodiment, the cancer is an epithelial cancer. In yet another embodiment, the cancer is breast cancer. In yet another embodiment, the therapeutically effective amount of the Globo-H moiety in the pharmaceutical composition/therapeutic composition can be from about 0.001 μg/kg to about 250 mg/kg. In yet another embodiment, the therapeutically effective amount of the Globo-H moiety in the pharmaceutical composition/therapeutic composition comprises a therapeutic conjugate at about 10 μg/kg to about 50 μg/kg per dose. In yet another embodiment, the therapeutically effective amount of the Globo-H moiety in the pharmaceutical composition/therapeutic composition comprises a therapeutic conjugate at about 0.10 μg/kg to about 0.75 μg/kg per dose.

In yet another embodiment, the therapeutically effective amount of the Globo-H-KLH complex in the therapeutic composition can be from about 0.001 μg/kg to about 250 mg/kg. In yet another embodiment, the therapeutically effective amount of the Globo-H-KLH complex in the therapeutic composition comprises about 10 μg/kg to about 50 μg/kg of a therapeutic conjugate per dose. In yet another embodiment, the therapeutically effective amount of the Globo-H-KLH complex in the therapeutic composition comprises about 0.60 μg/kg to about 4.50 μg/kg of a therapeutic conjugate per dose.

In yet another embodiment, the method can prolong progression-free survival by about or at least 1 week over a placebo control. In yet another embodiment, the method can prolong progression-free survival by about or at least 2 weeks over a placebo control. In yet another embodiment, the method can prolong progression-free survival by about or at least 1 month over a placebo control. In yet another embodiment, the method can prolong progression-free survival by about or at least 3 months over a placebo control. In yet another embodiment, the method can prolong progression-free survival by about or at least 6 months over a placebo control. In yet another embodiment, the method can prolong overall survival by about or at least 12 months over a placebo control.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more fully understood by reference to the accompanying drawings when considered in conjunction with the following detailed description.The embodiments shown in the drawings are intended only to illustrate the invention and are not to be construed as limiting the invention to the embodiments shown.

Figure 1A shows the chemical structures of Globo H and several exemplary Globo H analogs. Glc represents glucose, Gal represents galactose, GalNAc represents N-acetylgalactosamine, and Fuc represents fucose. Figure 1B shows exemplary Globo H-KLH subunits conjugated via an MMCCH linker.

Figure 2A shows an exemplary Globo H-KLH subunit conjugate synthesis pathway. Figure 2B shows Globo H-KLH dimers and trimers of the present invention compared to the Globo H conjugates disclosed in Slovin et al., (1999), Proc Natl Acad Sci USA 96:5710-5 and Gilewski et al., (2001), Proc Natl Acad Sci USA 98:3270-5.

FIG3 shows the results of multi-angle laser light scattering spectrometry (MALS) of native KLH (8.3 MDa).

FIG4 shows the results of size exclusion chromatography of native KLH (8.3 MDa).

Figure 5 shows the temporal expansion of B/CD3 ⁺ T/CD4 ⁺ T/CD8 ⁺ T cell populations in Lewis rats immunized with the Globo H-KLH glycoconjugate of the present invention. Panels A to D represent B cell, CD3+ T cell, CD4+ T cell, and CD8+ T cell populations, respectively. Data are presented as percentages of the number of cells in the indicated groups, normalized to the percentage of cells in the PBS group. Multiple comparisons were analyzed using a two-way ANOVA followed by a Bonferroni post hoc test. *, p <0.05; **, p <0.01; and ***, p < 0.001 compared to the PBS group.

FIG6 shows the time series changes in the reciprocal titers of (A) IgM antibodies and (B) IgG antibodies in the blood from Lewis rats immunized with the glycoconjugate of the present invention (Globo H-KLH).

Figure 7 shows IgM antibody titers in mice in response to a glycoconjugate of the invention (Globo H-KLH).

Figure 8 shows the immunogenicity of C57BL/6 mice immunized with PBS, adjuvant alone, or Globo H-KLH + adjuvant on days 0, 5, and 10. Sera were collected on day 14 for ELISA analysis to determine anti-Globo H IgG and IgM production.

Figure 9 shows complement-dependent cytotoxicity, where Globo H (+) or Globo H (-) TOV21G cells were plated in 96-well plates. Anti-Globo H serum or control serum was added at a dilution of 1:50 or 1:100. Complement was then added to the plate with or without the addition of complement. Complement-dependent cytotoxicity (CDC) was determined by the LDH assay.

Figure 10 shows the cytotoxicity of Globo H (+) or Globo H (-) TOV21G cells seeded in 96-well plates. Anti-Globo H serum or control serum was added at a dilution of 1:50 or 1:100. Human NK cells isolated from peripheral blood mononuclear cells (PBMC) and activated with anti-CD3 antibodies were used as effector cells. For complement-dependent cell-mediated cytotoxicity (ADCC) reactions, effector cells were added at an ET ratio of 4:1, 2:1, or 1:1 or without the addition of effector cells. The cytotoxicity of each cell at different ET ratios was normalized to a control without mouse serum.

Figure 11 shows irradiated NOD-SCID mice injected intraperitoneally with ^1x10 Globo H-positive TOV21G cells on day 0. Antisera were collected from C57BL/6 mice after three vaccinations with three different treatments (PBS, adjuvant alone, and Globo H-KLH/adjuvant). Each NOD-SCID mouse received 200 μl of the aforementioned antisera intraperitoneally on days 0, 2, 4, 6, 9, 11, 13, and 16. Tumor images were tracked using an IVIS imaging system on days 3, 7, and 9.

Figure 12 shows LLC1 (a lung cancer epithelial cancer cell line) tumor growth in Globo H KLH-immunized C57BL/6 mice that were vaccinated subcutaneously with PBS, adjuvant alone, or Globo H-KLH/adjuvant on days 0, 5, and 11. 1 x 10 ⁵ LLC1 cells were injected subcutaneously into each mouse on day 16. Treatment was subsequently administered subcutaneously on days 29 and 34. Tumor size was monitored on days 16, 21, 25, 29, 32, 34, and 37.

Figure 13 shows a tabular summary of peptide identifications.

FIG14 shows the identification details of Globo-H conjugated peptides of sample 1 (1 ^st LC-MS/MS) of KLH1 (a) and KLH2 (b).

FIG15 shows the identification details of Globo-H conjugated peptides of sample 2 (1 ^st LC-MS/MS) for KLH1 (a) and KLH2 (b).

FIG16 shows the identification details of Globo-H conjugated peptides of sample 3 (1 ^st LC-MS/MS) for KLH1 (a) and KLH2 (b).

FIG17 shows the identification details of Globo-H conjugated peptides of sample 4 (1 ^st LC-MS/MS) for KLH1 (a) and KLH2 (b).

FIG18 shows the identification details of Globo-H conjugated peptides of sample 1 (2 ^st LC-MS/MS) for KLH1 (a) and KLH2 (b).

FIG19 shows the identification details of Globo-H conjugated peptides of sample 2 (2 ^st LC-MS/MS) for KLH1 (a) and KLH2 (b).

FIG20 shows the identification details of Globo-H conjugated peptides of sample 3 (2 ^st LC-MS/MS) for KLH1 (a) and KLH2 (b).

FIG21 shows the identification details of Globo-H conjugated peptides of sample 4 (1 ^st LC-MS/MS) for KLH1 (a) and KLH2 (b).

FIG22 shows the identification details of the MMCCH-conjugated peptides of sample 1 (1 ^st LC-MS/MS) of KLH1 (a) and KLH2 (b).

FIG23 shows the identification details of the MMCCH-conjugated peptides of sample 2 (1 ^st LC-MS/MS) of KLH1 (a) and KLH2 (b).

FIG24 shows the identification details of the MMCCH-conjugated peptides of sample 3 (1 ^st LC-MS/MS) for KLH1 (a) and KLH2 (b).

FIG25 shows the identification details of the MMCCH-conjugated peptides of sample 4 (1 ^st LC-MS/MS) for KLH1 (a) and KLH2 (b).

FIG26 shows the identification details of the MMCCH-conjugated peptides of sample 1 (2 ^st LC-MS/MS) of KLH1 (a) and KLH2 (b).

FIG27 shows the identification details of the MMCCH-conjugated peptides of sample 2 (2 ^st LC-MS/MS) of KLH1 (a) and KLH2 (b).

FIG28 shows the identification details of the MMCCH-conjugated peptides of sample 3 (2 ^st LC-MS/MS) for KLH1 (a) and KLH2 (b).

FIG29 shows the identification details of the MMCCH-conjugated peptides of sample 4 (1 ^st LC-MS/MS) for KLH1 (a) and KLH2 (b).

FIG30 shows a summary of Globo-H conjugated lysine identification for KLH1 (a) and KLH2 (b) (1 ^st LC-MS/MS) and for KLH1 (c) and KLH2 (d) (2 ^nd LC-MS/MS).

FIG31 shows a summary of MMCCH-conjugated lysine identification for KLH1 (a) and KLH2 (b) (1 ^st LC-MS/MS) and for KLH1 (c) and KLH2 (d) (2 ^nd LC-MS/MS).

Figure 32 shows the summary of Globo-H conjugation analysis in the first (a) and second (b) rounds of LC-MS/MS.

Figure 33(a) shows the chemical formula: C(56)H(91)N(5)O(33)S(1), with a monoisotopic MW sum of 1393.5317 Da. Figure 33(b) shows the chemical formulas: 1. C(18)H(28)N(4)O(4)S(1), single isotope MW sum: 396.1831Da; 2. Chemical formula: C(24)H(38)N(4)O(9)S(1), single isotope MW sum: 558.2360Da; 3. Chemical formula: C(30)H(48)N(4)O(14)S(1), single isotope MW sum: 720.2888Da; 4. Chemical formula: C(36)H(58)N(4)O(19)S(1), single isotope MW sum: 882.3416Da; 5. Chemical formula: C(44)H(71)N(5)O(24)S(1), single isotope MW sum: 1085.4210Da.

Figure 34(a) shows the chemical structure of the MMCCH derivative. Chemical formula: C(16)H(24)N(4)O(3)S(1), monoisotopic MW sum: 352.1569 Da. Figure 34(b) shows the deamidated MMCCH derivative, chemical formula: C(16)H(22)N(2)O(4)S(1), monoisotopic MW sum: 338.1300 Da.

Detailed Description of the Invention

Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology, microbiology, and immunology, which are within the capabilities of those skilled in the art and are fully explained in the literature. See, e.g., Molecular Cloning A Laboratory Manual, 2nd ed., Sambrook, Fritsch and Maniatis, eds. (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Vols. 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); 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 & 155 (Wu et al., eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, Harlow and Lanes (Cold Spring Harbor Laboratory Press, 1988); and Handbook Of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986).

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or this specification can mean "one", but it also complies with the following meanings: "one or more", "at least one" and "one or more than one".

Throughout this application, the term "about" is used to indicate that a value includes, for example, the inherent error variation of the measuring device, the method being used to determine the value, or the variation that exists between study subjects. Generally, the term is intended to encompass about or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% variability, as the case may be.

As used herein, the term "alkyl" refers to a linear or branched monovalent hydrocarbon containing 1 to 20 carbon atoms (e.g., C1 _{- C8} or _C1 - _C4 ), which may be substituted or unsubstituted, unless otherwise specified. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.

While this disclosure supports definitions referring only to alternatives and "and/or," use of the term "or" in the claims is intended to mean "and/or" unless explicitly indicated referring only to alternatives or the alternatives are mutually exclusive.

As used in this specification and claims, the words "comprising" (and any form of comprising, such as "comprising" and "containing"), "having" (and any form of having, such as "having" and "having"), "including" (and any form of including, such as "including" and "including"), or "containing" (and any form of containing, such as "containing" and "containing") are inclusive or open-ended and do not exclude additional, unrecited agents or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Additionally, the compositions of the invention can be used to implement the methods of the invention.

"Treatment" or "treatment" refers herein to the administration of a therapeutic composition to a subject with the intent to cure, alleviate, relieve, correct, prevent, or ameliorate a condition, a symptom of a condition, a disease state secondary to a condition, or a predisposition to a condition.

An "effective amount" is an amount of a therapeutic composition that is capable of producing a medically desirable result, as described herein, in the subject being treated. A medically desirable result can be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject gives an indication of or feels an effect).

As referred to herein, "a disease treatable with a therapeutic composition" means any process, condition, disorder, illness, and/or disease that can be treated by administering a therapeutic composition disclosed herein.

A "proliferative disorder" is a disorder in which too many cells of a certain type are produced, leading to poor health. A proliferative disorder can be benign or malignant. A proliferative disorder can include, for example, cancer.

"Cancer," which can be treated by the therapeutic compositions disclosed herein, is an abnormal growth of cells. Cancer cells have lost their normal control mechanisms and are therefore able to continuously expand, invade adjacent tissues, migrate to distant parts of the body, and promote the growth of new blood vessels from which the cells extract nutrients. As used herein, cancer can be malignant or benign. Cancer can form from any tissue inside the body. As cells grow and proliferate, they form a mass of tissue called a tumor. The term tumor refers to an abnormal growth or mass. Tumors can be cancerous (malignant) or noncancerous (benign). Cancerous tumors can invade adjacent tissues and spread throughout the body (metastasis). However, benign tumors generally do not invade adjacent tissues and do not spread throughout the body. Cancer can be divided into cancers of the blood and blood-forming tissues (leukemias and lymphomas) and "solid" tumors. "Solid" tumors can be carcinomas or sarcomas.

Cancers that can be treated by the therapeutic compositions of the present invention include those classified by site, including cancers of the oral cavity and pharynx (lips, tongue, salivary glands, floor of mouth, gums and other oral sites, nasopharynx, tonsils, oropharynx, hypopharynx, other oral/pharyngeal sites); cancers of the digestive system (esophagus; stomach; small intestine; colon and rectum; anus, anal canal and anorectal region; liver; intrahepatic bile duct; gallbladder; other biliary sites; pancreas; retroperitoneum; peritoneum, omentum and mesentery; other digestive tract sites); cancers of the respiratory system (nasal cavity, middle ear and sinuses; larynx; lungs and bronchi; pleura; trachea, mediastinum and other respiratory sites); mesothelioma; cancers of the bones and joints and soft tissues (including the heart); skin cancers, including melanoma and other non-upper cutaneous skin cancer; Kaposi's sarcoma and breast cancer; cancers of the female genital system (cervix; uterine body; uterus, ovaries; vagina; vulva; and other female genital areas); cancers of the male genital system (prostate; testicles; penis; and other male genital areas); cancers of the urinary system (urinary bladder; kidneys and renal pelvis; ureters; and other urinary tract areas); cancers of the eye and orbit; cancers of the brain and nervous system (brain; and other nervous system areas); cancers of the endocrine system (thyroid and other endocrine areas, including the thymus); lymphomas (Hodgkin's disease and non-Hodgkin's lymphoma), multiple myeloma, and leukemias (lymphocytic leukemias; myeloid leukemias; monocytic leukemias; and other leukemias).

Other cancers classified by histological type that may be suitable targets for the therapeutic compositions of the present invention include, but are not limited to, malignant neoplasms; carcinoma (NOS, not specified); undifferentiated carcinoma (not specified); giant cell and spindle cell carcinoma; small cell carcinoma (not specified); papillary carcinoma (not specified); squamous cell carcinoma (not specified); lymphoepithelial carcinoma; basal cell carcinoma (not specified); pilomatricoma; transitional cell carcinoma (not specified); papillary transitional cell carcinoma; adenocarcinoma (not specified); gastrinoid carcinoma; cholangiocarcinoma; hepatocellular carcinoma (not specified); Mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous polyps in the adenocarcinoma; adenocarcinoma, familial polyposis; solid carcinoma (unspecified); carcinoid tumor (unspecified); bronchioloalveolar adenocarcinoma; papillary adenocarcinoma (unspecified); chromophobe cell carcinoma; oncocytic carcinoma; oncocytic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma (unspecified); granular cell carcinoma; follicular adenocarcinoma (unspecified); papillary and follicular adenocarcinomas; nonencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; cerumenous gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma (unspecified); papillary cystadenocarcinoma (unspecified); papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma (unspecified); mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma (unspecified); lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; malignant androblastoma; Sertoli cell carcinoma; Malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomus sarcoma; malignant melanoma (unspecified); amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma (unspecified); fibrosarcoma (unspecified); malignant fibrous histiocytoma; myxosarcoma; liposarcoma (unspecified); leiomyosarcoma (unspecified); rhabdomyosarcoma (unspecified); embryonal rhabdomyosarcoma Tumor; alveolar rhabdomyosarcoma; stromal sarcoma (unspecified); malignant mixed tumor (unspecified); mixed Müllerian tumor; Wilms' tumor; hepatoblastoma; carcinosarcoma (unspecified); malignant mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma (unspecified); malignant mesothelioma; dysgerminoma; embryonal carcinoma (unspecified); malignant teratoma (unspecified); malignant ovarian goiter; choriocarcinoma; malignant mesonephroblastoma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma Lymphangiosarcoma; Osteosarcoma (unspecified); Juxtacortical osteosarcoma; Chondrosarcoma (unspecified); Malignant chondroblastoma; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Malignant odontogenic tumor; Ameloblastic odontosarcoma; Malignant ameloblastoma; Ameloblastic fibrosarcoma; Malignant pinealoma; Chordoma; Malignant glioma; Ependymoma (unspecified); Astrocytoma (unspecified); Protoplasmic astrocytoma; Fibrillary astrocytoma; Astroblastoma; Glioblastoma (unspecified); Oligoblastoma Glioma (unspecified); Interstitial glioma; Primitive neuroectodermal tumor; Cerebellar sarcoma (unspecified); Ganglioblastoma; Neuroblastoma (unspecified); Retinoblastoma (unspecified); Olfactory neurogenic tumor; Malignant meningioma; Neurofibrosarcoma; Malignant neurilemmoma; Malignant granulocytic tumor; Malignant lymphoma (unspecified); Hodgkin's disease (unspecified); Hodgkin's disease; Paragranuloma (unspecified); Small lymphocytic malignant lymphoma; Diffuse large cell malignant lymphoma; Follicular malignant lymphoma unspecified); mycosis fungoides; other specified non-Hodgkin lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative intestinal disease; leukemia (unspecified); lymphoid leukemia (unspecified); plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia (unspecified); basophilic leukemia; eosinophilic leukemia; monocytic leukemia (unspecified); mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

As defined herein, "epithelial cancer" refers to cancers that form from the epithelium or related tissues of the skin, hollow internal organs, and other organs. Epithelial cancers include, but are not limited to, breast cancer, lung cancer, liver cancer, oral cancer, stomach cancer, colon cancer, nasopharyngeal cancer, skin cancer, kidney cancer, brain tumors, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, intestinal cancer, pancreatic cancer, and bladder cancer.

As used herein, "patient" or "subject" refers to a mammalian subject diagnosed with or suspected of having or developing a proliferative disease, such as cancer. Exemplary patients can be humans, monkeys, dogs, pigs, cows, cats, horses, goats, sheep, rodents, and other mammals that may benefit from developing a proliferative disease, such as cancer.

As used herein, "substantially purified" or "substantially isolated" refers to a molecule (e.g., a compound) that is in a state in which it is separated from substantially all other molecules with which it is normally associated in its natural state. Preferably, the substantially purified molecule is the predominant species present in the preparation. In particular, the substantially purified molecule may be greater than 60% free, preferably 75% free, more preferably 90% free, and most preferably 95% free of other molecules present in the natural mixture (excluding solvent). The term "substantially purified" or "substantially isolated" is not intended to include molecules or substances in their natural state. In certain embodiments, the term "substantially purified" or "substantially isolated" includes purifying one KLH portion from another KLH portion (e.g., substantially purifying or substantially separating a KLH dimer portion from a KLH trimer portion). In another embodiment, the term "substantially purified" or "substantially isolated" does not include purification of one KLH portion from another KLH portion (e.g., including KLH dimer and KLH trimer in a substantially purified or substantially isolated composition), but rather substantial removal of impurities.

"Administering" is referred to herein as providing a therapeutic composition of the present invention to a patient. By way of example and not limitation, administration of the composition, e.g., injection, can be performed by intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be used. Parenteral administration can be performed, for example, by bolus injection or by gradual perfusion over time. Alternatively or concurrently, administration can be performed orally. Additionally, administration can also be performed by surgically inserting a pellet or placing a medical device.

A "patient in need thereof" is referred to herein as a patient diagnosed with or suspected of having a proliferative disorder. In one embodiment, the patient has or is at risk of developing cancer.

As used herein, the term "antigen" is defined as any substance capable of stimulating an immune response with or without the aid of a protein carrier and/or adjuvant. Preferably, the antigen of the composition of the present invention comprises a carbohydrate and more preferably comprises a glycan-antigen and most preferably comprises a Globo H moiety.

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

As used herein, the term "immunotherapy" refers to a range of therapeutic strategies based on the concept of modulating the immune system for prophylactic and/or therapeutic purposes.

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

The "therapeutic composition" of the present invention preferably comprises a "therapeutic conjugate" and/or a "therapeutic antibody". The therapeutic conjugate comprises at least one antigen linked to a carrier. Preferably, the linking of the therapeutic conjugate is covalent. In one embodiment of the therapeutic conjugate, the antigen is a glycan such as a Globo H moiety, and the carrier is a KLH moiety and/or a KLH moiety subunit. Thus, the term "therapeutic conjugate" encompasses one or more KLH moiety subunits linked to one or more Globo H moieties. In one embodiment, the term "therapeutic conjugate" encompasses one or more KLH moieties linked to approximately or at least 1, 10, ^{10 2} or 10 ³ Globo H moieties. In another embodiment, the term "therapeutic conjugate" encompasses a therapeutic conjugate that is conjugated to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 , 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or more Globo H moieties. Another embodiment encompasses isolated dimers, trimers, tetramers, pentamers, or hexamers of such Globo H linked to KLH moiety subunits or combinations thereof.

In one embodiment, the therapeutic conjugate is: Fucα(1→2)Galβ(1→3)GalNAcβ(1→3)Galα(1→4)Galβ(1→4)Gluβ(1-O-ethylhydrazine-1-carbonyl-cyclohexyl-4-(methyl-N-maleimido)-3-(thiobutylimino)-keyhole limpet hemocyanin (KLH), also known as OPT-822.

A "therapeutic antibody" is defined as an antibody (as further defined below) that specifically binds to a therapeutic conjugate of the invention and preferably binds to a portion of the Globo H portion of the therapeutic conjugate.

As used herein, the term "vaccine" refers to a therapeutic composition containing a therapeutic conjugate that is used to confer immunity against diseases associated with antigens. Cancer vaccines are designed to enhance the body's ability to naturally protect itself from the dangers of damaged cells or abnormal cells such as cancer cells with the help of the immune system. A protective immune response is an immune response that reduces the severity of the disease, including but not limited to preventing the disease, delaying the onset of the disease, reducing the severity of symptoms, reducing the incidence rate, and delaying death. Preferably, the vaccine is capable of activating a humoral immune response (e.g., stimulating B lymphocytes to produce antibodies) and a cellular immune response (e.g., an immune response mediated by T lymphocytes and/or other cells, such as NK cells and macrophages). Standard assays for measuring immune responses have been developed, such as enzyme-linked immunosorbent assay (ELISA), flow cytometry, cell proliferation assays, CTL assays, and ADCC/CDC assays.

As used herein, the term "glycan" refers to a polysaccharide or oligosaccharide. Glycans are used herein to refer to the sugar portion of a glycoconjugate (such as a glycoprotein, glycolipid, glycopeptide, glycoprotein group, peptidoglycan, lipopolysaccharide or proteoglycan). Glycans are generally composed entirely of O-glycosidic bonds between monosaccharides. For example, cellulose is a sugar (or more specifically glucan) composed of β-1,4-linked D-glucose, and chitin is a glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can be homopolymers or heteropolymers of monosaccharide residues and can be linear or branched. Glycans linked to proteins can be found, such as in glycoproteins and proteoglycans. They are generally present on the outer surface of cells. O-linked and N-linked glycans are very common in eukaryotes, but can also exist in prokaryotes, but are less common. N-linked glycans exist by being linked to the R-group nitrogen (N) of asparagine in the sequence segment. The sequence stretch is an Asn-X-Ser or Asn-X-Thr sequence, wherein X is any amino acid except proline. A preferred glycan is the Globo H moiety.

Globo H is a hexasaccharide, a member of a family of antigenic carbohydrates that is highly expressed in various types of cancer, particularly breast, prostate, pancreatic, gastric, ovarian, colon, and lung cancers. In illustrative embodiments, certain patients exhibit no anti-Globo H antibody levels at time zero, and high titers are detected after immunization with the therapeutic composition of the present invention. In other illustrative embodiments, certain patients exhibit anti-Globo H antibody levels at time zero, and high titers are detected after immunization with the therapeutic composition of the present invention. In certain embodiments, anti-Globo H antibodies are expressed on the surface of cancer cells as glycolipids and possibly as glycoproteins. In other embodiments, sera from breast cancer patients contain high levels of antibodies against the Globo H epitope. In certain embodiments, this epitope is also targeted by the monoclonal antibodies Mbr1, VK9, and anti-SSEA-3 in immunohistochemical studies. Although certain normal tissues also react with Mbr1, including normal breast, pancreatic, small intestine, and prostate tissue, the prevalence of the antigen in these tissues is limited to the secretory rim, where immune system access is restricted.

"Globo H moiety" is defined as a glycan that is a fragment or analog thereof (i.e., a molecule containing a sugar moiety). Globo H is a glycan containing a hexasaccharide epitope (Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glc) and optionally containing a non-sugar moiety. Its fragments are glycans containing the hexasaccharide epitope fragment and, if applicable, a non-sugar moiety. These oligosaccharides can be prepared by routine methods. (See Huang et al., Proc. Natl. Acad. Sci. USA 103:15-20 (2006)). If desired, they can be linked to a non-sugar moiety. U.S. Patent Application Serial No. 12/485,546 relates to a method for producing antibodies specific for Globo H or a fragment thereof by administering the above-described immune composition to a non-human mammal (e.g., a mouse, rabbit, goat, sheep, or horse) and isolating antibodies that bind to Globo H or a fragment thereof from the mammal.

Analogs of Globo H can be generated using glycan microarrays and include those disclosed in Wang et al., Proc Natl Acad Sci USA. 2008 August 19; 105(33): 11661-11666 and shown in FIG1 .

Globo H analogs preferably bind to antibodies VK-9, Mbr1, and anti-SSEA-3. Preferably, Globo H analogs bind with a specific dissociation constant (K _D,surf ). A Langmuir isotherm can be used to analyze the binding curve to generate the dissociation constant (K _D,surf ) on the surface. Under equilibrium conditions during the incubation period, the average fluorescence (F _obs ) of replicate spots can be described by the following formula:

F _obs = F _max [P]/(K _{D, surf} + [P])

Wherein _Fmax is the maximum fluorescence intensity, a measure of the amount of active sugar on the surface, [P] is the total antibody concentration, and KD _,surf is the equilibrium dissociation constant between the surface sugar and the antibody. As described in Wang et al., in some embodiments, the preferred (KD _,surf ) for Globo H analogs relative to VK-9, Mbr1, and anti-SSEA-3 antibodies described in Wang et al. is at least about or exactly 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, or 1.6 nM.

Keyhole limpet hemocyanin (KLH) is a large, multi-subunit, oxygen-carrying metalloprotein found in the hemolymph of Megathura crenulata. KLH is a heterogeneously glycosylated protein composed of subunits ranging in molecular weight from approximately 350,000 to approximately 390,000, with aggregates ranging in molecular weight from approximately 400 kDa (e.g., KLH monomers) to approximately 8000 kDa (e.g., KLH icosomers). Each domain of a KLH subunit contains two copper atoms that together bind a single oxygen molecule. When oxygen is bound to hemocyanin, the molecule exhibits a striking, transparent, opalescent blue color. In certain embodiments, KLH protein is highly immunogenic in humans, yet remains safe. In certain embodiments, KLH can be purified from the hemolymph of Megathura crenulata through a series of steps that generally include ammonium sulfate precipitation and dialysis, and may involve column chromatography purification to achieve the highest purity. In certain embodiments, KLH purification may also include the removal of endotoxins, although this step may not be necessary, as endotoxins can act as adjuvants for antibody production upon injection. Preferably, a high-quality KLH preparation with a clear opalescent blue color is the best indicator of KLH solubility. In certain embodiments, KLH monomer units assemble into large multimers (decamers or icosomers) with a total molecular weight of approximately 4,000 kDa to 8,000 kDa. A "keyhole limpet hemocyanin portion" or "KLH portion" is defined herein as the KLH1 (SEQ ID NO: 1) and KLH2 (SEQ ID NO: 2) proteins, or proteins substantially identical thereto, or mixtures thereof. In this context, "substantially identical" means that each KLH portion has an amino sequence that is at least, approximately, or exactly 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, or 75% identical to the amino sequence of native, wild-type KLH. In certain embodiments, the KLH of the present invention have enhanced immunogenic activity, particularly enhanced anti-tumor activity. In certain embodiments, the KLH in the compositions of the present invention comprises intact, undegraded subunits of approximately 400,000 molecular weight. In other embodiments, the KLH of the present invention comprises higher-order KLH multimers.

In certain embodiments, the higher-order KLH multimers have a molecular weight of approximately 8-10 million and a sedimentation coefficient of approximately 92-107 s. The amount of higher-order KLH multimers present is determined based on sedimentation-equilibrium and/or sedimentation-velocity ultracentrifugation analysis. In other embodiments, the KLH of the present invention exhibits enhanced immunogenic activity, particularly enhanced anti-tumor activity. Enhanced immunogenic activity is seen, for example and without limitation, (a) when KLH is injected (in the absence of an adjuvant), (b) when KLH is used as an adjuvant, (c) when KLH is used as a carrier immunogen for a hapten or weakly immunogenic antigen, and (d) when KLH is used as an anti-tumor agent. The KLH compositions of the present invention exhibit enhanced anti-tumor activity against a variety of tumors, including, but not limited to, bladder, breast, and ovarian tumors. In certain embodiments, two KLH moieties can form a dimer via a covalent bond between the KLH monomers. Without being limited by theory, it is believed that the covalent bond between the KLH moieties is via a disulfide bond. In certain embodiments, two or more KLH moieties can form a dimer, trimer, tetramer, pentamer, etc. by means of covalent bonds between KLH monomers, dimers, trimers, etc. Without being limited by theory, it is believed that the covalent bonds between the KLH moieties are by means of disulfide bonds.

There are a variety of methods for linking the KLH moiety to the antigen, including direct conjugation and conjugation using a bifunctional linker group such as 4-(4-N-maleimidomethyl)cyclohexane-1-carboxyhydrazide (MMCCH). Such linking techniques are disclosed in U.S. Patent No. 6,544,952. In some embodiments, to prepare the therapeutic conjugates of the present invention, for example, Globo H allyl glycoside is converted to an aldehyde by ozonolysis and the aldehyde group is linked to the NH group on the cross-linker MMCCH to produce Globo H-MMCCH; the carrier protein KLH is thiolated to produce KLH-SH; and the sulfhydryl group on the thiolated KLH is subsequently linked to the maleimide group on MMCCH to produce the Globo H-KLH conjugate.

In one embodiment, Globo H allyl glycoside is prepared by chemical synthesis. The thiolating agent 2-iminothiolane, cGMP-grade KLH, and a 4-(4-N-maleimidomethyl)-cyclohexane-1-carboxyhydrazide (MMCCH) linker are also used. In some embodiments, the following steps are performed: 1) converting Globo H allyl glycoside to Globo H-aldehyde; 2) coupling Globo H-aldehyde with MMCCH to form Globo H-MMCCH; 3) chemical thiolation of KLH; 4) conjugation of Globo H-MMCCH to the thiolated KLH; and 5) purification of the Globo H-KLH conjugate (OPT-822). See, for example, Figure 2a.

In certain embodiments, during conjugation of the Globo H moiety protein to the KLH moiety, the KLH moiety protein, in certain embodiments, exhibits a decrease in molecular weight compared to the intact molecule, which is preferably attributed to dissociation of the Globo H moiety subunits. In other embodiments, the conjugation methods disclosed herein result in previously unreported dissociation of KLH subunits. While not wishing to be bound by any particular theory, it is contemplated that the high glycosylation level of the Globo H moiety-KLH moiety subunit conjugates of the present invention leads to the formation of hydrogen bonds between the Globo H moieties. Thus, in certain embodiments, van der Waals forces and hydrophobic interactions between the KLH moiety subunits are replaced by Globo H hydrogen bonds, and this results in the separation of the KLH moiety subunits. Following conjugation, the KLH moiety subunits of the Globo H moiety-KLH moiety conjugate preferably aggregate to form new monomers, dimers, trimers, tetramers, pentamers, or hexamers, or any combination thereof. The resulting therapeutic Globo H moiety-KLH moiety conjugates possess unexpectedly high epitope ratios, resulting in unexpectedly superior immunogenic properties. In certain embodiments, the Globo H moiety is conjugated to a lysine on KLH1 and KLH2. In other embodiments, the Globo H moiety is not conjugated to a lysine on KLH1 and KLH2. In certain embodiments, the lysine site to which Globo H is conjugated is conserved in peptide mapping analysis, suggesting that the Globo H-KLH composition is unique in its structure.

In one embodiment, a therapeutic composition of the invention comprises one or more KLH moiety subunits, wherein at least one such subunit is conjugated to at least, about, or exactly 1, 10, ^{10 2} , or 10 ^3- fold: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 8, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、1 29, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159 or 160 or more Globo H sections.

Using mass spectrometry analysis, the inventors discovered that the Globo H moiety is conjugated to a lysine residue of KLH. In certain embodiments, it is therefore preferred that the Globo H moiety be conjugated to a lysine residue.

In one embodiment, there are a total of exactly or approximately 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 lysine residues per KLH portion subunit. In another embodiment, there are exactly or approximately 150 or 156 lysine residues per KLH portion subunit. In another embodiment, there are exactly or approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 lysine conjugation sites available for or actually bound to the Globo H portion per KLH portion subunit. In another embodiment, there are 62, 66, 67, 68, 70, 72, 76, 86, 87, 88, 90, 92, 93, 100 such lysine conjugation sites per KLH moiety subunit. Lysine conjugation sites are those lysine residues in the KLH moiety that are available for binding or actually bind to the Globo H moiety and/or to a linker of the Globo H moiety, such as, for example, an MMCCH linker.

In certain therapeutic embodiments containing a mixture of partial subunits (e.g., KLH1 and KLH2 or variants thereof), the total number of available lysines (for both subunits) as counted collectively across the different subunit types can be exactly or approximately 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, or 310. In such embodiments, there are or may be exactly or approximately 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 lysine conjugation sites total across different subunits (e.g., KLH1 and KLH2 or variants thereof). In other such embodiments, there are 136, 137, 141, 140, 143, 147, or 155 lysine conjugation sites.

In a most preferred embodiment, there are 136, 137, 140, 141, 143, 147 or 155 lysine conjugation sites among the total 306 lysine residues in KLH1/KLH2.

In certain embodiments, the therapeutic compositions of the present invention contain a mixture of KLH partial subunit-Globo H portion conjugates, wherein such conjugates remain monomeric or form dimers, trimers, tetramers, pentamers, or hexamers, or any combination thereof. In another embodiment, the therapeutic compositions of the present invention comprise isolated KLH partial subunit-Globo H portion conjugate monomers, dimers, trimers, or tetramers, or any combination thereof. In yet another embodiment, the therapeutic compositions of the present invention comprise only KLH partial subunit-Globo H portion conjugate dimers and trimers.

In another embodiment, the therapeutic composition contains at least two KLH moiety subunits, wherein each of the two KLH moiety subunits is linked to a different glycan. Other tumor-associated glycan antigens that can be linked to the KLH moiety subunits include, but are not limited to, GM2, GD2, GD3, fucosyl, GM1, sTn, sialyl-Lewis ^x , Lewis ^x , sialyl Lewis ^a , Lewis ^a , sTn, TF, polysialic acid, Lewis ^y , mucin, T antigen, and the like. In some embodiments, only, at least, or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the KLH moiety subunits in the therapeutic composition are linked to the Globo H moiety, while the remaining KLH moiety subunits in the therapeutic composition are linked to other tumor-associated glycan antigens.

As used herein, "epitope ratio" in relation to the therapeutic conjugates disclosed herein refers, for example, to the relationship of the antigenic epitopes in the therapeutic conjugate relative to the carrier molecule. Preferably, it refers to the relationship of the Globo H moiety relative to the KLH moiety. Most preferably, the epitope ratio of the therapeutic conjugate is calculated using the following formula: (actual Globo H moiety weight/Globo H moiety molecular weight)/(actual KLH moiety weight/KLH moiety molecular weight). The epitope ratio is readily determinable by one skilled in the art. Preferably, the weight of Globo H is determined, for example, by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

Preferably, the epitope ratios of the therapeutic conjugates of the invention are about, at least, or exactly: 1, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1 550, 1575, 1600, 1625, 1650, 1675, 1700, 1725, 1750, 1775, 1800, 1825, 1850, 1875, 1900, 1925, 1950, 1975, 2000, 2025, 2050, 2075, 2100, 2125, 2150, 2175, 2200, 2225, 2250, 2275 5. 2300, 2325, 2350, 2375, 2400, 2425, 2450, 2475, 2500, 2525, 2550, 2575, 2600, 2625, 2650, 2675, 2700, 2725, 2750, 2775, 2800, 2825, 2850, 2875, 2900, 2925, 2950, 2975 or 3000.

In one embodiment, the therapeutic composition of the present invention comprises a mixture of therapeutic conjugates having a range of epitope ratios. In one embodiment, the range, mean, or median epitope ratio of the therapeutic conjugates in the therapeutic composition is about 10 to about 3200, about 800 to about 2500, about 1000 to about 2000, about 1250 to about 1750, or about 1400 to about 1600. In another embodiment, the range, mean, or median epitope ratio of the therapeutic conjugates in the therapeutic composition is about 10 to about 150, about 40 to about 125, about 50 to about 100, about 62 to about 87, or about 70 to about 80. In another embodiment, the range, mean, or median epitope ratio of the therapeutic conjugates in the therapeutic composition is about 20 to about 300, about 80 to about 250, about 100 to about 200, about 125 to about 175, or about 140 to about 160. In another embodiment, the range, mean, or median epitope ratio of the therapeutic conjugate in the therapeutic composition is about 30 to about 450, about 120 to about 375, about 150 to about 300, about 185 to about 260, or about 210 to about 240. In certain pharmaceutical compositions, at least or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the therapeutic conjugate is present as a monomer, or as a dimer, trimer, tetramer, pentamer, or a combination thereof.

Antibodies against therapeutic conjugates

In certain illustrative embodiments, the present invention also encompasses isolated therapeutic antibodies that specifically bind with high affinity to the therapeutic conjugates disclosed herein, and their use in treating and/or diagnosing proliferative diseases.

As used herein, the terms "antibody" and "antibody" (immunoglobulin) encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab') ₂ fragments, antibody fragments that exhibit the desired biological activity, disulfide-linked Fv (sdFv) and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to antibodies of the invention), intrabodies, and epitope-binding fragments of any of the foregoing. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

The "affinity" of an antibody to an epitope to be used in the therapies described herein (e.g., the Globo H portion of a therapeutic conjugate) is a term well understood in the art and refers to the degree or intensity with which an antibody binds to an epitope. Affinity can be measured and/or expressed in many ways known in the art, including but not limited to the equilibrium dissociation constant (KD or Kd), the apparent equilibrium dissociation constant (KD' or Kd'), and _IC50 (the amount required to achieve 50% inhibition in a competition assay). It will be understood that for the purposes of the present invention, affinity is the average affinity of a given population of antibodies that bind to an epitope. KD' values reported herein as mg IgG per mL or mg/mL represent mg Ig per mL of serum, although plasma may be used. When using antibody affinity as a basis for implementing or selecting a method of treating a disease described herein, antibody affinity can be measured before and/or during treatment, and the values obtained can be used by clinicians to assess whether a human patient is a suitable candidate for treatment.

As used herein, the term "specific binding" refers to the interaction between a binding pair (e.g., an antibody and an antigen). In various cases, specific binding can be embodied by an affinity constant of at least or about 10-6 mol/L, about 10-7 mol/L, or about 10-8 mol/L or less.

Biological assays

In one embodiment, when administered to a patient, a therapeutic composition containing a therapeutic conjugate of the invention is capable of inducing an anti-Globo H antibody titer that is at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 250, 500, 1000, 1500, 2000, 2500, 3000, 4000, or 5000-fold higher than the same anti-Globo H antibody titer prior to administration in the same experiment (i.e., pre-treatment baseline titer). In certain embodiments, the anti-Globo H antibody is an IgM antibody. In another embodiment, the anti-Globo H antibody is an IgG antibody.

The therapeutic composition of the present invention can induce humoral reactions and cellular reactions in subjects. In certain embodiments, the vaccine composition of the present invention induces the production of Globo H partial specific IgG antibodies and IgM antibodies and the expansion of B cells and T cells (such as CD3 ⁺ T cells, CD4 ⁺ T cells and/or CD8 ⁺ T cells). Generally, these immune responses occur in chronological order after administration. In a specific example, after administration, the production of B cells occurs at about the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 20th, 30th or 60th day, and then produces IgG antibodies and IgM antibodies at about the 10th, 20th, 30th, 60th or 90th day and subsequently produces T cells at about the 24th, 30th, 40th, 50th, 60th, 90th, 120th, 150th or 180th day. The vaccine composition of the present invention potentially provides a long-term immune protection that may prevent the growth of a small amount of cancer cells, and is therefore ideal for minimal residual disease, thereby achieving disease stabilization and survival improvement.

"Antibody-dependent cellular cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. In one embodiment, such cells are human cells. Although not wishing to be limited to any particular mechanism of action, these cytotoxic cells that mediate ADCC typically express Fc receptors (FcRs). The primary cells for mediating ADCC, NK cells, express FcγRIII, while monocytes express FCγRI, FCγRII, FcγRIII, and/or FcγRIV. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991). To assess the ADCC activity of a molecule, an in vitro ADCC assay, such as that described in U.S. Patent Nos. 5,500,362 or 5,821,337, can be performed. Effector cells used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the therapeutic conjugates of the invention can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA), 95: 652-656 (1998).

"Complement-dependent cytotoxicity" or "CDC" refers to molecules that initiate complement activation and lyse targets in the presence of complement. The complement activation pathway begins with the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed to a cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santaro et al., J. Immunol. Methods 202:163 (1996).

In another embodiment, when administered to a patient, a therapeutic composition containing a therapeutic conjugate of the invention is capable of inducing the production of anti-Globo H immune sera in the patient/subject that specifically binds to Globo H-positive cancer cell lines (e.g., MCF-7 cells).

combination

The therapeutic composition can include other anticancer/antiproliferative drugs and adjuvants and other immunomodulatory molecules such as cytokines or chemokines. These drugs can be delivered together in a kit in separate containers or a single container. The drugs can be combined at the time of administration or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 minutes, hours or days before administration.

Adjuvants are pharmacological or immunological substances that regulate the effects of other drugs. They can be inorganic or organic chemicals, macromolecules or complete cancer cells or parts thereof that enhance the immune response to a given antigen. Adjuvants include Freund's complete and Freund's incomplete adjuvants, Toll-like receptor molecules and their mimetics, LPS, lipoproteins, lipopeptides, flagella, double-stranded RNA, non-methylated CpG islands, levamisole, Bacillus Calmette-guerin, octreotide, isoprenoid and Zadaxin, various forms of DNA and RNA classically released by bacteria and viruses, PD-1 antagonists and CTLA antagonists. In one embodiment, the adjuvant is a saponin adjuvant.

In certain embodiments, the saponin adjuvant is OPT-821 saponin, which is substantially pure. In other embodiments, OPT-821 saponin is a biologically active fragment thereof. The adjuvant may also encompass impure forms of OPT-821 saponin. Purified OPT-821 saponin exhibits an enhanced adjuvant effect when administered with the vaccines described herein or mixed with other substantially pure saponins or non-saponin adjuvants.

OPT-821 saponin is a naturally occurring glycoside extracted from the Quillaja saponaria Molina tree in high purity by high pressure liquid chromatography (HPLC), low pressure liquid silica gel chromatography, and hydrophilic interaction chromatography (HILIC), as described, for example, in U.S. Pat. No. 5,057,540 and U.S. Pat. No. 6,524,584, the contents of which are incorporated by reference in their entirety. HPLC analysis showed that OPT-821 is a mixture of structurally related isomeric compounds. The purified different isomeric compounds of OPT-821 saponin have been identified and disclosed herein.

In certain embodiments, OPT-821 saponin comprises at least one isolated compound of Formula I as follows:

in

^R1 is β-D-apiose or β-D-xylose; and

^R2 and ^R3 are independently H, alkyl

(the fatty acyl portion of compound 1989), or

(Acyl moiety of compound 1857).

OPT-821 saponin may also comprise an isolated compound of formula I, wherein

(i) ^R1 is β-D-apiose, ^R2 is the fatty acyl moiety of the above-mentioned 1989 compound, and ^R3 is H (1989 compound V1A);

(ii) R1 is β-D-apiose, ^R2 is H, and ^R3 is the fatty acyl moiety of the above-mentioned 1989 compound (1989 compound V1B);

(iii) R ¹ is β-D-xylose, R ² is the fatty acyl moiety of the above-mentioned 1989 compound, and R ³ is H (1989 compound V2A); or

(iv) ^R1 is β-D-xylose, ^R2 is H, and ^R3 is the fatty acyl moiety of the above-described 1989 compound (1989 Compound V2B). Collectively, 1989 Compound V1A, 1989 Compound V1B, 1989 Compound V2A, and 1989 Compound V2B are referred to as the "1989 Compound Mixture."

Table 1 summarizes the functional groups of the 1989 compounds and the mole % of each 1857 compound in the 1857 compound mixture.

Table 1

OPT-821 saponin may comprise an isolated compound of formula I, wherein

(i) R ¹ is β-D-apiose, R ² is the fatty acyl moiety of the above-mentioned 1857 compound, and R ³ is H (1857 compound V1A);

(ii) R ¹ is β-D-apiose, R ² is H, and R ³ is the fatty acyl moiety of the above-mentioned 1857 compound (1857 compound V1B);

(iii) R ¹ is β-D-xylose, R ² is the fatty acyl moiety of the above-mentioned 1857 compound, and R ³ is H (1857 compound V2A); or

(iv) ^R1 is β-D-xylose, ^R2 is H, and ^R3 is the fatty acyl moiety of the above-described 1857 compound (1857 Compound V2B). Collectively, 1857 Compound V1A, 1857 Compound V1B, 1857 Compound V2A, and 1857 Compound V2B are referred to as the "1857 Compound Mixture."

Table 2 summarizes the functional groups of the 1857 compounds and the mole % of each 1857 compound in the 1857 compound mixture. HPLC

Table 2

OPT-821 Saponin contains one or more of the following compounds:

(i) 1857 compound V1A; (ii) 1857 compound V1B;

(ii) 1857 compound V2A;

(iii) 1857 compound V2B;

(iv) 1989 compound V1A;

(v) 1989 compound V1B;

(vi) 1989 Compound V2A; or

(vii) 1989 Compound V2B.

The percentages of the mixture of 1857 compounds and the mixture of 1989 compounds in OPT-821 saponin can be as follows:

(i) from about 1 mol % to about 15 mol % of OPT-821 of a mixture comprising 1857 compounds; and

(ii) from about 85 mole % to about 99 mole % of OPT-821 of a mixture comprising 1989 compounds.

All mole % can vary in 0.1% increments (eg, about 87% to about 90%, about 90.5% to about 97%, about 3.5% to about 11%, about 10% to about 14%).

The 1989 compound mixture can contain about 60-70 mole percent 1989 Compound V1A; about 1-5 mole percent 1989 Compound V1B; about 30-40 mole percent 1989 Compound V2A; and about 0.1-3 mole percent 1989 Compound V2B. The total mole percent can be varied in 0.1 increments (e.g., 65%, 2.5%, 35.6%).

The 1857 compound mixture can contain about 60-70 mol% of 1857 Compound V1A; about 1-5 mol% of 1857 Compound V1B; about 30-40 mol% of 1857 Compound V2A; and about 0.1-3 mol% of 1857 Compound V2B. The total mol% can be varied in 0.1 increments (e.g., 65%, 2.5%, 35.6%).

In another embodiment, substantially pure OPT-821 is purified from a crude extract of Quillaja saponaria, wherein the OPT-821 is characterized by a single predominant peak occupying 90% or more of the total area of all peaks in the chromatogram (excluding the solvent peak) when analyzed on a Symmetry C18 column having a 5 μm particle size, pores, 4.6 mm ID x 25 cm L by reverse phase-HPLC, wherein the elution program comprises mobile phase A:B 95%:5% to 75%:25% over 11 minutes, wherein mobile phase A is distilled water with 0.1% trifluoroacetic acid and mobile phase B is acetonitrile with 0.1% trifluoroacetic acid at a flow rate of 1 ml/min.

In one embodiment, the pharmaceutical composition comprises a compound of formula (I)

in,

^R1 is β-D-apiose or β-D-xylose; and

^R2 and ^R3 are independently H, alkyl or

and a pharmaceutically acceptable carrier.

The vaccine may comprise a saccharide antigen or an immunogenic fragment thereof and OPT-821 saponin. In yet another embodiment, the vaccine comprises a saccharide antigen or an immunogenic fragment thereof; a carrier protein; and OPT-821 saponin. In another embodiment, the vaccine comprises a saccharide antigen selected from Globo H, KLH, and OPT-821 saponin. Non-limiting examples of carrier proteins include KLH.

As used herein, the term "cytokine" refers to any of a number of secreted small proteins that regulate the intensity and duration of immune responses by affecting the immune cell differentiation process, which generally involves changes in gene expression by precursor cells that transform into different specialized cell types. Cytokines have been named lymphokines, interleukins, and chemokines based on their assumed functions, secretory cells, or targets. For example, some common interleukins include, but are not limited to, IL-2, IL-12, IL-18, IFN-γ, TNF, IL-4, IL-10, IL-13, IL-21, GM-CSF, and TGF-β.

As used herein, the term "chemokine" refers to any of a variety of small chemotactic cytokines that are provided in lymphocyte mobilization and activation means and are released at the site of infection. Chemokines attract leukocytes to the site of infection. Chemokines have conserved cysteine residues that allow them to be divided into four groups. These groups, together with representative chemokines, are C-C chemokines (RANTES, MCP-1, MIP-1α and MIP-1β), C-X-C chemokines (IL-8), C chemokines (lymphocyte chemoattractant factors) and CXXXC chemokines (fractalin).

The therapeutic composition of the present invention may also include PD-1/PD-L1 inhibitors (cytotoxic T lymphocyte (CTL) immunotherapeutics), CTLA-4 immunotherapeutics, CDK4/6 inhibitors (targeted therapeutics), PI3K inhibitors (targeted therapeutics), mTOR inhibitors (targeted therapeutics), AKT inhibitors (targeted therapeutics), and pan-Her inhibitors (targeted therapeutics). These inhibitors may also be modified to produce corresponding monoclonal antibodies. Such antibodies may be included in the therapeutic composition of the present invention.

The therapeutic composition may include other anticancer/antiproliferative drugs or chemotherapeutics. In some embodiments, examples of such drugs are found in V.T. Devita and S. Hellman (eds.), Cancer Principles and Practice of Oncology, 6th edition (February 15, 2001), Lippincott Williams & Wilkins Publishers. Such anticancer drugs include, but are not limited to, the following: hormone therapy drugs (e.g., selective estrogen receptor modulators, androgen receptor modulators), monoclonal antibody therapy drugs, chemotherapeutics, retinoid receptor modulators, cytotoxic/cytostatic drugs, antitumor drugs, antiproliferative drugs, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, nitrogen mustards, nitrosoureas, angiogenesis inhibitors (e.g., bevacizumab), cell proliferation and survival signaling pathway inhibitors, apoptosis inducers, drugs that interfere with cell cycle checkpoints, drugs that interfere with receptor tyrosine kinases drugs that can be used to treat anemia, drugs that can be used to treat neutropenia, immune-enhancing drugs, bisphosphonates, aromatase inhibitors, drugs that induce terminal differentiation of tumor cells, gamma-secretase inhibitors, cancer vaccines, and any combination thereof.

Preparations of the present invention

Therapeutic compositions (also referred to herein as pharmaceutical compositions) typically include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Supplementary active compounds may also be incorporated into the composition. The pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, e.g., intravenous, intradermal, subcutaneous, intramuscular, intraarterial, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: a sterile diluent such as water for injection, saline solution, phosphate-buffered saline, tris-buffered saline, fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; complexing agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and substances for adjusting tonicity such as sodium chloride or dextrose. The pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral formulation may be placed in ampoules, disposable syringes or multiple-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable applications include sterile aqueous solutions (which are water-soluble) or dispersions and sterile powders for preparing sterile injection solutions or dispersants on site. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be kept flowing to a degree that allows easy injectability. It should be stable under manufacturing and storage conditions and should be protected from the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium, which, for example, includes water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions and by using a surfactant. Various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc., can be used to prevent the effect of microorganisms. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent along with one or a combination of ingredients listed above, as needed, followed by sterilization by filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and the desired other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze drying to produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof.

Oral compositions typically include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and provided in the form of a tablet, lozenge, or capsule (e.g., gelatin capsule). Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. A pharmaceutically compatible binder or auxiliary material can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. can contain any of the following ingredients or compounds with similar properties: a binder such as microcrystalline cellulose, tragacanth gum, or gelatin; an excipient such as starch or lactose, a disintegrant such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterote; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring such as peppermint, methyl salicylate, or an orange flavoring.

For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be transmucosal or transdermal administration.For transmucosal or transdermal administration, use a penetrant suitable for the barrier to be penetrated in the preparation. This type of penetrant is generally known in the art, and for example, for transmucosal administration, includes detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be completed by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels or creams, as known in the art. Compound can also be formulated for rectal delivery with suppositories (for example, with conventional suppository bases such as cocoa butter and other glycerides) or retention enema forms.

According to an embodiment, the active compound is prepared together with a carrier that will protect the compound from being quickly eliminated from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. The method for preparing such preparations will be apparent to those skilled in the art. These materials can also be commercially available. Liposomal suspensions (comprising liposomes targeted to infected cells for cell-specific antigen monoclonal antibodies) can also be used as pharmaceutically acceptable carriers. These suspensions can be prepared according to methods known to those skilled in the art (for example, as described in U.S. Patent number 4,522,811), which are incorporated herein by reference.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Dosage form

Toxicity and therapeutic efficacy of such therapeutic compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the _LD50 (the dose lethal to 50% of the population) and the _ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the _LD50 / _ED50 ratio. Therapeutic compositions that exhibit high therapeutic indices are preferred. Although compounds that exhibit toxic side effects can be used, care should be taken to design delivery systems that direct such compounds to the site of disease to minimize potential damage to uninfected cells and thereby reduce side effects.

The data obtained from cell culture assays and animal studies can be used when formulating dosage ranges for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the _ED50 in the presence of low or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration used. For any compound used in the disclosed methods, a preliminary estimate of the therapeutically effective dose can be made from cell culture assays. A dose can be formulated in an animal model to achieve a circulating plasma concentration range that includes the _IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms), as determined in cell culture. This information can be used to more accurately determine dosages for use in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In some embodiments, a therapeutically effective amount (i.e., an effective dose) of the therapeutic composition can be about 0.001 μg/kg to about 250 g/kg, 0.01 μg/kg to 10 g/kg, or 0.1 μg/kg to 1 g/kg, or about or at least: 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009 , 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2 5, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225 or 250 grams or micrograms per kilogram of patient body weight, or other ranges that will be apparent and understood by the skilled artisan without undue experimentation. The skilled artisan will understand that certain factors may influence the dose and time course required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatments, the overall health or age of the subject, and the presence of other diseases.

In other embodiments, the therapeutically effective amount (i.e., effective dose) of the Globo-H moiety in the therapeutic composition can be about 0.001 μg/kg to about 250 g/kg, 0.01 μg/kg to 10 g/kg, or 0.1 μg/kg to 1 g/kg, or about or at least: 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.010, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.020, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.030, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.040, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.050, 0.051, 0.052, 0.053, 0.054, 0.055, 0.056, 0.057, 0.058, 0.059, 0.061, 0.06 8, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09; 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, , 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225 or 250 grams or micrograms per kilogram of patient body weight, or other ranges that will be apparent and understood by the skilled artisan without undue experimentation. The skilled artisan will understand that certain factors may influence the dose and time course required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatments, the overall health or age of the subject, and the presence of other diseases.

In certain embodiments, the therapeutic compositions disclosed herein contain or are associated with at least one therapeutic conjugate or therapeutic antibody such that each of the at least one therapeutic conjugate or therapeutic antibody is present at a concentration of about, at least, or greater than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 times the dose of 10 Preferably, the therapeutic conjugate is present at a concentration of between about ^1-100 , ^2-60 , ^3-50 , ^4-40 , ^5-30 , ^6-20 , ^7-15 , ^8-10 , 2-18, 3-16, ^{4-14, 5-12} , ^6-10 , or 7-8 μM in a single administration.

In some embodiments, the therapeutic compositions disclosed herein contain or are associated with at least one therapeutic conjugate or therapeutic antibody such that each of the at least one therapeutic conjugate or therapeutic antibody is present at a concentration of about, at least, or greater than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, ⁷⁰ , 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 times 10 ^-3 , 10 -2 , 10 ^-1 or 10 micrograms. In certain embodiments, each dose comprises about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 micrograms or more of a therapeutic conjugate or therapeutic antibody.

In certain embodiments, the therapeutic compositions disclosed herein are administered in a dose of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 times daily, weekly, or monthly, or more, over a period of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days, weeks, months, or years, or longer.

Reagent test kit

According to another aspect, one or more kits of parts can be conceived by those skilled in the art, wherein the kit of parts will implement at least one method disclosed herein, and the kit of parts comprises one or more therapeutic conjugates, anticancer drugs/antiproliferative drugs, adjuvants, cytokines and/or chemokines. According to at least one method mentioned above, the therapeutic composition comprises an effective amount of the therapeutic composition disclosed herein, alone or in combination. The aforementioned drugs can be present in a single container or in different containers in the kit.

The kit may also contain an identifier of a biological event or other compound recognizable by a skilled artisan upon reading this disclosure. The kit may also contain at least one composition comprising an effective amount of a therapeutic composition disclosed herein. The therapeutic composition of the kit will implement at least one method disclosed herein according to a procedure recognizable to a skilled artisan.

The disclosure also includes methods of treating a proliferative disease using the therapeutic compositions disclosed herein. In one embodiment, the methods involve treating cancer, e.g., breast cancer. The methods generally involve providing a therapeutic composition disclosed herein to a patient in need thereof in an amount effective to treat the proliferative disorder.

In some embodiments, the therapeutic compositions of the invention are administered to a subject in need thereof (e.g., a subject having cancer, such as breast cancer) in a method that prolongs, on average, progression-free survival or overall survival over that of a control placebo (e.g., phosphate-buffered saline placebo) by about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 days, weeks, months, or years.

In some embodiments, the therapeutic composition is administered subcutaneously at weeks 0-2, 6, 14, and 26 in the absence of unacceptable toxicity or disease progression.

In some embodiments, a therapeutic composition of the invention is administered to a subject in need thereof (e.g., a subject having cancer, such as breast cancer) in a method that provides, on average, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 5, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 days, weeks, months or years to reduce tumor volume by about or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, ... 4%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 74%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

In some embodiments, tumor volume can be accurately measured in at least one dimension (the longest diameter in the measurement plane will be recorded), where the minimum dimension of a CT scan is 10 mm (CT scan slice thickness is recommended to be between 2.5 mm and 5 mm).

Methods for synthesizing the compositions of the present invention

The Globo H hexasaccharide portion of the therapeutic composition of the present invention is chemically synthesized as an allyl glycoside and subsequently prepared for conjugation to KLH.

In an illustrative embodiment, the chemical synthesis of Globo H involves the following general steps:

KLH was treated with 2-iminothiolane in aqueous buffer. The thiolated KLH was then separated from the unreacted 2-iminothiolane by size exclusion on a Sephadex G-15 column. The thiolated KLH was stored under an inert gas (nitrogen or argon) atmosphere and used immediately for conjugation with Globo HMMCCH.

Example

Example 1: Preparation of the glycoconjugate of the present invention (Globo H-KLH)

Globo H allyl glycoside (commercially available) is converted to an aldehyde by ozonolysis. Globo H aldehyde is reacted with M ₂ C ₂ H (linker) and NaCNBH ₃ to produce Globo H-MMCCH. The mixture is purified using a column that receives Globo H-MMCCH. Fractions positive for Globo H-MMCCH are confirmed by high-performance liquid chromatography (HPLC) and then pooled. KLH is dissolved in thiolation buffer and 2-iminothiolane is added to the reaction portionwise. The reaction is incubated to completion and KLH-SH is then purified by column. Globo H-MMCCH and KLH-SH are combined. The reaction is stirred until completion. Globo H-KLH is then purified to provide the final product.

Example 2: Analysis of the Weight Ratio of Globo H to KLH in Glycoconjugates

The weight ratio of Globo H to KLH in the glycoconjugate prepared as above was confirmed by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The results are shown in Table 3.

Table 3: Weight ratio of Globo H to KLH in glycoconjugates

Example 3: Analysis of the epitope ratio of Globo H to KLH in glycoconjugates

The molecular weight of the KLH 20-mer (native aggregated form) is approximately 7.5 to 8.6 MDa, as described in the literature, such as Micron 30 (1999) 597-623. Size exclusion chromatography and multi-angle laser light scattering spectrometry (MALS) confirmed that native KLH had a molecular weight of approximately 8.6 MDa (see Figures 3 and 4). Glycoconjugates of the present invention (sample No. 5, Globo H to KLH weight ratio of 0.17:1) were analyzed by size exclusion chromatography. The results showed that the glycoconjugates of the present invention exhibited a reduced molecular weight compared to the native aggregated KLH 20-mer. See Figure 4. The molecular ratios were then calculated as shown in Table 4.

Table 4: Calculation of the molecular ratio of Globo H to KLH

*The above molecular ratios are calculated based on the following formula:

**KLH molecular weight depends on whether it is in monomeric, dimer or icosamer form

In view of the above, it is concluded that in the glycoconjugates of the present invention, KLH monomer subunits form monomers, dimers and/or trimers after conjugation to Globo H.

Example 4: Preparation of vaccine composition and immunization in rats

Different samples of the glycoconjugate (Globo H-KLH) prepared as in Example 1 were stored at 4°C and mixed with saponin adjuvant under a laminar flow cabinet. The resulting vaccine composition was placed on ice and transported to the animal facility for subsequent immunization.

Three groups of Lewis rats were immunized with various vaccine compositions as shown in Table 5.

Table 5: Grouping of immunized rats

*GH:KLH＝0.17:1(w/w)

GH: Globo H

SC: subcutaneous

PBS: Phosphate buffered saline

Rats were immunized on days 0, 7, 14, and 21. Peripheral blood mononuclear cells (PBMCs) and plasma were collected on days 0, 3, 10, 17, 24, and 31. Spleens, lymph nodes, and peritoneal washes were harvested on day 31.

Example 5: Assays for the induction of humoral and cellular immune responses in rats

Example 5.1 Analysis of immune effector cell subsets by flow cytometry

Peripheral blood mononuclear cells (PBMCs) were isolated from the animals and various immune effector cell subsets in the PBMCs were subsequently identified by flow cytometry using specific antibodies against different cell markers. PBMCs isolated from immunized rats on days 0, 3, 10, 17, 24, and 31 were stained with different fluorescent (FITC)-conjugated antibodies and placed on ice for 30 minutes. After incubation, the cells were washed with wash buffer (1% bovine serum albumin (Sigma) and 0.1% _NaN3 (Sigma) in phosphate-buffered saline (UniRegion Biotech)) and centrifuged at 350 g for 5 minutes. The cells were resuspended in wash buffer for fluorescence measurement by FACS Canto (BD Bioscience). The data were analyzed using BD FACSDiva (BD Bioscience) software. The results showed that in rats immunized with the glycoconjugates of the present invention, T cells, B cells, CD4+ T cells, and CD8+ T cells were significantly expanded compared to the PBS control group. Specifically, the B cell population first appeared on day 3 and was followed by CD3 ⁺ T cells, CD4+ T cells, and CD8 ⁺ T cells. T cells and CD8+ T cells appeared on day 24. See Figure 5(A)-(D).

Example 5.2 Testing for Globo H-specific antibodies by ELISA

The production of Globo H-specific antibodies in the plasma of immunized rats was determined by ELISA. The results showed that the titer of Globo H-specific IgG began to rise on day 10 after immunization and reached a peak on day 17. A similar pattern of Globo H-specific IgM antibody production was observed. See Figures 5(A)-(B). There was no Globo H-specific IgG and IgM antibody response in rats treated with PBS or KLH alone as an adjuvant (data not shown).

In summary, in immunized rats, the production of B cells appeared on day 3, followed by the production of IgM and IgG antibodies against Globo H, which appeared on day 10, and followed by the appearance of CD3 ⁺ T cells, CD4 ⁺ T cells, and CD8 ⁺ T cells on day 24. The glycoconjugate of the present invention (Globo H-KLH) effectively induced humoral and cellular responses.

Example 6: Immunization in mice and detection of antibodies by ELISA

Different samples of the glycoconjugate (Globo H-KLH) were stored at 4°C and mixed with saponin adjuvant under a laminar flow cabinet. The resulting vaccine composition was placed on ice and transported to the animal facility for subsequent immunization.

Approximately 8-week-old Balb/c mice were administered Globo H-KLH at varying ratios of carbohydrate to protein (Globo H:KLH) via subcutaneous injection once weekly for 4 weeks (days 0, 7, 14, and 21). Blood samples were collected from the retroorbital or facial vein without anticoagulants before immunization or on day 0 and 3 days after each vaccination (days 10, 17, and 24). Samples were then centrifuged to separate serum and red blood cells. Serum was collected and stored at -20°C and subsequently analyzed by ELISA. The Mann-Whitneyt test was used for statistical analysis.

As shown in Figure 6, the glycoconjugate of the present invention (Globo H-KLH) combined with a saponin adjuvant has been shown to significantly induce Globo H-specific IgM antibody responses in an animal model compared to a PBS control group. Specifically, the glycoconjugate at a weight ratio of 0.17:1 (Globo H:KLH) induced better antibody titers than the glycoconjugate at a weight ratio of 0.07:1 (Globo H:KLH).

In summary, it has been shown that the glycoconjugate of the present invention (Globo H-KLH) in combination with a suitable adjuvant induces unexpectedly superior humoral and cellular immune responses in animal models, in particular inducing expansion of B cells and T cells (including CTLs) as well as IgM and IgG responses, which are important in cancer immunotherapy.

Example 7: Immunogenicity study of Globo H-KLH vaccine with or without adjuvant in mice

The ability of the Globo H compositions of the present invention to elicit an immune response in mice when paired with an adjuvant has been investigated. The amount of Globo-H specific antibodies induced by immunotherapy was quantified by ELISA and FACS.

Groups of 6-week-old female C57BL/6 mice were immunized subcutaneously with Globo H-KLH and adjuvant. Globo H-KLH dose levels were equivalent to the amount of Globo-H in Globo H-KLH (in μg). Each injection contained a range of doses equivalent to 0.6 μg to 5.0 μg Globo-H and 20 μg adjuvant in Globo H-KLH. Immunizations were performed on days 0, 8, and 14, and serum was collected on days 0 (before injection) and 24 for comparative analysis by ELISA and FACS. Hematological responses were measured by ELISA to determine the titer of antibodies against Globo H ceramide and by FACS to determine cell surface reactivity against Globo H-positive MCF-7 cells.

Throughout the immunization period, there were no noticeable changes in behavior, appetite, overall appearance, or grooming habits following vaccination. Ten days after the third immunization, sera were collected to determine IgG and IgM anti-Globo H antibody titers by ELISA, using titers ≥8 times pre-treatment values as the criterion for a positive reaction. As shown in Figure 2, no response was observed in mice treated with Globo H alone, Globo H KLH conjugate, or adjuvant alone. In contrast, 14/15 mice treated with Globo H-KLH plus adjuvant responded with significant IgG anti-Globo H titers that were dose-independent across doses of Globo H-KLH from 0.6 to 5 μg. The mean IgG titer for each dose increased 13- to 17.5-fold compared to pre-treatment values. Regarding IgM anti-Globo H, one of 15 mice exhibited an 8-fold increase in titer following immunization with adjuvant plus Globo H-KLH. However, 5/15 mice had IgM titers ≥4 times those of pre-immune sera, and a total of 6/15 mice had titers ≥2 times those of pre-immune sera. The mean IgM titer increased 2.5-fold at each dose compared to pre-treatment values.

At a 1:25 serum dilution, the ability of immune sera to bind to the Globo H-expressing MCF-7 breast cancer cell line was determined by FACS analysis. Post-treatment values that were 30% greater than pre-treatment values (i.e., a 1.3-fold increase or greater) were considered positive in this assay. As shown in Figure 3, immune sera from all adjuvant + Globo H-KLH-treated groups contained IgM antibodies that reacted with MCF-7 cells, with the reactions being 5-6-fold above the pre-treatment baseline. In addition, immune sera from 3/5 mice treated with 0.6 μg or 2 μg Globo H-KLH + adjuvant and from 2/5 mice treated with 5 μg Globo H-KLH + adjuvant showed IgG antibodies that could bind to MCF-7 cells. The average binding capacity increased by 1.3 to 2.0-fold over the pre-treatment baseline.

Vaccination with Globo H-KLH and adjuvant has been shown to elicit IgG anti-Globo H and IgM anti-Globo H responses in female C57BL/6 mice. The immune serum has the ability to bind to Globo H-expressing breast cancer MCF-7 cells.

Example 8: LC-MS/MS Analysis of Globo-H Conjugation Sites on KLH

Globo H conjugation sites were identified in four KLH samples using multi-enzyme digestion and LC-MS/MS. These four Globo H-conjugated KLH samples were first digested with four different enzymes and then analyzed by LC-MS/MS and Mascot database search. Two types of derivatives were identified: Globo H derivatives (Globo H + MMCCH) and MMCCH derivatives (MMCCH alone). Globo H derivatives and their neutral loss forms were considered for Globo H conjugation site identification. MMCCH formation and its deamidated form were considered for MMCCH conjugation site identification. Only peptides with high-quality MS/MS spectra and high Mascot scores were considered. For Globo H conjugation analysis, 31 and 28 conjugated lysines were observed from two replicate LC-MS/MS analyses of OPT-822-13001-DP (sample 1); 19 and 21 conjugated lysines were observed from two replicate LC-MS/MS analyses of OPT-822-13002-DP (sample 2); 10 and 11 conjugated lysines were observed from two replicate LC-MS/MS analyses of OPT-822-13003-DP (sample 3); and 18 and 19 conjugated lysines were observed from two replicate LC-MS/MS analyses of OPT-822-13004-DS (sample 4). For MMCCH conjugation analysis, 155 and 141 conjugated lysines were found from two replicate LC-MS/MS analyses of OPT-822-13001-DP (sample 1); 143 and 137 conjugated lysines were found from two replicate LC-MS/MS analyses of OPT-822-13002-DP (sample 2); 147 and 143 conjugated lysines were found from two replicate LC-MS/MS analyses of OPT-822-13003-DP (sample 3); and 140 and 136 conjugated lysines were found from two replicate LC-MS/MS analyses of OPT-822-13004-DS (sample 4).

Example 8 Materials and Methods:

Abbreviations in this section are as follows: K = lysine; LC-MS/MS = liquid chromatography-tandem mass spectrometry; DTT = dithiothreitol; IAM = iodoacetamide; ACN = acetonitrile; FA = formic acid; Glu-C = endoproteinase Glu-C; ABC = ammonium bicarbonate; RT = room temperature; MW = molecular weight.

Four KLH samples, samples 1-4, were first processed by buffer exchange into 50 mM ammonium bicarbonate buffer using a 100 kDa cutoff Amicon Ultra centrifugal filter and denaturation with 6 M urea. The samples were then reduced with 10 mM DTT for 1 hour at 37°C, alkylated with 50 mM IAM for 30 minutes in the dark at room temperature, and quenched with 50 mM DTT for 5 minutes at room temperature. The resulting protein was diluted to a 1 M urea concentration and digested in solution with various enzymes as described in the following sections.

In-solution digestion with different enzymes was performed using the following digestion conditions: (1) trypsin digestion at 37°C for 24 hours (protein:enzyme = 40:1); (2) Glu-C digestion at 37°C for 24 hours (protein:enzyme 25:1); (3) chymotrypsin digestion at room temperature for 24 hours (protein:enzyme = 25:1); (4) thermolysin digestion at 37°C for 24 hours (protein:enzyme = 25:1).

The digestion reaction was stopped by adding formic acid and all four digested samples were subjected to LC-MS/MS analysis.

The samples were analyzed using a Q Exactive mass spectrometer (Thermo Scientific) coupled to an Ultimate 3000 RSLC system (Dionex). LC separations were performed using a C18 column (Acclaim PepMap RSLC, 75 μm x 150 mm, 2 μm) with the gradient shown below:

Mobile phase A: 5% CAN/0.1% FA

Mobile phase B: 95% ACN/0.1% FA

The in-source CID was set at 45 eV. A full MS scan was performed over the m/z range of 350-2000, and the ten most intense ions from the MS scan were fragmented for MS/MS spectra. The raw data were processed by Proteome Discoverer 1.4 into peak lists for Mascot database searches.

Database searches were performed using Mascot version 2.4.1 and Thermo Proteome Discoverer version 1.4 for KLH1 and KLH2 [KLH1, EMBL accession number CAG28307.1; KLH2, EMBL accession number CAG28308.1]. The following parameters were used: Enzymes: trypsin, Glu-C, chymotrypsin, and thermolysin, depending on the digestion method; Fixed modification: carbamidomethyl (C).

Variable modifications of MMCCH derivatives (MMCCH alone): deamidation (NQ), oxidation (M), dK_MMCCH-1 (K), dK_MMCCH-2 (K).

Variable modifications of Globo H derivatives (Globo H+MMCCH): Deamidation (NQ), Oxidation (M), Globo_H_MMCCH (K), dK_MMCCH_NL997 (K), dK_MMCCH_NL835 (K), dK_MMCCH_NL673 (K), dK_MMCCH_NL511 (K), dK_MMCCH_NL308 (K), Deamidation (NQ), Oxidation (M), Peptide mass tolerance: ±10 ppm; Fragment mass tolerance: ±0.05 Da; Maximum missed cleavage: 5; Instrument type: ESI-TRAP; Ion cutoff score: 13.

"dK_MMCCH-1" in the search parameters indicates MMCCH-conjugated lysine with an increased MW of 352.1569 Da.

"dK_MMCCH-2" in the search parameters indicates the deamidated form of MMCCH-conjugated lysine with an increased MW of 338.1300 Da.

"Globo_H_MMCCH(K)" in the search parameters indicates Globo H-conjugated lysine with an increased MW of 1393.5317 Da.

"dK_MMCCH_NL997" in the search parameters indicates the neutral loss form of Globo H-conjugated lysine with an increased MW of 396.1831 Da.

"dK_MMCCH_NL835" in the search parameters indicates the neutral loss form of Globo H-conjugated lysine with an increased MW of 558.2360 Da.

"dK_MMCCH_NL873" in the search parameters indicates the neutral loss form of Globo H-conjugated lysine with an increased MW of 720.2888 Da.

"dK_MMCCH_NL511" in the search parameters indicates the neutral loss form of Globo H-conjugated lysine with an increased MW of 882.3416 Da.

"dK_MMCCH_NL308" in the search parameters indicates the neutral loss form of Globo H-conjugated lysine with an increased MW of 1085.4210 Da.

"MW increase" indicates an increase in molecular weight compared to an intact lysine residue.

Example 8: Results

LC-MS/MS-based techniques are tools for identifying proteins and characterizing amino acid modifications. Detailed information about peptide sequences and modification sites can be obtained by assigning fragment ions from MS/MS spectra. Mascot is a search engine with a well-established probability-based scoring algorithm. In this study, Mascot scores were used as a confidence reference for protein sequencing and identification of Globo H or MMCCH conjugation sites. To fully analyze the distribution of Globo H or MMCCH conjugation sites in samples 1-4, these samples were digested with various enzymes and subsequently subjected to LC-MS/MS analysis.

The expected chemical structure of the Globo H derivative (Globo H + MMCCH) is shown in FIG33A , and a corresponding MW increase of 1393.53 Da can be observed on the lysine-containing peptide in these results. In addition, unstable polysaccharides tend to be shed by neutral loss during electrospray ionization in LC-MS analysis. Therefore, molecular weight increases of 396.18 Da, 558.24 Da, 720.29 Da, 882.34 Da, and 1085.42 Da due to neutral loss can also be observed for the glycoconjugate peptide, as shown in FIG33B . To identify the Globo H conjugation site, all derivatized forms were considered.

In addition, (MMCCH alone) to MMCCH derivatives were also observed in these Globo H-conjugated KLH samples. The expected chemical structures of the MMCCH derivatives and their deamidated forms are shown in Figures 34A and 34B, and the corresponding MW increases of 352.16 Da and 338.13 Da, respectively, on the lysine-containing peptides can be observed in these results. To identify the MMCCH conjugation site, two derivatization forms were considered. The conversion from the Globo H derivative to MMCCH is unclear, but it is assumed to occur during sample processing.

Precursor ion mass accuracy of ±10 ppm and fragment ion mass accuracy of ±0.05 Da were used as criteria for protein identification and spectral interpretation. Globo H derivatives and their neutral loss forms were selected as variable modifications for Globo H conjugation site identification, and MMCCH derivatives and their deamidated forms were selected as variable modifications for MMCCH conjugation site identification. A database search was performed on the sponsor-provided sequences of KLH1 and KLH2. Only peptides with high-quality MS/MS spectra (ion score ≥13, p < 0.05) were reported.

To demonstrate reproducible results, LC-MS/MS analysis was performed twice, followed by independent Mascot database searches for the Globo H conjugation site identification results and the MMCCH conjugation site identification results, as summarized in FIG17 .

In the Globo H conjugation site analysis, 31 and 28 lysines were identified in the first and second LC-MS/MS runs of sample 1, respectively; 19 and 21 lysines were identified in the first and second LC-MS/MS runs of sample 2, respectively; 10 and 11 lysines were identified in the first and second LC-MS/MS runs of sample 3, respectively; and 18 and 19 lysines were identified in the first and second LC-MS/MS runs of sample 4. Details of the identification are listed in Figures 14-21. In the MMCCH conjugation site analysis, 155 and 141 lysines were found in the first and second LC-MS/MS runs of sample 1, respectively; 143 and 137 lysines were found in the first and second LC-MS/MS runs of sample 2, respectively; 147 and 143 lysines were found in the first and second LC-MS/MS runs of sample 3, respectively; and 140 and 136 lysines were found in the first and second LC-MS/MS runs of sample 4. Details of the identification are listed in Figures 22-29.

The Globo H conjugation sites from the multienzyme experiment are summarized in Figure 30 and the MMCCH conjugation sites are summarized in Figure 31. The overall results of the conjugation site analysis of the Globo H conjugated samples are summarized in Figure 32.

Example 8 Conclusion:

The mass spectrometry signals for Globo H-derivatized conjugated peptides were lower than those for MMCCH-conjugated peptides due to multiple neutral loss forms and the lower ionization efficiency of the polysaccharide, which makes direct identification of Globo H conjugation more difficult. This is why the number of identified peptides was higher for the MMCCH conjugation analysis. Therefore, the MMCCH results can be used to represent Globo H conjugation.

Conjugation site analysis showed that there were 155, 143, 147, and 140 lysine conjugation sites out of a total of 306 lysine residues in KLH1/KLH2 for samples 1-4, respectively. In replicate analyses, 141, 137, 143, and 136 conjugation sites were identified for samples 1-4, respectively.

Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of the present invention. Although any compositions, methods, kits, and means similar or equivalent to those described herein can be used to practice the present invention, preferred compositions, methods, kits, and means are described herein.

All references cited herein are incorporated herein by reference to the full extent permitted by law. The discussion of these references is intended only to summarize the assertions of their authors. No admission is made that any reference (or portion of a reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and relevance of any cited reference.

Claims (47)

1. A compound comprising the structure: in: n is an independent integer from 1 to 800. m is an independent integer from 1 to 5; The Globo H moiety is covalently bonded to the KLH moiety on a basic amino acid residue via a 4-(4-N-maleimidemethyl)cyclohexane-1-carboxyhydrazide (MMCCH) linker group; The epitope ratio of the compound ranges from 750 to 3000, and the epitope ratio is calculated using the following formula: = (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 2. The compound according to claim 1, wherein m is 1 and n is from 1 to 150. 3. The compound according to claim 1, wherein m is 2 and n is from 1 to 300. 4. The compound according to claim 1, wherein m is 3 and n is from 1 to 450. 5. The compound according to claim 1, wherein m is 4 and n is from 1 to 600. 6. The compound according to claim 1, wherein m is 5 and n is from 1 to 750. 7. A therapeutic composition comprising a plurality of Globo H moieties covalently linked to one or more keyhole hemocyanin (KLH) moieties via MMCCH linker groups, wherein the ratio of the number of Globo H moieties to KLH moieties is from 1:1 to 150:1, expressed as the number of Globo H molecules relative to a single KLH moiety; The epitope ratio of the therapeutic composition is in the range of 750 to 3000, and the epitope ratio is calculated using the following formula: (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 8. A therapeutic composition comprising a plurality of GloboH moieties covalently linked to a keyhole hemocyanin (KLH) moieties via an MMCCH linker group, wherein the number of GloboH moieties is 1 to 150, expressed as the number of GloboH molecules relative to a monomeric KLH moieties; The epitope ratio of the therapeutic composition is in the range of 750 to 3000, and the epitope ratio is calculated using the following formula: (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 9. A therapeutic composition comprising a plurality of GloboH moieties covalently linked to two keyhole hemocyanin (KLH) moieties via MMCCH linker groups, wherein the number of GloboH moieties is 1 to 300, expressed relative to the number of GloboH molecules in the dimer KLH moieties; The epitope ratio of the therapeutic composition is in the range of 750 to 3000, and the epitope ratio is calculated using the following formula: (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 10. A therapeutic composition comprising a plurality of Globo H moieties covalently linked to three keyhole hemocyanin (KLH) moieties via MMCCH linker groups, wherein the number of Globo H moieties is 1 to 450, expressed relative to the number of Globo H molecules of the trimer KLH moieties; The epitope ratio of the therapeutic composition is in the range of 750 to 3000, and the epitope ratio is calculated using the following formula: (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 11. A therapeutic composition comprising a plurality of Globo H moieties covalently linked to four keyhole hemocyanin (KLH) moieties via MMCCH linker groups, wherein the number of Globo H moieties is 1 to 600, expressed relative to the number of Globo H molecules of the tetrameric KLH moieties; The epitope ratio of the therapeutic composition is in the range of 750 to 3000, and the epitope ratio is calculated using the following formula: (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 12. A therapeutic composition comprising a plurality of Globo H moieties covalently linked to five keyhole hemocyanin (KLH) moieties via MMCCH linker groups, wherein the number of Globo H moieties is 1 to 750, expressed relative to the number of Globo H molecules in the pentamer KLH moieties; The epitope ratio of the therapeutic composition is in the range of 750 to 3000, and the epitope ratio is calculated using the following formula: (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 13. A pharmaceutical composition comprising a compound and optionally a pharmaceutically acceptable carrier, said compound comprising the structure: Where n is an independent integer from 1 to 800, and m is an independent integer from 1 to 5; The Globo H moiety is covalently bonded to the KLH moiety on a basic amino acid residue via the MMCCH linker group; The epitope ratio of the compounds ranges from 750 to 3000, and the epitope ratio is calculated using the following formula: = (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 14. The composition according to claim 13, wherein m is 1 and n is from 1 to 150. 15. The composition according to claim 13, wherein m is 2 and n is from 1 to 300. 16. The composition according to claim 13, wherein m is 3 and n is from 1 to 450. 17. The composition of claim 13, wherein m is 4 and n is from 1 to 600. 18. The composition according to claim 13, wherein m is 5 and n is from 1 to 750. 19. A pharmaceutical composition comprising a therapeutic conjugate, said therapeutic conjugate comprising a plurality of Globo H moieties covalently linked to one or more keyhole hemocyanin (KLH) partial subunits via an MMCCH linker group, wherein the number of Globo H moieties relative to KLH partial subunits is from 1:1 to 150:1; The epitope ratio of the therapeutic conjugate ranges from 750 to 3000, and the epitope ratio is calculated using the following formula: (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 20. A pharmaceutical composition comprising a therapeutic conjugate, said therapeutic conjugate comprising a plurality of Globo H moieties covalently linked to one or more keyhole hemocyanin (KLH) partial subunits via an MMCCH linker group, wherein the KLH moieties exist as monomers, dimers, trimers, tetramers, and pentamers; The epitope ratio of the therapeutic conjugate ranges from 750 to 3000, and the epitope ratio is calculated using the following formula: (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 21. A pharmaceutical composition comprising a therapeutic conjugate, said therapeutic conjugate comprising a plurality of Globo H moieties covalently linked to one or more keyhole hemocyanin (KLH) partial subunits via an MMCCH linker group, wherein the number of Globo H moieties relative to KLH partial subunits is expressed as 1:1 to 300:1 relative to the number of Globo H molecules in the dimer KLH moieties; The epitope ratio of the therapeutic conjugate ranges from 750 to 3000, and the epitope ratio is calculated using the following formula: (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 22. A pharmaceutical composition comprising a therapeutic conjugate comprising a plurality of Globo H moieties covalently linked to one or more keyhole hemocyanin (KLH) partial subunits via an MMCCH linker group, wherein the number of Globo H moieties relative to KLH partial subunits is expressed as 1:1 to 450:1 relative to the number of Globo H molecules of a trimer KLH moieties; The epitope ratio of the therapeutic conjugate ranges from 750 to 3000, and the epitope ratio is calculated using the following formula: (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 23. A pharmaceutical composition comprising a therapeutic conjugate comprising a plurality of Globo H moieties covalently linked to one or more keyhole hemocyanin (KLH) partial subunits via an MMCCH linker group, wherein the number of Globo H moieties relative to KLH partial subunits is expressed as 1:1 to 600:1 relative to the number of Globo H molecules of a tetrameric KLH moieties; The epitope ratio of the therapeutic conjugate ranges from 750 to 3000, and the epitope ratio is calculated using the following formula: (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 24. A pharmaceutical composition comprising a therapeutic conjugate, said therapeutic conjugate comprising a plurality of Globo H moieties covalently linked to one or more keyhole hemocyanin (KLH) partial subunits via an MMCCH linker group, wherein the number of Globo H moieties relative to the number of KLH partial subunits, expressed as a number of Globo H molecules relative to one pentamer KLH, is from 1:1 to 750:1. The epitope ratio of the therapeutic conjugate ranges from 750 to 3000, and the epitope ratio is calculated using the following formula: (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 25. A pharmaceutical composition comprising a monomer, dimer, trimer, tetramer, or pentamer or a combination thereof comprising a KLH moiety, wherein each KLH moiety comprises one or more Globo H moieties covalently linked to the KLH moiety via an MMCCH linker group; The epitope ratio of the pharmaceutical composition is in the range of 750 to 3000, and the epitope ratio is calculated using the following formula: = (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 26. The pharmaceutical composition of claim 25, wherein 1% to 99% of the therapeutic conjugate in the composition is a monomer. 27. The pharmaceutical composition of claim 25, wherein 1% to 99% of the therapeutic conjugate in the composition is a dimer. 28. The pharmaceutical composition of claim 25, wherein 1% to 99% of the therapeutic conjugate in the composition is a trimer. 29. The pharmaceutical composition of claim 25, wherein 1% to 99% of the therapeutic conjugate in the composition is a tetramer. 30. The pharmaceutical composition of claim 25, wherein 1% to 99% of the therapeutic conjugate in the composition is a pentamer. 31. A pharmaceutical composition comprising a therapeutic conjugate, said therapeutic conjugate comprising a plurality of Globo H moieties covalently linked to one or more keyhole hemocyanin (KLH) partial subunits via an MMCCH linker group, wherein 1% to 99% of the KLH moieties in the composition are monomers, dimers, trimers, tetramers, pentamers or combinations thereof; The epitope ratio of the therapeutic conjugate ranges from 750 to 3000, and the epitope ratio is calculated using the following formula: (actual Globo H fraction weight / Globo H fraction molecular weight) / (actual KLH fraction weight / KLH fraction molecular weight); and Before being conjugated with the Globo H portion, the thiohydroxylated KLH portion is stored in an inert gas environment. 32. The pharmaceutical composition of claim 25, further comprising an adjuvant, wherein the adjuvant is a Freund's complete and incomplete adjuvant, a Toll-like receptor molecule, LPS, lipoprotein, lipopeptide, flagella, double-stranded RNA, viral DNA, unmethylated CpG islands, levamisole, Bacillus calmette-guerin, isopyrosine, Zadaxin, a PD-1 antagonist, a PD-1 antibody, a CTLA antagonist, a CTLA antibody, interleukin, cytokines, GM-CSF, an aluminum-based glycolipid, aluminum phosphate, alum, aluminum hydroxide, liposomes, a TLR2 agonist, nanoparticles, monophosphate lipid A, OPT-821 saponin, an oil-in-water nanoemulsion, and bacterial-like particles. 33. The pharmaceutical composition of claim 25 further comprises a cytokine selected from IL-2, IL-12, IL-18, IFN-γ, TNF, IL-4, IL-10, IL-13, IL-21, GM-CSF, and TGF-β. 34. The pharmaceutical composition according to claim 25 further comprises a chemokine. 35. The pharmaceutical composition of claim 25, further comprising a drug selected from: hormone therapy drugs, monoclonal antibody therapy drugs, chemotherapeutic drugs, retinoid receptor modulators, cytotoxic/cell-inhibiting drugs, antitumor drugs, antiproliferative drugs, isopentenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, nitrogen mustard, nitrosoureas, angiogenesis inhibitors, cell proliferation and survival signaling pathway inhibitors, apoptosis inducers, drugs that interfere with cell cycle checkpoints, drugs that interfere with receptor tyrosine kinases (RTKs), mammalian target of rapamycin (mTOR) inhibitors, and human epidermal growth factor receptors. HER2 inhibitors, epidermal growth factor receptor (EGFR) inhibitors, integrin blockers, NSAIDs, PPAR agonists, intrinsic multidrug resistance (MDR) inhibitors, antiemetics, drugs for treating anemia, drugs for treating neutropenia, immune enhancers, bisphosphonates, aromatase inhibitors, drugs for inducing terminal differentiation of tumor cells, γ-secretase inhibitors, cancer vaccines, PD-1/PD-L1 inhibitors, CTLA-4 immunotherapy, CDK4/6 inhibitors, PI3K inhibitors, AKT inhibitors, pan-HER inhibitors, and any combination thereof. 36. The pharmaceutical composition of claim 25 further comprises a pharmaceutically acceptable carrier. 37. The pharmaceutical composition of claim 36, wherein the pharmaceutical composition is a cancer vaccine. 38. The pharmaceutical composition according to claim 25, formulated for subcutaneous injection, intravenous administration or intramuscular administration. 39. Use of the composition of claim 25 in the preparation of a medicament for treating cancer in patients in need, wherein a therapeutically effective amount of the composition of claim 25 is administered to the patient. 40. The use according to claim 39, wherein the cancer is ovarian cancer or breast cancer. 41. The use according to claim 39, wherein the cancer is epithelial carcinoma. 42. The use according to claim 41, wherein the epithelial carcinoma is breast cancer, lung cancer, liver cancer, oral cancer, gastric cancer, colon cancer, nasopharyngeal carcinoma, skin cancer, kidney cancer, brain tumor, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, intestinal cancer, pancreatic cancer, or bladder cancer. 43. The use according to claim 39, wherein the therapeutically effective amount of the Globo-H portion in the composition comprises 0.01 μg/kg to 250 mg/kg. 44. The use according to claim 39, wherein the therapeutically effective amount of the Globo-H portion in the composition comprises 0.10 μg/kg to 0.75 μg/kg. 45. The use according to claim 39, wherein the therapeutically effective amount of the Globo-H-KLH complex in the composition comprises 0.01 μg/kg to 250 mg/kg. 46. The use according to claim 39, wherein the therapeutically effective amount of the Globo-H-KLH complex in the composition comprises 0.60 μg/kg to 4.50 μg/kg. 47. Use of the composition according to claim 25 in the preparation of a medicament for inducing antibodies in animals or humans, wherein an effective amount of the composition is administered to the animal or human.