γ polyglutamated tetrahydrofolates and uses thereof

Abstract

To provide a γ polyglutamated tetrahydrofolate (THF) composition for treating hyperproliferative disorders (e.g., cancer) and disorders of the immune system (e.g., inflammation and autoimmune diseases).SOLUTION: A composition comprising a liposome encapsulating γ polyglutamated tetrahydrofolate, in which the γ polyglutamated tetrahydrofolate comprises 2 to 15 glutamyl groups linked via γ carboxyl group linkages, and is selected from the group consisting of: (a) γ polyglutamated 5-formyl-THF; (b) γ polyglutamated 10-formyl-THF; (c) γ polyglutamated 5,10-methenyl-THF; (d) γ polyglutamated 5-methyl-THF; (e) γ polyglutamated 5,10-methylene-THF; and (f) γ polyglutamated 5-formimino-THF, the liposome composition having a diameter of 50 to 150 nm.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure generally relates to compositions of gamma-polyglutamylated tetrahydrofolic acid, including delivery vehicles such as liposomes containing gamma-polyglutamylated tetrahydrofolic acid compositions, and methods of manufacturing and using the compositions for treating diseases including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria. The polyglutamylated gamma-tetrahydrofolic acid composition also has use in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effectiveness of the therapeutic agent(s), or as a "chemoprotectant" (e.g., in combination with folate antagonists such as methotrexate) to reduce the toxic side effects associated with the therapeutic agent(s).

Background Art

[0002] Folic acid is an essential cofactor that mediates the transfer of one-carbon units involved in biosynthesis and DNA repair, remethylation of homocysteine (Hcy), and methylation of DNA, proteins, and lipids. The only circulating form of folic acid in the blood is monoglutamic acid, and folic acid monoglutamate is the only form of folic acid that is transported across cell membranes. Similarly, monoglutamic acid form of tetrahydrofolic acid is also transported across cell membranes. Once taken up into cells, intracellular tetrahydrofolic acid is polyglutamylated by the enzyme folylpoly-gamma-glutamate synthetase (FPGS). Polyglutamylation of tetrahydrofolic acid by FPGS serves at least two major therapeutic purposes: (1) it greatly enhances the affinity of tetrahydrofolic acid for DHFR; and (2) it promotes the accumulation of polyglutamylated tetrahydrofolic acid, which, unlike tetrahydrofolic acid (monoglutamic acid), is not readily transported out of cells by cellular efflux pumps.

[0003] The provided gamma-polyglutamylated tetrahydrofolic acid composition provides a strategy for improving the therapeutic effect of tetrahydrofolic acid.

Summary of the Invention

[0004] The present disclosure generally relates to γ-polyglutamyl oxidized tetrahydrofolate (THF) compositions, and methods of making and using the compositions for treating diseases including hyperproliferative diseases such as cancer, disorders of the immune system such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria. The α-polyglutamyl oxidized tetrahydrofolate compositions also have use in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effectiveness of the therapeutic agent(s), or as a "chemoprotectant" (e.g., in combination with a folate antagonist such as methotrexate) to reduce the toxic side effects associated with the therapeutic agent(s).

[0005] In some embodiments, the present disclosure provides the following: [1] A composition comprising γ-polyglutamyl oxidized tetrahydrofolate; [2] The composition of [1], wherein the γ-polyglutamyl oxidized tetrahydrofolate is selected from the group consisting of: (a) Polyglutamyl oxidized 5-formyl-THF (e.g., polyglutamyl oxidized [6S]-5-formyl-THF); (b) Polyglutamyl oxidized 10-formyl-THF (e.g., polyglutamyl oxidized [6R]-10-formyl-THF); (c) Polyglutamyl oxidized 5,10-methenyl-THF (e.g., polyglutamyl oxidized [6R]-5,10-methenyl-THF); (d) Polyglutamyl oxidized 5-methyl-THF (e.g., polyglutamyl oxidized [6S]-5-methyl-THF); (e) Polyglutamyl oxidized tetrahydrofolate (e.g., polyglutamyl oxidized [6S]-tetrahydrofolate); (f) Polyglutamyl oxidized 5,10-methylene-THF (e.g., polyglutamyl oxidized [6R]-5,10-methylene-THF); and (g) Polyglutamyl oxidized 5-formimino-THF (e.g., polyglutamyl oxidized [6S]-5-formimino-THF); [3]γ-Polyglutamyl oxidized tetrahydrofolate is a composition according to [1] or [2], comprising a glutamyl group having a γ-carboxyl group bond of 4, 5, 2-10, 4-6, or more than 5; [4]γ-Polyglutamyl oxidized tetrahydrofolate is a composition according to any one of [1]-[3], which is γ-tetraglutamyl oxidized tetrahydrofolate; [5]γ-Polyglutamyl oxidized tetrahydrofolate is a composition according to any one of [1]-[3], which is γ-pentaglutamyl oxidized tetrahydrofolate; [6]γ-Polyglutamyl oxidized tetrahydrofolate is a composition according to any one of [1]-[3], which is γ-hexaglutamyl oxidized tetrahydrofolate; [7]A composition according to any one of [1]-[6], which is as follows: (a)γ-Polyglutamyl oxidized tetrahydrofolate contains two or more L-type glutamyl groups having a γ-carboxyl group bond, (b)Each of the glutamyl groups of γ-polyglutamyl oxidized tetrahydrofolate is of the L-type and has a γ-carboxyl group bond, (c)At least one of the glutamyl groups of γ-polyglutamyl oxidized tetrahydrofolate is of the D-type and has a γ-carboxyl group bond, (d)Each of the glutamyl groups of γ-polyglutamyl oxidized tetrahydrofolate other than the glutamyl group of tetrahydrofolate is of the D-type and has a γ-carboxyl group bond, or (e)γ-Polyglutamyl oxidized tetrahydrofolate contains two or more L-type glutamyl groups and at least one D-type glutamyl group having a γ-carboxyl group bond; [8]A composition according to [4], which is as follows: (a)Each of the glutamyl groups is of the L-type and has a γ-carboxyl group bond, or (b)Each of the glutamyl groups other than the glutamyl group of tetrahydrofolate is of the D-type, and each of the glutamyl (gluytamyl) groups has a γ-carboxyl group bond; [9]A composition according to [5], which is as follows: (a)Each of the glutamyl groups is of the L-type and has a γ-carboxyl group bond, or (b) Each of the glutamyl groups other than the glutamyl group of tetrahydrofolic acid is of the D type, and each of the glutamyl groups has a γ-carboxyl group bond;

[10] The composition of [6] as described below: (a) Each of the glutamyl groups is of the L type and has a γ-carboxyl group bond, or (b) Each of the glutamyl groups other than the glutamyl group of tetrahydrofolic acid is of the D type, and each of the glutamyl groups has a γ-carboxyl group bond;

[11] γ-Polyglutamylated tetrahydrofolic acid is polyglutamylatable by FGPS under physiological conditions, and / or the polyglutamylated THF has a lower uptake rate (<30%) by hepatocytes than THF, a composition according to any of [1]-

[10] ;

[12] A liposomal composition (Lp-γPTHF) containing γ-polyglutamylated tetrahydrofolic acid according to any of [1]-

[11] ;

[13] The Lp-γPTHF composition according to

[12] , wherein γ-polyglutamylated tetrahydrofolic acid contains two or more L-type glutamyl groups;

[14] The Lp-γPTHF composition according to

[12] or

[13] , wherein each of the glutamyl groups of γ-polyglutamylated tetrahydrofolic acid is of the L type;

[15] The Lp-γPTHF composition according to

[12] or

[13] , wherein at least one of the glutamyl groups of γ-polyglutamylated tetrahydrofolic acid is of the D type;

[16] The liposome is the Lp-γPTHF composition according to any of

[12] -

[15] , containing γ-polyglutamylated tetrahydrofolic acid having 1-10 glutamyl groups with γ-carboxyl group bonds;

[17] The liposome is the Lp-γPTHF composition according to any of

[12] -

[16] , containing γ-polyglutamylated tetrahydrofolic acid having 4, 5, 2-10, 4-6, or more than 5 glutamyl groups;

[18] The liposome is the Lp-γPTHF composition according to any of

[12] -

[17] , containing γ-tetraglutamylated tetrahydrofolic acid;

[19] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[17] , containing γ-pentaglutamyl oxidized tetrahydrofolic acid;

[20] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[17] , containing γ-hexaglutamyl oxidized tetrahydrofolic acid;

[21] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[20] , which is not pegylated (PγLp-γPTHF);

[22] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[20] , which is pegylated (PγLp-γPTHF);

[23] The liposome contains at least 1% weight / weight (w / w) of γ-polyglutamyl oxidized tetrahydrofolic acid, or at least 1% of the starting material of γ-polyglutamyl oxidized THF is encapsulated (trapped) within the Lp-γPTHF during the process of preparing the Lp-γPTHF, and it is an Lp-γPTHF composition according to any one of

[12] -

[22] ;

[24] The liposome has a diameter in the range of 20 nm to 500 nm, and it is an Lp-γPTHF composition according to any one of

[12] -

[23] ;

[25] The liposome has a diameter in the range of 20 nm to 200 nm, and it is an Lp-γPTHF composition according to any one of

[12] -

[24] ;

[26] The liposome has a diameter in the range of 80 nm to 120 nm, and it is an Lp-γPTHF composition according to any one of

[12] -

[25] ;

[27] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[26] , formed from liposome components;

[28] The liposome components include at least one of an anionic lipid and a neutral lipid, and it is an Lp-γPTHF composition according to

[27] ;

[29] The liposome components include at least one selected from the group consisting of the following, and it is an Lp-γPTHF composition according to

[27] or

[28] : DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide;

[30] The liposome component contains at least one selected from the group consisting of the following, and the Lp-γPTHF composition according to any one of

[27] -

[29] : DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC;

[31] The Lp-γPTHF composition according to any one of

[27] -

[30] , wherein one or more liposome components further contain a steric stabilizer;

[32] The steric stabilizer is at least one selected from the group consisting of the following, and the Lp-γPTHF composition according to

[31] : polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidylpolygycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymer; oligoglycerol, polyethylene glycol and polypropylene oxide-containing copolymer, poloxamer 188, and polyvinyl alcohol;

[33] The steric stabilizer is PEG, and PEG has a number average molecular weight (Mn) of 200 to 5000 daltons, and the Lp-γPTHF composition according to

[32] ;

[34] The liposome is anionic or neutral, and the Lp-γPTHF composition according to any one of

[12] -

[33] ;

[35] The liposome has a zeta potential that is zero or less, and the Lp-γPTHF composition according to any one of

[12] -

[33] ;

[36] The liposome has a zeta potential of 0 to -150 mV, and the Lp-γPTHF composition according to any one of

[12] -

[33] ;

[37] The liposome has a zeta potential of -30 to -50 mV, and the Lp-γPTHF composition according to any one of

[12] -

[33] ;

[38] The liposome is cationic, and the Lp-γPTHF composition according to any one of

[12] -

[33] ;

[39] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[38] , having an internal space containing γ-polyglutamylated tetrahydrofolic acid and an aqueous, pharmaceutically acceptable carrier;

[40] The pharmaceutically acceptable carrier is an Lp-γPTHF composition according to

[39] , containing an isotonic agent such as dextrose, mannitol, glycerin, potassium chloride, sodium chloride, etc. at a concentration exceeding 1%;

[41] The aqueous, pharmaceutically acceptable carrier is trehalose, an Lp-γPTHF composition according to

[39] ;

[42] The pharmaceutically acceptable carrier is an Lp-γPTHF composition according to

[41] , containing 1% - 50% trehalose;

[43] The pharmaceutically acceptable carrier is an Lp-γPTHF composition according to any one of

[39] -

[42] , containing a 1% - 50% dextrose solution;

[44] The internal space of the liposome is an Lp-γPTHF composition according to any one of

[39] -

[43] , containing 5% dextrose suspended in HEPES buffer;

[45] The pharmaceutically acceptable carrier is an Lp-γPTHF composition according to any one of

[39] -

[44] , containing a buffer such as HEPES buffered saline (HBS) or the like at a concentration of 1 - 200 mM and a pH of 2 - 8;

[46] The pharmaceutically acceptable carrier is an Lp-γPTHF composition according to any one of

[39] -

[45] , containing sodium acetate and calcium acetate at a total concentration of 50 mM to 500 mM;

[47] The internal space of the liposome has a pH of 5 - 8 or 6 - 7, or any range therebetween, an Lp-γPTHF composition according to any one of

[12] -

[46] ;

[48] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[47] , containing less than 500,000 or less than 200,000 molecules of γ-polyglutamylated tetrahydrofolic acid;

[49] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[48] , containing 10 - 100,000 molecules of γ-polyglutamylated tetrahydrofolic acid, or any range therebetween;

[50] An Lp-γPTHF composition according to any one of

[12] -

[49] , further comprising a targeting moiety, wherein the targeting moiety has specific affinity for a surface antigen on a target cell of interest;

[51] An Lp-γPTHF composition according to

[50] , wherein the targeting moiety is attached to one or both of the outer surface of PEG and the liposome, and optionally, the targeting moiety is attached to one or both of the outer surface of PEG and the liposome by a covalent bond;

[52] An Lp-γPTHF composition according to

[50] or

[51] , wherein the targeting moiety is a polypeptide;

[53] An Lp-γPTHF composition according to any one of

[50] -

[52] , wherein the targeting moiety is an antibody or an antigen-binding fragment of an antibody;

[54] An Lp-γPTHF composition according to any one of

[50] -

[53] , wherein the targeting moiety binds to the surface antigen with an equilibrium dissociation constant (Kd) in the range of 0.5x10 -10 ~10x10 -6 ;

[55] An Lp-γPTHF composition according to any one of

[50] -

[54] , wherein the targeting moiety specifically binds to one or more folate receptors selected from the group consisting of: folate receptor α (FR-α), folate receptor β (FR-β), and folate receptor δ (FR-δ);

[56] An Lp-γPTHF composition according to any one of

[50] -

[55] , wherein the targeting moiety comprises one or more selected from the group consisting of: antibody, humanized antibody, antigen-binding fragment of an antibody, single-chain antibody, single-domain antibody, bispecific antibody, synthetic antibody, pegylated antibody, and multimeric antibody;

[57] An Lp-γPTHF composition according to any one of

[50] -

[56] , wherein each pegylated liposome comprises 1 to 1000 or 30 to 200 targeting moieties;

[58] An Lp-γPTHF composition according to any one of

[39] -

[57] , further comprising one or more of an immunostimulant, a detectable marker, and maleimide, wherein the immunostimulant, detectable marker, or maleimide is attached to the outer surface of the PEG or liposome;

[59] The immunostimulant is at least one selected from the group consisting of the Lp-γPTHF composition of

[58] : protein immunostimulant; nucleic acid immunostimulant; chemical immunostimulant; hapten; and adjuvant;

[60] The immunostimulant is at least one selected from the group consisting of the Lp-γPTHF composition of

[58] or

[59] : fluorescein; fluorescein isothiocyanate (FITC); DNP; β-glucan; β-1,3-glucan; β-1,6-glucan; resorcin (e.g., D n-6DPA or D n-3DPA such as resorcin D, resorcin E, or T-series resorcin; and toll-like receptor (TLR) regulators such as oxidized low-density lipoprotein (e.g., OXPAC, PGPC), and erythran lipids (e.g., E5564);

[61] The immunostimulant and the detectable marker are the same in the Lp-γPTHF composition according to any of

[58] -

[60] ;

[62] The Lp-γPTHF composition according to any of

[58] -

[61] further comprising a hapten;

[63] The hapten in the Lp-γPTHF composition of

[62] comprises one or more of fluorescein or β1,6-glucan;

[64] The Lp-γPTHF composition according to any of

[12] -

[63] further comprising at least one cryoprotective substance selected from the group consisting of mannitol; trehalose; sorbitol; and sucrose;

[65] A targeted composition comprising the composition according to any of [1]-

[64] ;

[66] A non-targeted composition comprising the composition according to any of [1]-

[49] ;

[67] The Lp-γPTHF composition according to any of

[12] -

[66] further comprising carboplatin and / or pembrolizumab;

[68] A pharmaceutical composition comprising the liposome γ-polyglutamine oxidized tetrahydrofolic acid composition according to any of

[12] -

[67] ;

[69] A pharmaceutical composition comprising the γ-polyglutamine oxidized tetrahydrofolic acid composition according to any of [1]-[7];

[70] A composition according to any one of [1]-

[69] for use in the treatment of a disease;

[71] Use of a composition according to any one of [1]-

[70] for enhancing the effectiveness of a therapeutic agent(s) in the manufacture of a medicament for the treatment of a disease and / or in combination therapy with one or more therapeutic agents such as chemotherapeutic agents (e.g., 5-fluorouracil), or for reducing the toxic side effects associated with a therapeutic agent(s) as a "chemoprotective agent" (e.g., in combination with a folic acid antagonist such as methotrexate);

[72] A method for treating (e.g., treating or preventing) a disease or chemical-induced toxicity in a subject in need of such treatment or prevention, the method comprising administering to the subject a composition according to any one of [1]-

[70] ;

[73] A method for treating (e.g., treating or preventing) a disease or chemical-induced toxicity in a subject in need of such treatment or prevention, the method comprising administering to the subject a liposomal γ-polyglutamylated tetrahydrofolic acid composition according to any one of

[12] -

[69] ;

[74] A method for killing proliferative cells, comprising contacting the proliferative cells with a composition according to any one of [1]-

[69] ;

[75] A method for killing proliferative cells, comprising contacting the proliferative cells with a liposomal γ-polyglutamylated tetrahydrofolic acid composition according to any one of

[12] -

[69] ;

[76] The method of

[74] or

[75] , wherein the proliferative cells are cancer cells, mammalian cells, and / or human cells;

[77] A method for treating cancer, comprising administering to a subject having or at risk of having cancer an effective amount of a composition according to any one of [1]-

[69] ;

[78] A method for treating (e.g., treating or preventing) cancer, comprising administering to a subject having or at risk of having cancer an effective amount of a liposomal γ-polyglutamylated tetrahydrofolic acid composition according to any one of

[12] -

[68] ;

[79] The composition is administered for treating or preventing cancer, and the cancer is selected from the group consisting of the methods of

[77] or

[78] : for example, non - hematological tumors including lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors such as leukemia, lymphoma and other B - cell malignancies, multiple myeloma and other plasma cell dyscrasias;

[80] The composition is administered for treating or preventing cancer, and the cancer is a member selected from the group consisting of the methods of

[77] or

[78] : lung cancer, breast cancer, colon cancer, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, ovarian cancer, and cervical cancer;

[81] The composition is administered for treating or preventing cancer, and the cancer is a member selected from the group consisting of the methods of

[77] or

[78] : colorectal cancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non - small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma;

[82] The composition is administered for treating or preventing colorectal cancer, by the method of

[77] or

[78] ;

[83] A method for treating (e.g., treating or preventing) cancer, comprising administering an effective amount of any of the Lp - γPTHF compositions of

[50] -

[66] to a subject having or at risk of having cancer cells expressing folate receptor on its surface, which is bound by a targeting moiety;

[84] Maintenance therapy, comprising administering an effective amount of any of the compositions of [1] -

[69] to a subject who is undergoing or has undergone cancer therapy;

[85] Maintenance therapy, comprising administering an effective amount of any of the liposomal γ - polyglutamylated tetrahydrofolate compositions of

[12] -

[69] to a subject who is undergoing or has undergone cancer therapy;

[86] A method for treating (e.g., treating or preventing) an immune system disorder, comprising administering to a subject having or at risk of having an immune system disorder an effective amount of any of the compositions of [1]-

[69] , wherein optionally the immune system disorder is selected from the following: inflammation (e.g., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, systemic lupus erythematosus, and Takayasu disease, and psoriasis;

[87] A method for treating (e.g., treating or preventing) an immune system disorder, comprising administering to a subject having or at risk of having an immune system disorder an effective amount of any of the liposomal γ-polyglutamylated tetrahydrofolate compositions of [8]-

[69] , wherein optionally the immune system disorder is selected from the following: inflammation (e.g., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, systemic lupus erythematosus, and Takayasu disease, and psoriasis;

[88] A method for treating (e.g., treating or preventing) as follows: (a) A method for treating (e.g., treating or preventing) leukopenia, comprising administering to a subject having or at risk of having leukopenia an effective amount of a composition according to any of [1]-

[59] ; (b) A method for treating (e.g., treating or preventing) an infectious disease, comprising administering to a subject having or at risk of having an infectious disease an effective amount of a composition according to any of [1]-

[69] ; (c) A method for treating (e.g., treating or preventing) a cardiovascular disease or a metabolic disease, comprising administering to a subject having or at risk of having an infectious disease, a cardiovascular disease, or another disease, wherein the disease is a member selected from the following: atherosclerosis, cardiovascular disease (CVD), coronary artery disease, myocardial infarction, stroke, metabolic syndrome, gestational trophoblastic disease, and ectopic pregnancy, an effective amount of a composition according to any of [1]-

[59] ; (d) A method for treating (e.g., treating or preventing) an autoimmune disease, comprising administering to a subject having or at risk of having an autoimmune disease an effective amount of a composition according to any one of [1]-

[59] ; (e) A method for treating rheumatoid arthritis (e.g., treating or preventing), comprising administering to a subject having or at risk of having rheumatoid arthritis an effective amount of a composition according to any one of [1]-

[59] ; (f) A method for treating (e.g., treating or preventing) an inflammatory condition, comprising administering to a subject having or at risk of having inflammation an effective amount of a composition according to any one of [1]-

[59] , wherein optionally the inflammation is acute, chronic, and / or systemic inflammation; or (g) A method for treating (e.g., treating or preventing) a skin disease, comprising administering to a subject having or at risk of having a skin disease an effective amount of a composition according to any one of [1]-

[59] , wherein optionally the skin disease is psoriasis;

[89] A method for treating (e.g., treating or preventing) an infectious disease, comprising administering to a subject having or at risk of having an infectious disease an effective amount of a liposomal γ-polyglutamylated tetrahydrofolic acid composition according to any one of

[12] -

[69] ;

[90] A method for delivering γ-polyglutamylated tetrahydrofolic acid to a tumor expressing a folate receptor on its surface, the method comprising: administering to a subject having a tumor an Lp-γPTHF composition according to any one of [1]-

[69] in an amount effective to deliver a therapeutically effective amount of γ-polyglutamylated tetrahydrofolic acid to the tumor;

[91] A method for preparing a γ-polyglutamylated tetrahydrofolic acid composition comprising a liposomal γ-polyglutamylated tetrahydrofolic acid composition according to any one of

[12] -

[69] , the method comprising: forming a mixture comprising a liposomal component and a γ-polyglutamylated folic acid antagonist in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes containing γ-polyglutamylated tetrahydrofolic acid;

[92] A method for preparing a composition of any one of

[12] -

[69] , comprising the following steps: forming a mixture comprising liposome components and γ-polyglutamylated tetrahydrofolic acid in solution; homogenizing the mixture in solution to form liposomes; treating the mixture to form liposomes that capture and / or encapsulate γ-polyglutamylated tetrahydrofolic acid; and providing a targeting moiety on the surface of the liposomes, the targeting moiety having specific affinity for at least one of folate receptor α (FR-α), folate receptor β (FR-β), and folate receptor δ (FR-δ);

[93] The treatment step comprises one or more of the following steps of the method according to

[92] : thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw technique, reverse phase evaporation, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, freeze-dried double emulsion, 3D printing, membrane contactor method, and stirring; and / or

[94] The treatment step comprises one or more steps of changing the size of the liposomes by one or more of extrusion, high pressure microfluidization, and / or sonication of the method according to

[92] .

[0006] In some embodiments, the present disclosure provides a γ-polyglutamylated tetrahydrofolic acid (γPTHF) composition, wherein at least two of the glutamyl residues of γ-polyglutamylated tetrahydrofolic acid have γ-carboxyl group linkages. In some embodiments, γPTHF contains 2-20, 2-15, 2-10, 2-5, or more than 5 glutamyl groups (including the glutamyl groups in tetrahydrofolic acid). In some embodiments, γPTHF contains two or more L-type glutamyl groups. In other embodiments, γPTHF contains a D-type glutamyl group. In further embodiments, γPTHF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0007] In one embodiment, the γPTHF composition contains a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid (i.e., tetraglutamine oxidized tetrahydrofolic acid). In some embodiments, the tetraglutamine oxidized THF contains two or more L-type glutamyl groups. In other embodiments, the tetraglutamine oxidized THF contains a D-type glutamyl group. In further embodiments, the tetraglutamine oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0008] In one embodiment, the γPTHF composition contains a chain of four γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid (e.g., γ-pentaglutamine oxidized tetrahydrofolic acid). In some embodiments, the γ-pentaglutamine oxidized THF contains two or more L-type glutamyl groups. In other embodiments, the γ-pentaglutamine oxidized THF contains a D-type glutamyl group. In further embodiments, the γ-pentaglutamine oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0009] In one embodiment, the γPTHF composition contains a chain of five γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid (e.g., γ-hexaglutamine oxidized tetrahydrofolic acid). In some embodiments, the γ-hexaglutamine oxidized THF contains two or more L-type glutamyl groups. In other embodiments, the γ-hexaglutamine oxidized THF contains a D-type glutamyl group. In further embodiments, the γ-hexaglutamine oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0010] In additional embodiments, the present disclosure provides compositions containing delivery vehicles such as liposomes filled with (e.g., encapsulating) and / or otherwise conjugated to γ-polyglutamylated tetrahydrofolate, as well as methods of making γPTHF-filled / conjugated delivery vehicle compositions (DV-γPTHF) and methods of using the same to deliver γ-polyglutamylated tetrahydrofolate to diseased (e.g., cancerous) and / or target cells. These compositions have uses including, but not limited to, treating (e.g., treating or preventing) diseases including hyperproliferative diseases such as cancer, immune system disorders such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria. In some embodiments, the γ-polyglutamylated tetrahydrofolate in DV-γPTHF contains 2-20, 2-15, 2-10, 2-5, greater than 5, or greater than 20 glutamyl groups (including the glutamyl group in tetrahydrofolate). The DV-γPTHF-filled / conjugated delivery vehicle compositions provide an improvement in the efficacy and safety of delivering tetrahydrofolate to cancer cells by providing preferential delivery of a more cytotoxic payload (e.g., polyglutamylated tetrahydrofolate) compared to the cytotoxicity of tetrahydrofolate administered in its monoglutamate form (THF).

[0011] In some embodiments, the present disclosure provides for the use of a composition containing a delivery vehicle, such as a liposome filled with (e.g., encapsulating) and / or otherwise conjugated with γ-polyglutamylated tetrahydrofolic acid, for enhancing the efficacy of one or more therapeutic agents (e.g., chemotherapeutic agents such as 5-fluorouracil) in combination therapy with one or more therapeutic agents or for reducing the toxic side effects associated with the therapeutic agent(s) (e.g., in combination with a folic acid antagonist such as methotrexate) as a “chemoprotectant.” In some embodiments, the γ-polyglutamylated tetrahydrofolic acid in the DV-αPTHF contains 2-20, 2-15, 2-10, 2-5, greater than 5, or greater than 20 glutamyl groups (including the glutamyl group in tetrahydrofolic acid). The DV-αPTHF-filled / conjugated delivery vehicle composition provides an improvement in the efficacy and safety of delivering tetrahydrofolic acid to cancer cells by providing preferential delivery of a more cytotoxic payload (e.g., polyglutamylated tetrahydrofolic acid) compared to the cytotoxicity of tetrahydrofolic acid administered in its monoglutamic acid state (THF).

[0012] In additional embodiments, the present disclosure provides a composition comprising γ-polyglutamylated tetrahydrofolic acid (γPTHF).

[0013] In some embodiments, the present disclosure provides a composition comprising γ-polyglutamylated 5-formyl-THF. In some embodiments, the γ-polyglutamylated 5-formyl-THF is γ-polyglutamylated [6S]-5-formyl-THF. In some embodiments, the composition comprises γ-polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the composition comprises γ-polyglutamylated [6R]-5-formyl-THF. In some embodiments, the composition contains γ-polyglutamylated 5-formyl-THF having 2-20, 2-15, 2-10, 2-5, or more than 20 glutamyl groups (including the glutamyl group in 5-formyl-THF). In some embodiments, the composition contains γ-polyglutamylated 5-formyl-THF having two or more L-type glutamyl groups. In other embodiments, the composition contains γ-polyglutamylated 5-formyl-THF having a D-type glutamyl group. In further embodiments, the composition contains γ-polyglutamylated 5-formyl-THF having a D-type glutamyl group and two or more L-type glutamyl groups.

[0014] In some embodiments, the present disclosure provides a composition comprising γ-polyglutamylated 5-formyl-THF (i.e., tetraglutamylated 5-formyl-THF) containing a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises γ-tetraglutamylated [6S]-5-formyl-THF. In some embodiments, the composition comprises γ-tetraglutamylated [6R,S]-5-formyl-THF. In some embodiments, the composition comprises γ-tetraglutamylated [6R]-5-formyl-THF. In some embodiments, γ-tetraglutamylated 5-formyl-THF contains two or more L-type glutamyl groups. In other embodiments, γ-tetraglutamylated 5-formyl-THF contains a D-type glutamyl group. In further embodiments, γ-tetraglutamylated 5-formyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0015] In some embodiments, the present disclosure provides a composition comprising γ-pentaglutamylated 5-formyl-THF (i.e., pentaglutamylated 5-formyl-THF) containing a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises γ-pentaglutamylated [6S]-5-formyl-THF. In some embodiments, the composition comprises γ-pentaglutamylated [6R,S]-5-formyl-THF. In some embodiments, the composition comprises γ-pentaglutamylated [6R]-5-formyl-THF. In some embodiments, pentaglutamylated 5-formyl-THF contains two or more L-type glutamyl groups. In other embodiments, pentaglutamylated 5-formyl-THF contains a D-type glutamyl group. In further embodiments, pentaglutamylated 5-formyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0016] In some embodiments, the present disclosure provides a composition comprising γ-hexaglutamylated 5-formyl-THF (i.e., hexaglutamylated 5-formyl-THF) containing a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises γ-hexaglutamylated [6S]-5-formyl-THF. In some embodiments, the composition comprises γ-hexaglutamylated [6R,S]-5-formyl-THF. In some embodiments, the composition comprises γ-hexaglutamylated [6R]-5-formyl-THF. In some embodiments, γ-hexaglutamylated 5-formyl-THF contains two or more L-type glutamyl groups. In other embodiments, hexaglutamylated THF contains a D-type glutamyl group. In further embodiments, γ-hexaglutamylated 5-formyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0017] In some embodiments, the present disclosure provides a composition comprising γ-polyglutamylated 5,10-methenyl-THF. In some embodiments, the composition comprises γ-polyglutamylated [6R]-5,10-methenyl-THF. In some embodiments, the composition comprises γ-polyglutamylated [6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprises γ-polyglutamylated [6S]-5,10-methenyl-THF. In some embodiments, the composition contains γ-polyglutamylated 5,10-methenyl-THF having 2-20, 2-15, 2-10, 2-5, or more than 20 glutamyl groups (including the glutamyl group in 5,10-methenyl-THF). In some embodiments, the composition contains γ-polyglutamylated 5-formyl-THF having two or more L-type glutamyl groups. In other embodiments, the composition contains γ-polyglutamylated 5,10-methenyl-THF having a D-type glutamyl group. In further embodiments, the composition contains γ-polyglutamylated 5,10-methenyl-THF having a D-type glutamyl group and two or more L-type glutamyl groups.

[0018] In some embodiments, the present disclosure provides a composition comprising γ-polyglutamylated 5,10-methenyl-THF (i.e., tetraglutamylated 5,10-methenyl-THF) containing a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises γ-tetraglutamylated [6R]-5,10-methenyl-THF. In some embodiments, the composition comprises γ-tetraglutamylated [6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprises γ-tetraglutamylated [6S]-5,10-methenyl-THF. In some embodiments, γ-tetraglutamylated 5,10-methenyl-THF contains two or more L-type glutamyl groups. In other embodiments, γ-tetraglutamylated 5,10-methenyl-THF contains a D-type glutamyl group. In further embodiments, γ-tetraglutamylated 5,10-methenyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0019] In some embodiments, the present disclosure provides a composition comprising γ-pentaglutamylated 5,10-methenyl-THF (i.e., pentaglutamylated 5,10-methenyl-THF) containing a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises γ-pentaglutamylated [6R]-5,10-methenyl-THF. In some embodiments, the composition comprises γ-pentaglutamylated [6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprises γ-pentaglutamylated [6S]-5,10-methenyl-THF. In some embodiments, γ-pentaglutamylated 5,10-methenyl-THF contains two or more L-type glutamyl groups. In other embodiments, γ-pentaglutamylated 5,10-methenyl-THF contains a D-type glutamyl group. In further embodiments, γ-pentaglutamylated 5,10-methenyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0020] In some embodiments, the present disclosure provides a composition comprising γ-hexaglutamylated 5,10-methenyl-THF (i.e., hexaglutamylated 5,10-methenyl-THF) containing a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises γ-hexaglutamylated [6R]-5,10-methenyl-THF. In some embodiments, the composition comprises γ-hexaglutamylated [6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprises γ-hexaglutamylated [6S]-5,10-methenyl-THF. In some embodiments, γ-hexaglutamylated 5,10-methenyl-THF contains two or more L-type glutamyl groups. In other embodiments, γ-hexaglutamylated 5,10-methenyl-THF contains a D-type glutamyl group. In further embodiments, γ-hexaglutamylated 5,10-methenyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0021] In some embodiments, the present disclosure provides a composition comprising γ-polyglutamylated 5-methyl-THF. In some embodiments, the composition comprises γ-polyglutamylated [6S]-5-methyl-THF. In some embodiments, the composition comprises γ-polyglutamylated [6R,S]-5-methyl-THF. In some embodiments, the composition comprises γ-polyglutamylated [6R]-5-methyl-THF. In some embodiments, the composition contains γ-polyglutamylated 5-methyl-THF having 2-20, 2-15, 2-10, 2-5, or more than 20 glutamyl groups (including the glutamyl group in 5-methyl-THF). In some embodiments, the composition contains γ-polyglutamylated 5-methyl-THF having two or more L-type glutamyl groups. In other embodiments, the composition contains γ-polyglutamylated 5-methyl-THF having a D-type glutamyl group. In further embodiments, the composition contains γ-polyglutamylated 5-methyl-THF having a D-type glutamyl group and two or more L-type glutamyl groups.

[0022] In some embodiments, the present disclosure provides a composition comprising γ-polyglutamylated 5-methyl-THF (i.e., tetraglutamylated 5-methyl-THF) containing a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises γ-tetraglutamylated [6S]-5-methyl-THF. In some embodiments, the composition comprises γ-tetraglutamylated [6R,S]-5-methyl-THF. In some embodiments, the composition comprises γ-tetraglutamylated [6R]-5-methyl-THF. In some embodiments, γ-tetraglutamylated 5-methyl-THF contains two or more L-type glutamyl groups. In other embodiments, γ-tetraglutamylated 5-methyl-THF contains a D-type glutamyl group. In further embodiments, γ-tetraglutamylated 5-methyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0023] In some embodiments, the present disclosure provides a composition comprising γ-pentaglutamylated 5-methyl-THF (i.e., pentaglutamylated 5-methyl-THF) containing a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises γ-pentaglutamylated [6S]-5-methyl-THF. In some embodiments, the composition comprises γ-pentaglutamylated [6R,S]-5-methyl-THF. In some embodiments, the composition comprises γ-pentaglutamylated [6R]-5-methyl-THF. In some embodiments, γ-pentaglutamylated 5-methyl-THF comprises two or more L-type glutamyl groups. In other embodiments, γ-pentaglutamylated 5-methyl-THF comprises a D-type glutamyl group. In further embodiments, γ-pentaglutamylated 5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0024] In some embodiments, the present disclosure provides a composition comprising γ-hexaglutamylated 5-methyl-THF (i.e., hexaglutamylated 5-methyl-THF) containing a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises γ-hexaglutamylated [6S]-5-methyl-THF. In some embodiments, the composition comprises γ-hexaglutamylated [6R,S]-5-methyl-THF. In some embodiments, the composition comprises γ-hexaglutamylated [6R]-5-methyl-THF. In some embodiments, γ-hexaglutamylated 5-methyl-THF comprises two or more L-type glutamyl groups. In other embodiments, γ-hexaglutamylated 5-methyl-THF comprises a D-type glutamyl group. In further embodiments, γ-hexaglutamylated 5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0025] In some embodiments, the present disclosure provides a composition comprising γ-polyglutamylated THF. In some embodiments, the composition comprises γ-polyglutamylated [6S]-THF. In some embodiments, the composition comprises γ-polyglutamylated [6R,S]-THF. In some embodiments, the composition comprises γ-polyglutamylated [6R]-THF. In some embodiments, the composition contains γ-polyglutamylated THF having 2 - 20, 2 - 15, 2 - 10, 2 - 5, or more than 20 glutamyl groups (including glutamyl groups in THF). In some embodiments, the composition contains γ-polyglutamylated THF having two or more L-type glutamyl groups. In other embodiments, the composition contains γ-polyglutamylated THF having a D-type glutamyl group. In further embodiments, the composition contains γ-polyglutamylated THF having a D-type glutamyl group and two or more L-type glutamyl groups.

[0026] In some embodiments, the present disclosure provides a composition comprising γ-polyglutamylated tetrahydrofolic acid (i.e., tetraglutamylated tetrahydrofolic acid THF) containing a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid THF. In some embodiments, the composition comprises γ-tetraglutamylated [6S] tetrahydrofolic acid THF. In some embodiments, the composition comprises γ-tetraglutamylated [6R,S]-tetrahydrofolic acid THF. In some embodiments, the composition comprises γ-tetraglutamylated [6R]-tetrahydrofolic acid THF. In some embodiments, γ-tetraglutamylated tetrahydrofolic acid THF contains two or more L-type glutamyl groups. In other embodiments, γ-tetraglutamylated tetrahydrofolic acid THF contains a D-type glutamyl group. In further embodiments, γ-tetraglutamylated tetrahydrofolic acid THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0027] In some embodiments, the present disclosure provides a composition comprising γ-polyglutamylated tetrahydrofolic acid (i.e., pentaglutamylated tetrahydrofolic acid THF) containing a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid THF. In some embodiments, the composition comprises γ-pentaglutamylated [6S]tetrahydrofolic acid. In some embodiments, the composition comprises γ-pentaglutamylated [6R,S]-tetrahydrofolic acid THF. In some embodiments, the composition comprises γ-pentaglutamylated [6R]-tetrahydrofolic acid THF. In some embodiments, γ-pentaglutamylated tetrahydrofolic acid THF comprises two or more L-type glutamyl groups. In other embodiments, γ-pentaglutamylated tetrahydrofolic acid THF comprises a D-type glutamyl group. In further embodiments, γ-pentaglutamylated tetrahydrofolic acid THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0028] In some embodiments, the present disclosure provides a composition comprising γ-polyglutamylated tetrahydrofolic acid (i.e., hexaglutamylated tetrahydrofolic acid THF) containing a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid THF. In some embodiments, the composition comprises γ-hexaglutamylated [6S]tetrahydrofolic acid THF. In some embodiments, the composition comprises γ-hexaglutamylated [6R,S]-tetrahydrofolic acid THF. In some embodiments, the composition comprises γ-hexaglutamylated [6R]-tetrahydrofolic acid THF. In some embodiments, γ-hexaglutamylated tetrahydrofolic acid THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamylated tetrahydrofolic acid THF comprises a D-type glutamyl group. In further embodiments, γ-hexaglutamylated tetrahydrofolic acid THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0029] In some embodiments, the present disclosure provides a composition comprising γ-polyglutamylated 5,10-methylene-THF. In some embodiments, the composition comprises γ-polyglutamylated [6R]-5,10-methylene-THF. In some embodiments, the composition comprises γ-polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises γ-polyglutamylated [6S]-5,10-methylene-THF. In some embodiments, the composition contains γ-polyglutamylated 5,10-methylene-THF having 2-20, 2-15, 2-10, 2-5, or more than 20 glutamyl groups (including the glutamyl group in 5,10-methylene-THF). In some embodiments, the composition contains γ-polyglutamylated 5,10-methylene-THF having two or more L-type glutamyl groups. In other embodiments, the composition contains γ-polyglutamylated 5,10-methylene-THF having a D-type glutamyl group. In further embodiments, the composition contains γ-polyglutamylated 5,10-methylene-THF having a D-type glutamyl group and two or more L-type glutamyl groups.

[0030] In some embodiments, the present disclosure provides a composition comprising γ-polyglutamylated 5,10-methylene-THF (i.e., tetraglutamylated 5,10-methylene-THF) containing a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises γ-tetraglutamylated [6R]-5,10-methylene-THF. In some embodiments, the composition comprises γ-tetraglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises γ-tetraglutamylated [6S]-5,10-methylene-THF. In some embodiments, γ-tetraglutamylated 5,10-methylene-THF contains two or more L-type glutamyl groups. In other embodiments, γ-tetraglutamylated 5,10-methylene-THF contains a D-type glutamyl group. In further embodiments, γ-tetraglutamylated 5,10-methylene-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0031] In some embodiments, the present disclosure provides a composition comprising γ-pentaglutamylated 5,10-methylene-THF (i.e., pentaglutamylated 10-methylene-THF) containing a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises γ-pentaglutamylated [6R]5,10-methylene-THF. In some embodiments, the composition comprises γ-pentaglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises γ-pentaglutamylated [6S]-5,10-methylene-THF. In some embodiments, γ-pentaglutamylated 5,10-methylene-THF contains two or more L-type glutamyl groups. In other embodiments, γ-pentaglutamylated 5,10-methylene-THF contains a D-type glutamyl group. In further embodiments, γ-pentaglutamylated 5,10-methylene-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0032] In some embodiments, the present disclosure provides a composition comprising γ-hexaglutamylated 5,10-methylene-THF (i.e., hexaglutamylated 5,10-methylene-THF) containing a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises γ-hexaglutamylated [6R]5,10-methylene-THF. In some embodiments, the composition comprises γ-hexaglutamylated [6R]5,10-methylene-THF. In some embodiments, the composition comprises γ-hexaglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises γ-hexaglutamylated [6S]-5,10-methylene-THF. In some embodiments, γ-hexaglutamylated 5,10-methylene-THF contains two or more L-type glutamyl groups. In other embodiments, γ-hexaglutamylated 5,10-methylene-THF contains a D-type glutamyl group. In further embodiments, γ-hexaglutamylated 5,10-methylene-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0033] In some embodiments, the present disclosure provides a composition comprising γ-polyglutamylated 5-formiminotetrahydrofolate. In some embodiments, the composition comprises γ-polyglutamylated [6S]-5-formiminotetrahydrofolate. In some embodiments, the composition comprises γ-polyglutamylated [6R,S]-5-formiminotetrahydrofolate. In some embodiments, the composition comprises γ-polyglutamylated [6R]-5-formiminotetrahydrofolate. In some embodiments, the composition contains γ-polyglutamylated 5-formiminotetrahydrofolate having 2-20, 2-15, 2-10, 2-5, or more than 20 glutamyl groups (including the glutamyl group in 5-formiminotetrahydrofolate). In some embodiments, the composition contains γ-polyglutamylated 5-formiminotetrahydrofolate having two or more L-type glutamyl groups. In other embodiments, the composition contains γ-polyglutamylated 5-formiminotetrahydrofolate having a D-type glutamyl group. In further embodiments, the composition contains γ-polyglutamylated 5-formiminotetrahydrofolate having a D-type glutamyl group and two or more L-type glutamyl groups.

[0034] In some embodiments, the present disclosure provides a composition comprising: (a) γ-polyglutamyl oxidized (b) polyglutamyl oxidized 5-formyl-THF; (c) polyglutamyl oxidized 5,10-methenyl-THF; (d) polyglutamyl oxidized 5-methyl-THF; (e) polyglutamyl oxidized-tetrahydrofolate; (f) polyglutamyl oxidized 5,10-methylene-THF; and (g) polyglutamyl oxidized-5-formimino-THF (i.e., tetraglutamyl oxidized 5-formimino-THF) containing a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises γ-tetraglutamyl oxidized [6S]-5-formimino-THF. In some embodiments, the composition comprises γ-tetraglutamyl oxidized [6S] 5-formimino-THF. In some embodiments, the composition comprises γ-tetraglutamyl oxidized [6R,S]-5-formimino-THF. In some embodiments, the composition comprises γ-tetraglutamyl oxidized [6R]-5-formimino-THF. In some embodiments, γ-tetraglutamyl oxidized 5-formimino-THF comprises two or more L-type glutamyl groups. In other embodiments, γ-tetraglutamyl oxidized 5-formimino-THF comprises a D-type glutamyl group. In further embodiments, γ-tetraglutamyl oxidized 5-formimino-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0035] In one embodiment, the composition comprises γ-pentaglutamylated 5-formiminotetrahydrofolate (i.e., pentaglutamylated 5-formiminotetrahydrofolate) containing a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises γ-pentaglutamylated [6S]-5-formiminotetrahydrofolate. In some embodiments, the composition comprises γ-pentaglutamylated [6R,S]-5-formiminotetrahydrofolate. In some embodiments, the composition comprises γ-pentaglutamylated [6R]5-formiminotetrahydrofolate. In some embodiments, γ-pentaglutamylated 5-formiminotetrahydrofolate comprises two or more L-type glutamyl groups. In other embodiments, γ-pentaglutamylated 5-formiminotetrahydrofolate comprises a D-type glutamyl group. In further embodiments, γ-pentaglutamylated 5-formiminotetrahydrofolate comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0036] In one embodiment, the composition comprises γ-hexaglutamylated 5-formiminotetrahydrofolate (i.e., hexaglutamylated 5-formiminotetrahydrofolate) containing a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises γ-hexaglutamylated [6S]-5-formiminotetrahydrofolate. In some embodiments, the composition comprises γ-hexaglutamylated [6R,S]-5-formiminotetrahydrofolate. In some embodiments, the composition comprises γ-hexaglutamylated [6R]5-formiminotetrahydrofolate. In some embodiments, γ-hexaglutamylated 5-formiminotetrahydrofolate comprises two or more L-type glutamyl groups. In other embodiments, γ-hexaglutamylated 5-formiminotetrahydrofolate comprises a D-type glutamyl group. In further embodiments, γ-hexaglutamylated 5-formiminotetrahydrofolate comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0037] In additional embodiments, the present disclosure provides a composition comprising liposomes encapsulating (filling) γ-pentaglutamylated tetrahydrofolic acid (Lp-γPTHF).

[0038] In some embodiments, the present disclosure provides a composition comprising liposomes encapsulating (filling) γ-polyglutamylated 5-formyl-THF. In some embodiments, the liposomes comprise γ-polyglutamylated [6S]-5-formyl-THF. In some embodiments, the liposomes comprise γ-polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposomes comprise γ-polyglutamylated [6R]-5-formyl-THF. In some embodiments, the liposomes contain γ-polyglutamylated 5-formyl-THF having 2 - 20, 2 - 15, 2 - 10, 2 - 5, or more than 20 glutamyl groups (including the glutamyl group in 5-formyl-THF). In some embodiments, the liposomes contain γ-polyglutamylated 5-formyl-THF having two or more L-type glutamyl groups. In other embodiments, the liposomes contain γ-polyglutamylated 5-formyl-THF having a D-type glutamyl group. In further embodiments, the liposomes contain γ-polyglutamylated 5-formyl-THF having a D-type glutamyl group and two or more L-type glutamyl groups.

[0039] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated 5-formyl-THF (i.e., tetraglutamylated 5-formyl-THF) containing a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposomes comprise γ-tetraglutamylated [6S]-5-formyl-THF. In some embodiments, the liposomes comprise γ-tetraglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposomes comprise γ-tetraglutamylated [6R]-5-formyl-THF. In some embodiments, γ-tetraglutamylated 5-formyl-THF contains two or more L-type glutamyl groups. In other embodiments, γ-tetraglutamylated 5-formyl-THF contains a D-type glutamyl group. In further embodiments, γ-tetraglutamylated 5-formyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0040] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated 5-formyl-THF (i.e., pentaglutamylated 5-formyl-THF) containing a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-pentaglutamylated [6S]-5-formyl-THF. In some embodiments, the liposome comprises γ-pentaglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposome comprises γ-pentaglutamylated [6R]-5-formyl-THF. In some embodiments, pentaglutamylated 5-formyl-THF contains two or more L-type glutamyl groups. In other embodiments, pentaglutamylated 5-formyl-THF contains a D-type glutamyl group. In further embodiments, pentaglutamylated 5-formyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0041] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated 5-formyl-THF (i.e., hexaglutamylated 5-formyl-THF) containing a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-hexaglutamylated [6S]-5-formyl-THF. In some embodiments, the liposome comprises γ-hexaglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposome comprises γ-hexaglutamylated [6R]-5-formyl-THF. In some embodiments, γ-hexaglutamylated 5-formyl-THF contains two or more L-type glutamyl groups. In other embodiments, hexaglutamylated THF contains a D-type glutamyl group. In further embodiments, γ-hexaglutamylated 5-formyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0042] In some embodiments, the present disclosure provides a composition comprising liposomes encapsulating (filled with) γ-polyglutamyl oxidized 5,10-methenyl-THF. In some embodiments, the liposomes comprise γ-polyglutamyl oxidized [6R]-5,10-methenyl-THF. In some embodiments, the liposomes comprise γ-polyglutamyl oxidized [6R,S]-5,10-methenyl-THF. In some embodiments, the liposomes comprise γ-polyglutamyl oxidized [6S]-5,10-methenyl-THF. In some embodiments, the liposomes contain γ-polyglutamyl oxidized 5,10-methenyl-THF having 2 - 20, 2 - 15, 2 - 10, 2 - 5, or more than 20 glutamyl groups (including the glutamyl groups in 5,10-methenyl-THF). In some embodiments, the liposomes contain γ-polyglutamyl oxidized 5-formyl-THF having two or more L-type glutamyl groups. In other embodiments, the liposomes contain γ-polyglutamyl oxidized 5,10-methenyl-THF having D-type glutamyl groups. In further embodiments, the liposomes contain γ-polyglutamyl oxidized 5,10-methenyl-THF having D-type glutamyl groups and two or more L-type glutamyl groups.

[0043] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated 5,10-methenyl-THF (i.e., tetraglutamylated 5,10-methenyl-THF) containing a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-tetraglutamylated [6R]-5,10-methenyl-THF. In some embodiments, the liposome comprises γ-tetraglutamylated [6R,S]-5,10-methenyl-THF. In some embodiments, the liposome comprises γ-tetraglutamylated [6S]-5,10-methenyl-THF. In some embodiments, γ-tetraglutamylated 5,10-methenyl-THF contains two or more L-type glutamyl groups. In other embodiments, γ-tetraglutamylated 5,10-methenyl-THF contains a D-type glutamyl group. In further embodiments, γ-tetraglutamylated 5,10-methenyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0044] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated 5,10-methenyl-THF (i.e., pentaglutamylated 5,10-methenyl-THF) containing a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-pentaglutamylated [6R]-5,10-methenyl-THF. In some embodiments, the liposome comprises γ-pentaglutamylated [6R,S]-5,10-methenyl-THF. In some embodiments, the liposome comprises γ-pentaglutamylated [6S]-5,10-methenyl-THF. In some embodiments, γ-pentaglutamylated 5,10-methenyl-THF contains two or more L-type glutamyl groups. In other embodiments, γ-pentaglutamylated 5,10-methenyl-THF contains a D-type glutamyl group. In further embodiments, γ-pentaglutamylated 5,10-methenyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0045] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated 5,10-methenyl-THF (i.e., hexaglutamylated 5,10-methenyl-THF) containing a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-hexaglutamylated [6R]-5,10-methenyl-THF. In some embodiments, the liposome comprises γ-hexaglutamylated [6R,S]-5,10-methenyl-THF. In some embodiments, the liposome comprises γ-hexaglutamylated [6S]-5,10-methenyl-THF. In some embodiments, γ-hexaglutamylated 5,10-methenyl-THF comprises two or more L-type glutamyl groups. In other embodiments, γ-hexaglutamylated 5,10-methenyl-THF comprises a D-type glutamyl group. In further embodiments, γ-hexaglutamylated 5,10-methenyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0046] In some embodiments, the present disclosure provides a composition comprising liposomes encapsulating (filled with) γ-polyglutamylated 5-methyl-THF. In some embodiments, the liposome comprises γ-polyglutamylated [6S]-5-methyl-THF. In some embodiments, the liposome comprises γ-polyglutamylated [6R,S]-5-methyl-THF. In some embodiments, the liposome comprises γ-polyglutamylated [6R]-5-methyl-THF. In some embodiments, the liposome contains γ-polyglutamylated 5-methyl-THF having 2 - 20, 2 - 15, 2 - 10, 2 - 5, or more than 20 glutamyl groups (including the glutamyl group in 5-methyl-THF). In some embodiments, the liposome contains γ-polyglutamylated 5-methyl-THF having two or more L-type glutamyl groups. In other embodiments, the liposome contains γ-polyglutamylated 5-methyl-THF having a D-type glutamyl group. In further embodiments, the liposome contains γ-polyglutamylated 5-methyl-THF having a D-type glutamyl group and two or more L-type glutamyl groups.

[0047] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated 5-methyl-THF (i.e., tetraglutamylated 5-methyl-THF) containing a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-tetraglutamylated [6S]-5-methyl-THF. In some embodiments, the liposome comprises γ-tetraglutamylated [6R,S]-5-methyl-THF. In some embodiments, the liposome comprises γ-tetraglutamylated [6R]-5-methyl-THF. In some embodiments, γ-tetraglutamylated 5-methyl-THF comprises two or more L-type glutamyl groups. In other embodiments, γ-tetraglutamylated 5-methyl-THF comprises a D-type glutamyl group. In further embodiments, γ-tetraglutamylated 5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0048] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated 5-methyl-THF (i.e., pentaglutamylated 5-methyl-THF) containing a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-pentaglutamylated [6S]-5-methyl-THF. In some embodiments, the liposome comprises γ-pentaglutamylated [6R,S]-5-methyl-THF. In some embodiments, the liposome comprises γ-pentaglutamylated [6R]-5-methyl-THF. In some embodiments, γ-pentaglutamylated 5-methyl-THF comprises two or more L-type glutamyl groups. In other embodiments, γ-pentaglutamylated 5-methyl-THF comprises a D-type glutamyl group. In further embodiments, γ-pentaglutamylated 5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0049] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated 5-methyl-THF (i.e., hexaglutamylated 5-methyl-THF) containing a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-hexaglutamylated [6S]-5-methyl-THF. In some embodiments, the liposome comprises γ-hexaglutamylated [6R,S]-5-methyl-THF. In some embodiments, the liposome comprises γ-hexaglutamylated [6R]-5-methyl-THF. In some embodiments, γ-hexaglutamylated 5-methyl-THF comprises two or more L-type glutamyl groups. In other embodiments, γ-hexaglutamylated 5-methyl-THF comprises a D-type glutamyl group. In further embodiments, γ-hexaglutamylated 5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0050] In some embodiments, the present disclosure provides a composition comprising liposomes encapsulating (filling) γ-polyglutamylated THF. In some embodiments, the liposome comprises γ-polyglutamylated [6S]-THF. In some embodiments, the liposome comprises γ-polyglutamylated [6R,S]-THF. In some embodiments, the liposome comprises γ-polyglutamylated [6R]-THF. In some embodiments, the liposome contains γ-polyglutamylated THF having 2-20, 2-15, 2-10, 2-5, or more than 20 glutamyl groups (including the glutamyl group in THF). In some embodiments, the liposome contains γ-polyglutamylated THF having two or more L-type glutamyl groups. In other embodiments, the liposome contains γ-polyglutamylated THF having a D-type glutamyl group. In further embodiments, the liposome contains γ-polyglutamylated THF having a D-type glutamyl group and two or more L-type glutamyl groups.

[0051] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated tetrahydrofolic acid (i.e., tetraglutamylated tetrahydrofolic acid) containing a chain of three glutamyl groups attached to the glutamyl group of THF. In some embodiments, the liposome comprises γ-tetraglutamylated [6S]tetrahydrofolic acid. In some embodiments, the liposome comprises γ-tetraglutamylated [6R,S]-THF. In some embodiments, the liposome comprises γ-tetraglutamylated [6R]-THF. In some embodiments, γ-tetraglutamylated THF comprises two or more L-type glutamyl groups. In other embodiments, γ-tetraglutamylated THF comprises a D-type glutamyl group. In further embodiments, γ-tetraglutamylated THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0052] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated tetrahydrofolic acid (i.e., pentaglutamylated tetrahydrofolic acid) containing a chain of four glutamyl groups attached to the glutamyl group of THF. In some embodiments, the liposome comprises γ-pentaglutamylated [6S]tetrahydrofolic acid. In some embodiments, the liposome comprises γ-pentaglutamylated [6R,S]-THF. In some embodiments, the liposome comprises γ-pentaglutamylated [6R]-THF. In some embodiments, γ-pentaglutamylated THF comprises two or more L-type glutamyl groups. In other embodiments, γ-pentaglutamylated THF comprises a D-type glutamyl group. In further embodiments, γ-pentaglutamylated THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0053] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated tetrahydrofolic acid (i.e., hexaglutamylated tetrahydrofolic acid) containing a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-hexaglutamylated [6S] tetrahydrofolic acid. In some embodiments, the liposome comprises γ-hexaglutamylated [6R,S]-THF. In some embodiments, the liposome comprises γ-hexaglutamylated [6R]-THF. In some embodiments, γ-hexaglutamylated THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamylated THF comprises a D-type glutamyl group. In further embodiments, γ-hexaglutamylated THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0054] In some embodiments, the present disclosure provides a composition comprising liposomes encapsulating (filled with) γ-polyglutamylated 5,10-methylene-THF. In some embodiments, the liposome comprises γ-polyglutamylated [6R]-5,10-methylene-THF. In some embodiments, the liposome comprises γ-polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the liposome comprises γ-polyglutamylated [6S]-5,10-methylene-THF. In some embodiments, the liposome contains γ-polyglutamylated 5,10-methylene-THF having 2-20, 2-15, 2-10, 2-5, or more than 20 glutamyl groups (including the glutamyl groups in 5,10-methylene-THF). In some embodiments, the liposome contains γ-polyglutamylated 5,10-methylene-THF having two or more L-type glutamyl groups. In other embodiments, the liposome contains γ-polyglutamylated 5,10-methylene-THF having a D-type glutamyl group. In further embodiments, the liposome contains γ-polyglutamylated 5,10-methylene-THF having a D-type glutamyl group and two or more L-type glutamyl groups.

[0055] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated 5,10-methylene-THF (i.e., tetraglutamylated 5,10-methylene-THF) containing a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-tetraglutamylated [6R]-5,10-methylene-THF. In some embodiments, the liposome comprises γ-tetraglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the liposome comprises γ-tetraglutamylated [6S]-5,10-methylene-THF. In some embodiments, γ-tetraglutamylated 5,10-methylene-THF comprises two or more L-type glutamyl groups. In other embodiments, γ-tetraglutamylated 5,10-methylene-THF comprises a D-type glutamyl group. In further embodiments, γ-tetraglutamylated 5,10-methylene-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0056] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated 5,10-methylene-THF (i.e., pentaglutamylated 10-methylene-THF) containing a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-pentaglutamylated [6R]5,10-methylene-THF. In some embodiments, the liposome comprises γ-pentaglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the liposome comprises γ-pentaglutamylated [6S]-5,10-methylene-THF. In some embodiments, γ-pentaglutamylated 5,10-methylene-THF comprises two or more L-type glutamyl groups. In other embodiments, γ-pentaglutamylated 5,10-methylene-THF comprises a D-type glutamyl group. In further embodiments, γ-pentaglutamylated 5,10-methylene-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0057] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated 5,10-methylene-THF (i.e., hexaglutamylated 5,10-methylene-THF) containing a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-hexaglutamylated [6R]5,10-methylene-THF. In some embodiments, the liposome comprises γ-hexaglutamylated [6R]5,10-methylene-THF. In some embodiments, the liposome comprises γ-hexaglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the liposome comprises γ-hexaglutamylated [6S]-5,10-methylene-THF. In some embodiments, γ-hexaglutamylated 5,10-methylene-THF comprises two or more L-type glutamyl groups. In other embodiments, γ-hexaglutamylated 5,10-methylene-THF comprises a D-type glutamyl group. In further embodiments, γ-hexaglutamylated 5,10-methylene-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0058] In some embodiments, the present disclosure provides a composition comprising liposomes encapsulating (filling) γ-polyglutamyl oxidized 5-formiminotetrahydrofolate. In some embodiments, the liposomes comprise γ-polyglutamyl oxidized [6S]-5-formiminotetrahydrofolate. In some embodiments, the liposomes comprise γ-polyglutamyl oxidized [6R,S]-5-formiminotetrahydrofolate. In some embodiments, the liposomes comprise γ-polyglutamyl oxidized [6R]-5-formiminotetrahydrofolate. In some embodiments, the liposomes contain γ-polyglutamyl oxidized 5-formiminotetrahydrofolate having 2-20, 2-15, 2-10, 2-5, or more than 20 glutamyl groups (including the glutamyl group in 5-formiminotetrahydrofolate). In some embodiments, the liposomes contain γ-polyglutamyl oxidized 5-formiminotetrahydrofolate having two or more L-type glutamyl groups. In other embodiments, the liposomes contain γ-polyglutamyl oxidized 5-formiminotetrahydrofolate having a D-type glutamyl group. In further embodiments, the liposomes contain γ-polyglutamyl oxidized 5-formiminotetrahydrofolate having a D-type glutamyl group and two or more L-type glutamyl groups.

[0059] In one embodiment, the Lp-γPTHF composition comprises: (a) γ-polyglutamyl oxidized (b) polyglutamyl oxidized 5-formyl-THF; (c) polyglutamyl oxidized 5,10-methenyl-THF; (d) polyglutamyl oxidized 5-methyl-THF; (e) polyglutamyl oxidized-tetrahydrofolic acid; (f) polyglutamyl oxidized 5,10-methylene-THF; and (g) polyglutamyl oxidized-5-formimino-THF (i.e., tetraglutamyl oxidized 5-formimino-THF) containing a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-tetraglutamyl oxidized [6S]-5-formimino-THF. In some embodiments, the liposome comprises γ-tetraglutamyl oxidized [6S]5-formimino-THF. In some embodiments, the liposome comprises γ-tetraglutamyl oxidized [6R,S]-5-formimino-THF. In some embodiments, the liposome comprises γ-tetraglutamyl oxidized [6R]-5-formimino-THF. In some embodiments, γ-tetraglutamyl oxidized 5-formimino-THF comprises two or more L-type glutamyl groups. In other embodiments, γ-tetraglutamyl oxidized 5-formimino-THF comprises a D-type glutamyl group. In further embodiments, γ-tetraglutamyl oxidized 5-formimino-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0060] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated 5-formiminotetrahydrofolate (i.e., pentaglutamylated 5-formiminotetrahydrofolate) containing a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-pentaglutamylated [6S]-5-formiminotetrahydrofolate. In some embodiments, the liposome comprises γ-pentaglutamylated [6R,S]-5-formiminotetrahydrofolate. In some embodiments, the liposome comprises γ-pentaglutamylated [6R]5-formiminotetrahydrofolate. In some embodiments, γ-pentaglutamylated 5-formiminotetrahydrofolate contains two or more L-type glutamyl groups. In other embodiments, γ-pentaglutamylated 5-formiminotetrahydrofolate contains a D-type glutamyl group. In further embodiments, γ-pentaglutamylated 5-formiminotetrahydrofolate contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0061] In one embodiment, the Lp-γPTHF composition comprises γ-polyglutamylated 5-formiminotetrahydrofolate (i.e., hexaglutamylated 5-formiminotetrahydrofolate) containing a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises γ-hexaglutamylated [6S]-5-formiminotetrahydrofolate. In some embodiments, the liposome comprises γ-hexaglutamylated [6R,S]-5-formiminotetrahydrofolate. In some embodiments, the liposome comprises γ-hexaglutamylated [6R]5-formiminotetrahydrofolate. In some embodiments, γ-hexaglutamylated 5-formiminotetrahydrofolate contains two or more L-type glutamyl groups. In other embodiments, γ-hexaglutamylated 5-formiminotetrahydrofolate contains a D-type glutamyl group. In further embodiments, γ-hexaglutamylated 5-formiminotetrahydrofolate contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0062] In some embodiments, the Lp-γPTHF composition is cationic. In some embodiments, the Lp-γPTHF liposomes are cationic and have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-γPTHF liposomes are cationic and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the cationic Lp-γPTHF composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or greater than 75% w / w of γ-polyglutamylated THF. In some embodiments, during the process of preparing Lp-γPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or greater than 75% of the starting material of γ-polyglutamylated THF is encapsulated (trapped) in the cationic Lp-γPTHF. In additional embodiments, the γ-polyglutamylated tetrahydrofolic acid encapsulated by the liposome is present in the HEPES buffer within the liposome.

[0063] In other embodiments, the Lp-γPTHF composition is anionic or neutral. In some embodiments, the Lp-γPTHF liposomes are anionic or neutral and have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-γPTHF liposomes are anionic or neutral and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the Lp-γPTHF liposomes are anionic and have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-γPTHF liposomes are anionic and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the Lp-γPTHF liposomes are neutral and have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In some embodiments, the anionic or neutral Lp-γPTHF composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of γ-polyglutamylated THF. In some embodiments, during the process of preparing Lp-γPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of γ-polyglutamylated THF is encapsulated (trapped) in the anionic or neutral Lp-γPTHF. In some embodiments, the anionic or neutral Lp-γPTHF composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of γ-tetraglutamylated THF.In some embodiments, the anionic or neutral Lp-γPTHF composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of γ-pentaglutamylated THF. In some embodiments, the anionic or neutral Lp-γPTHF composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of γ-hexaglutamylated THF. In additional embodiments, the γ-polyglutamylated tetrahydrofolic acid encapsulated by the liposome is present in the HEPES buffer within the liposome.

[0064] In additional embodiments, the liposomal γ-polyglutamylated tetrahydrofolic acid composition is pegylated (PLp-γPTHF).

[0065] In some embodiments, the liposomal γ-polyglutamylated tetrahydrofolic acid composition is not targeted (NTLp-γPTHF). That is, the NTLp-γPTHF composition does not have a specific affinity for an epitope (e.g., an epitope of a surface antigen) expressed on the surface of the target cell of interest. In further embodiments, the non-targeted liposomal γ-polyglutamylated tetrahydrofolic acid composition is pegylated (NTPLp-γPTHF).

[0066] In other embodiments, the liposomal γ-polyglutamylated tetrahydrofolic acid composition is targeted (TLp-γPTHF). That is, the TLp-γPTHF composition contains a targeting moiety that has a specific affinity for an epitope (surface antigen) on the target cell of interest. In some embodiments, the targeting moiety of TLp-γPTHF or TPLp-γPTHF is not attached to the liposome via a covalent bond. In other embodiments, the targeting moiety of TLp-γPTHF or TPLp-γPTHF is attached to one or both of PEG and the outer surface of the liposome. The targeted liposomal γ-polyglutamylated tetrahydrofolic acid compositions (TLp-γPTHF and TPLp-γPTHF) provide further improvement in the efficacy and safety profile of tetrahydrofolic acid by specifically delivering γ-polyglutamylated (e.g., γ-pentaglutamylated and / or γ-hexaglutamylated) tetrahydrofolic acid to target cells such as cancer cells. In some embodiments, the targeted liposomal γ-polyglutamylated tetrahydrofolic acid composition is pegylated (TPLp-γPTHF). In some embodiments, the targeting moiety of TLp-γPTHF or TPLp-γPTHF is attached to one or both of PEG and the outer surface of the liposome. In some embodiments, the targeting moiety of TLp-γPTHF or TPLp-γPTHF is attached to the liposome via a covalent bond. Functions of the targeting moiety of the TLp-γPTHF and / or TPLp-γPTHF composition include, but are not limited to: targeting the liposome to the target cell of interest in vivo or in vitro; interacting with the surface antigen to which the targeting moiety has a specific affinity; and delivering the liposome payload (γPTHF) into the cell. Suitable targeting moieties are known in the art and include, but are not limited to, antibodies, antigen-binding antibody fragments, scaffold proteins, polypeptides, and peptides. In some embodiments, the targeting moiety is a polypeptide.In a further embodiment, the targeting moiety is a polypeptide comprising at least 3, 5, 10, 15, 20, 30, 40, 50, or 100 amino acid residues.

[0067] In some embodiments, the targeting moiety of TLp-γPTHF or TPLp-γPTHF is an antibody or an antigen-binding antibody fragment. In a further embodiment, the targeting moiety comprises one or more of an antibody, a humanized antibody, an antigen-binding fragment of an antibody, a single-chain antibody, a single-domain antibody, a bispecific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody. In some embodiments, the targeting moiety of TLp-γPTHF or TPLp-γPTHF has specific affinity for an epitope that is selectively expressed on target cells such as tumor cells as compared to normal or non-tumor cells. In some embodiments, the targeting moiety has specific affinity for an epitope on a tumor cell surface antigen that is present on tumor cells but not present on or difficult to access on non-tumor cells. In some embodiments, the targeting moiety binds to the target epitope with an equilibrium dissociation constant (Kd) in the range of 0.5x10 -10 ~10x10 -6 as measured by BIACORE® analysis.

[0068] In certain embodiments, the TLp-γPTHF or TPLp-γPTHF targeting moiety comprises a polypeptide that specifically binds to a folate receptor. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the folate receptor to which the targeting moiety binds is one or more folate receptors selected from the group consisting of folate receptor alpha (FR-α, FOLR1), folate receptor beta (FR-β, FOLR2), and folate receptor delta (FR-δ, FOLR4). In some embodiments, the folate receptor to which the targeting moiety binds is folate receptor alpha (FR-α). In some embodiments, the folate receptor to which the targeting moiety binds is folate receptor beta (FR-β). In some embodiments, the targeting moiety specifically binds to FR-α and FR-β.

[0069] In additional embodiments, the Lp-γPTHF composition comprises one or more of an immunostimulant, a detectable marker, and a maleimide disposed on at least one of the outer surface of the PEG and the liposome. In some embodiments, the liposomal γPTHF composition (e.g., Lp-γPTHF, PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF, or TPLp-γPTHF) is cationic. In other embodiments, the liposomal γPTHF composition (e.g., Lp-γPTHF, PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF or TPLp-γPTHF) is anionic or neutral. In additional embodiments, the liposomes of the liposomal γPTHF composition (e.g., Lp-γPTHF, PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF or TPLp-γPTHF) have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, 50 nm to 150 nm, or any range therebetween. In some embodiments, the liposomes of the liposome-γPTHF composition have a diameter in the range of 30 nm to 175 nm or 50 nm to 150 nm, or any range therebetween. In further embodiments, the liposomes of the liposomal γPTHF composition have a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the liposomal γPTHF composition is pegylated (e.g., PLp-γPTHF, NTPLp-γPTHF, or TPLp-γPTHF). In some embodiments, the liposomal γPTHF composition comprises a targeting moiety (e.g., TLp-γPTHF or TPLp-γPTHF). In further embodiments, the liposomal γPTHF composition is pegylated and targeted (e.g., TPLp-γPTHF). In some embodiments, the liposomal γPTHF composition comprises γ-polyglutamylated tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomal γPTHF composition comprises γ-tetraglutamylated tetrahydrofolate. In some embodiments, the liposomal γPTHF composition comprises γ-pentaglutamylated tetrahydrofolate.In other embodiments, the liposomal γPTHF composition comprises γ-hexaglutamyl oxidized tetrahydrofolate.

[0070] In some embodiments, the liposomal composition comprises γ-polyglutamyl oxidized tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of γ-polyglutamyl oxidized THF. In some embodiments, the Lp-γPTHF composition comprises γ-polyglutamyl oxidized tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups and 1%-98.5% w / w of γ-polyglutamyl oxidized THF. In some embodiments, the liposome comprises γ-polyglutamyl oxidized tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups, and during the process of preparing Lp-γPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of γ-polyglutamyl oxidized THF is encapsulated (trapped) in the Lp-γPTHF.

[0071] In some embodiments, the liposomal composition comprises γ-tetraglutamyl oxidized tetrahydrofolate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of γ-tetraglutamyl oxidized THF. In some embodiments, the Lp-γPTHF composition comprises γ-tetraglutamyl oxidized tetrahydrofolate and 1%-98.5% w / w of γ-tetraglutamyl oxidized THF. In some embodiments, the liposome comprises γ-tetraglutamyl oxidized tetrahydrofolate, and during the process of preparing Lp-γPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of γ-tetraglutamyl oxidized THF is encapsulated (trapped) in the Lp-γPTHF.

[0072] In some embodiments, the liposome composition comprises γ-pentaglutamyl oxidized tetrahydrofolic acid and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of γ-pentaglutamyl oxidized THF. In some embodiments, the Lp-γPTHF composition comprises γ-pentaglutamyl oxidized tetrahydrofolic acid and 1%-98.5% w / w of γ-pentaglutamyl oxidized THF. In some embodiments, the liposome comprises γ-pentaglutamyl oxidized tetrahydrofolic acid, and during the process of preparing Lp-γPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of γ-pentaglutamyl oxidized THF is encapsulated (trapped) in Lp-γPTHF.

[0073] In some embodiments, the liposome composition comprises γ-hexaglutamyl oxidized tetrahydrofolic acid and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of γ-hexaglutamyl oxidized THF. In some embodiments, the Lp-γPTHF composition comprises γ-hexaglutamyl oxidized tetrahydrofolic acid and 1%-98.5% w / w of γ-hexaglutamyl oxidized THF. In some embodiments, the liposome comprises γ-hexaglutamyl oxidized tetrahydrofolic acid, and during the process of preparing Lp-γPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of γ-pentaglutamyl oxidized THF is encapsulated (trapped) in Lp-γPTHF.

[0074] Liposome compositions comprising γPTHF-encapsulated liposomes are also provided. In some embodiments, the liposome composition comprises a pegylated γPTHF composition. In some embodiments, the liposome composition comprises a γPTHF composition linked or otherwise conjugated to a targeting moiety. In further embodiments, the liposome composition comprises a pegylated γPTHF composition linked or otherwise conjugated to a targeting moiety. In some embodiments, the liposome composition comprises γPTHF containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome composition comprises γ-tetraglutamyl oxidized tetrahydrofolate. In some embodiments, the liposome composition comprises γ-pentaglutamyl oxidized tetrahydrofolate. In other embodiments, the liposome composition comprises γ-hexaglutamyl oxidized tetrahydrofolate.

[0075] In some embodiments, the liposome composition comprises liposomal γPTHF (e.g., Lp-γPTHF, PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF, and TPLp-γPTHF). In some embodiments, the liposomal γPTHF is pegylated (e.g., NTPLp-γPTHF, and TPLp-γPTHF). In some embodiments, the liposomal γPTHF comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TLp-γPTHF or TPLp-γPTHF)). In further embodiments, the liposome composition comprises a liposomal γPTHF that is pegylated and further comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TPLp-γPTHF). In some embodiments, the liposome composition comprises a liposomal γPTHF that is cationic. In other embodiments, the liposome composition comprises a liposomal γPTHF that is anionic or neutral. In additional embodiments, the liposome composition comprises a liposomal γPTHF having a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposomal γPTHF has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

[0076] There is also provided γ-polyglutamyl oxidized tetrahydrofolic acid (γPTHF) comprising a delivery vehicle such as liposomal γPTHF. In some embodiments, the pharmaceutical composition comprises a pegylated γPTHF composition. In some embodiments, the pharmaceutical composition comprises a γPTHF composition linked or otherwise conjugated to a targeting moiety. In further embodiments, the pharmaceutical composition comprises a pegylated γPTHF composition linked or otherwise conjugated to a targeting moiety. In some embodiments, the pharmaceutical composition comprises γPTHF containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition comprises γ-tetraglutamyl oxidized tetrahydrofolic acid. In some embodiments, the pharmaceutical composition comprises γ-pentaglutamyl oxidized tetrahydrofolic acid. In other embodiments, the pharmaceutical composition comprises γ-hexaglutamyl oxidized tetrahydrofolic acid.

[0077] In some embodiments, the pharmaceutical composition comprises liposomal γPTHF (e.g., Lp-γPTHF, PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF, and TPLp-γPTHF). In some embodiments, the liposomal γPTHF composition is pegylated (e.g., NTPLp-γPTHF, and TPLp-γPTHF). In some embodiments, liposomal γPTHF comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TLp-γPTHF or TPLp-γPTHF)). In further embodiments, the pharmaceutical composition comprises a liposomal γPTHF composition that is pegylated and further comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TPLp-γPTHF). In some embodiments, the pharmaceutical composition comprises a liposomal γPTHF that is cationic. In other embodiments, the pharmaceutical composition comprises a liposomal γPTHF that is anionic or neutral. In additional embodiments, the pharmaceutical composition comprises a liposomal γPTHF having a diameter in the range of 20 nm to 500 nm or 20 nm to 500 nm, or any range therebetween. In further embodiments, the liposomal γPTHF composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

[0078] In additional embodiments, the present disclosure provides a method of killing cells comprising contacting the cells with a composition comprising a gamma polyglutamylated tetrahydrofolate composition (γPTHF) (e.g., γPTHF as disclosed herein). In some embodiments, the cells being contacted are mammalian cells. In further embodiments, the cells being contacted are human cells. In some embodiments, the cells being contacted are hyperproliferative cells. In further embodiments, the hyperproliferative cells are cancer cells. In further embodiments, the cancer cells being contacted are primary cells or cell line-derived cells obtained from / derived from cancer selected from the group consisting of: for example, non-hematological tumors including lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and, for example, hematological tumors such as leukemia, lymphoma and other B cell malignancies, multiple myeloma and other plasma cell dyscrasias or malignancies. In further embodiments, the cancer cells being contacted are primary cells or cell line-derived cells obtained from / derived from cancer selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, and villous adenoma, aleukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis, bladder cancer, and central nervous system (CNS) lymphoma. In yet another embodiment, the cancer cells are primary cells or cell line-derived cells obtained from / derived from cancer selected from colorectal cancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In certain embodiments, the cancer cells are primary cells or cell line-derived cells obtained from / derived from colorectal cancer. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the γPTHF contains 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the γPTHF contains 5-formyl-THF containing 4 γ-glutamyl groups.In some embodiments, γPTHF contains 5-formyl-THF containing five γ-glutamyl groups. In some embodiments, γPTHF contains six γ-glutamyl groups. In some embodiments, γPTHF contains D-type γ-glutamyl groups. In some embodiments, γPTHF contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, γPTHF contains L-type γ-glutamyl groups. In some embodiments, γPTHF contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, γPTHF contains both L- and D-type γ-glutamyl groups. In some embodiments, γPTHF contains 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the γPTHF composition contains γ-tetraglutamine oxidized tetrahydrofolic acid. In some embodiments, the γPTHF composition contains γ-pentaglutamine oxidized tetrahydrofolic acid. In other embodiments, the γPTHF composition contains γ-hexaglutamine oxidized tetrahydrofolic acid.

[0079] In additional embodiments, the present disclosure provides a method of killing cells, comprising contacting the cells with liposomes containing γ-polyglutamylated tetrahydrofolate (e.g., Lp-γPTHF such as PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF or TPLp-γPTHF). In some embodiments, the cells being contacted are mammalian cells. In further embodiments, the cells being contacted are human cells. In some embodiments, the cells being contacted are hyperproliferative cells. In yet another embodiment, the hyperproliferative cells being contacted are cancer cells. In further embodiments, the cancer cells are primary cells or cell line-derived cells obtained from / derived from cancers selected from the group consisting of: for example, non-hematological tumors including lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors such as, for example, leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias or malignancies. In further embodiments, the cancer cells being contacted are primary cells or cell line-derived cells obtained from / derived from cancers selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, and villous adenoma, aleukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis, bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the cancer cells are primary cells or cell line-derived cells obtained from / derived from cancers selected from colorectal cancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In certain embodiments, the cancer cells are primary cells or cell line-derived cells obtained from / derived from colorectal cancer. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the liposomes contain γPTHF containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups.In some embodiments, the liposome contains γ-tetraglutamyl oxidized tetrahydrofolic acid. In some embodiments, the liposome contains γ-pentaglutamyl oxidized tetrahydrofolic acid. In other embodiments, the liposome contains γ-hexaglutamyl oxidized tetrahydrofolic acid.

[0080] In some embodiments, the liposome comprises γPTHF containing 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the liposome comprises γPTHF containing 4 γ-glutamyl groups. In some embodiments, the liposome comprises γPTHF containing 5 γ-glutamyl groups. In some embodiments, the liposome comprises γPTHF containing 6 γ-glutamyl groups. In some embodiments, the liposome comprises γPTHF containing D-type γ-glutamyl groups. In some embodiments, the liposome comprises γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the liposome comprises γPTHF containing L-type γ-glutamyl groups. In some embodiments, the liposome comprises γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposome comprises γPTHF containing both L-type and D-type γ-glutamyl groups. In some embodiments, the liposome comprises γPTHF containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the liposome comprises γ-pentaglutamyl oxidized tetrahydrofolic acid. In other embodiments, the liposome comprises γ-hexaglutamyl oxidized tetrahydrofolic acid.

[0081] In additional embodiments, the present disclosure provides a method for treating cancer, comprising administering to a subject having or at risk of having cancer a delivery vehicle (e.g., an immune complex or liposome) comprising an effective amount of γ-polyglutamyl oxidized tetrahydrofolate. In some embodiments, the delivery vehicle is an antibody-containing immune complex (e.g., comprising a full-length IgG antibody, a bispecific antibody, or a scFv). In some embodiments, the delivery vehicle is a liposome (e.g., an Lp-γPTHF such as PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF, or TPLp-γPTHF). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is not pegylated. In additional embodiments, the administered delivery vehicle comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of cancer cells.In additional embodiments, the delivery vehicle comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRα., PDGFRβ, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery vehicle comprises a targeting moiety that specifically binds to a cell surface antigen(s) determined to be derived from or expressed on a cancer (tumor) of a specific subject, such as a neoantigen. In some embodiments, the targeting moiety has a specific affinity for an epitope of a cell surface antigen(s) determined to be derived from or expressed on a tumor of a specific subject, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the administered delivery vehicle comprises a γPTHF containing 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the administered delivery vehicle comprises a γPTHF containing 4 γ-glutamyl groups. In some embodiments, the administered delivery vehicle comprises a γPTHF containing 5 γ-glutamyl groups. In some embodiments, the administered delivery vehicle comprises a γPTHF containing 6 γ-glutamyl groups. In some embodiments, the γPTHF is a member selected from the following: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5-methyl-THF (e.g., polyglutamylated [6S]-5-methyl-THF); (e) polyglutamylated tetrahydrofolic acid THF (e.g., polyglutamylated [6S]-tetrahydrofolic acid THF); (f) polyglutamylated 5,10-methylene-THF (e.g., polyglutamylated [6R]-5,10-methylene-THF); and (g) polyglutamylated 5-formimino-THF (e.g., polyglutamylated [6S]-5-formimino-THF). In some embodiments, the γPTHF is polyglutamylated 5,10-methylene-THF. In a further embodiment, the γPTHF is polyglutamylated [6R]-5,10-methylene-THF.In other embodiments, γPTHF is polyglutaminylated [6R,S]-5,10-methylene-THF. In some embodiments, γPTHF is polyglutaminylated 5-methyl-THF. In further embodiments, γPTHF is [6S]-5-methyl-THF. In other embodiments, γPTHF is [6R,S]-5-methyl-THF. In some embodiments, γPTHF is polyglutaminylated 5-formyl-THF. In further embodiments, γPTHF is polyglutaminylated [6S]-5-formyl-THF. In other embodiments, γPTHF is polyglutaminylated [6R,S]-5-formyl-THF.

[0082] In some embodiments, the delivery vehicle comprises γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the delivery vehicle comprises γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the delivery vehicle comprises γPTHF containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5 or more than 5 D-type γ-glutamyl groups. In some embodiments, the administered delivery vehicle comprises γ-tetraglutamine oxidized tetrahydrofolic acid. In some embodiments, the administered delivery vehicle comprises γ-pentaglutamine oxidized tetrahydrofolic acid. In other embodiments, the administered delivery vehicle comprises γ-hexaglutamine oxidized tetrahydrofolic acid. In some embodiments, the administered delivery vehicle comprises γPTHF selected from the following: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5-methyl-THF (e.g., polyglutamylated [6S]-5-methyl-THF); (e) polyglutamylated tetrahydrofolic acid THF (e.g., polyglutamylated [6S]-tetrahydrofolic acid THF); (f) polyglutamylated 5,10-methylene-THF (e.g., polyglutamylated [6R]-5,10-methylene-THF); and (g) polyglutamylated 5-formimino-THF (e.g., polyglutamylated [6S]-5-formimino-THF). In some embodiments, the administered delivery vehicle comprises polyglutamylated 5,10-methylene-THF. In a further embodiment, the administered delivery vehicle comprises polyglutamylated [6R]-5,10-methylene-THF. In other embodiments, the administered delivery vehicle comprises polyglutamylated [6R,S]-5,10-methylene-THF.In some embodiments, the delivery vehicle to be administered comprises polyglutamylated 5-methyl-THF. In further embodiments, the delivery vehicle to be administered comprises [6S]-5-methyl-THF. In other embodiments, the delivery vehicle to be administered comprises [6R,S]-5-methyl-THF. In some embodiments, the delivery vehicle to be administered comprises polyglutamylated 5-formyl-THF. In further embodiments, the delivery vehicle to be administered comprises polyglutamylated [6S]-5-formyl-THF. In other embodiments, the delivery vehicle to be administered comprises polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the delivery vehicle to be administered comprises Lγ polyglutamylated tetrahydrofolate. In some embodiments, the delivery vehicle to be administered comprises Dγ polyglutamylated tetrahydrofolate. In further embodiments, the delivery vehicle to be administered comprises L and Dγ polyglutamylated tetrahydrofolate. In some embodiments, the cancer is selected from the group consisting of: for example, non-blood system tumors including lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma, brain cancer, central nervous system cancer, and melanoma; and, for example, blood system tumors such as leukemia, lymphoma and other B cell malignancies, multiple myeloma and other plasma cell dysplasias or cachexia. In some embodiments, the cancer is selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, and villous adenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis, bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the cancer is selected from the group consisting of: colorectal cancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In certain embodiments, the cancer is colorectal cancer.

[0083] In additional embodiments, the present disclosure provides a method for treating cancer, comprising administering to a subject having or at risk of having cancer an effective amount of liposomes (e.g., Lp-γPTHF such as PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF, or TPLp-γPTHF) comprising γ-polyglutamyl oxidized tetrahydrofolate. In some embodiments, the liposomes are pegylated. In some embodiments, the liposomes are not pegylated. In additional embodiments, the liposomes comprise a targeting moiety having specific affinity for an epitope of an antigen on the surface of cancer cells.In additional embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRα., PDGFRβ, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.This also includes the use of cancer stem cell targeting moieties such as those targeting CD34, CD133 and CD44, CD138, and CD15. In some embodiments, the liposome comprises a targeting moiety having a specific affinity for an epitope of a cell surface antigen(s) determined to be derived from or expressed on the tumor of a particular subject, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the liposome comprises γPTHF containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered liposome comprises a γPTHF selected from the following: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5-methyl-THF (e.g., polyglutamylated [6S]-5-methyl-THF); (e) polyglutamylated tetrahydrofolic acid THF (e.g., polyglutamylated [6S]-tetrahydrofolic acid THF); (f) polyglutamylated 5,10-methylene-THF (e.g., polyglutamylated [6R]-5,10-methylene-THF); and (g) polyglutamylated 5-formimino-THF (e.g., polyglutamylated [6S]-5-formimino-THF). In some embodiments, the administered liposome comprises polyglutamylated 5,10-methylene-THF. In a further embodiment, the administered liposome comprises polyglutamylated [6R]-5,10-methylene-THF. In other embodiments, the administered liposome comprises polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the administered liposome comprises polyglutamylated 5-methyl-THF. In a further embodiment, the administered liposome comprises [6S]-5-methyl-THF. In other embodiments, the administered liposome comprises [6R,S]-5-methyl-THF.In some embodiments, the liposomes to be administered contain polyglutamylated 5-formyl-THF. In further embodiments, the liposomes to be administered contain polyglutamylated [6S]-5-formyl-THF. In other embodiments, the liposomes to be administered contain polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposomes of the liposome composition to be administered contain γPTHF containing an L-type γ-glutamyl group. In some embodiments, the liposomes of the liposome composition to be administered contain γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposomes of the liposome composition to be administered contain γPTHF containing both L- and D-type γ-glutamyl groups. In some embodiments, the liposomes of the liposome composition to be administered contain γPTHF containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the liposome composition to be administered contains tetraglutamylated γPTHF. In some embodiments, the liposome composition to be administered contains pentaglutamylated γPTHF. In some embodiments, the liposome composition to be administered contains hexaglutamylated γPTHF. In some embodiments, the cancer is selected from the group consisting of: lung (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, and hematological tumors (e.g., leukemia or lymphoma). In yet another embodiment, the cancer cells are primary cells or cell line-derived cells obtained from / derived from a cancer selected from colorectal cancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In certain embodiments, the cancer cells are primary cells or cell line-derived cells obtained from / derived from colorectal cancer.

[0084] In an additional embodiment, the present disclosure provides a method for treating cancer, comprising administering to a subject having or at risk of having cancer, a liposomal composition comprising a liposome comprising an effective amount of γ-polyglutamylated tetrahydrofolic acid and a targeting moiety having specific affinity for an epitope of an antigen on the surface of the cancer.In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRα., PDGFRβ, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen(s) determined to be derived from or expressed on the cancer (tumor) of a particular subject, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the liposome comprises γPTHF containing 4, 5, 2 - 10, 4 - 6, or more than 5 γ-glutamyl groups. In some embodiments, the administered liposome comprises γPTHF containing 4 γ-glutamyl groups. In some embodiments, the administered liposome comprises γPTHF containing 5 γ-glutamyl groups. In some embodiments, the administered liposome comprises γPTHF containing 6 γ-glutamyl groups. In some embodiments, the administered liposome comprises a γPTHF selected from the following: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5-methyl-THF (e.g., polyglutamylated [6S]-5-methyl-THF); (e) polyglutamylated tetrahydrofolic acid THF (e.g., polyglutamylated [6S]-tetrahydrofolic acid THF); (f) polyglutamylated 5,10-methylene-THF (e.g., polyglutamylated [6R]-5,10-methylene-THF); and (g) polyglutamylated 5-formimino-THF (e.g., polyglutamylated [6S]-5-formimino-THF). In some embodiments, the administered liposome comprises polyglutamylated 5,10-methylene-THF. In a further embodiment, the administered liposome comprises polyglutamylated [6R]-5,10-methylene-THF. In other embodiments, the administered liposome comprises polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the administered liposome comprises polyglutamylated 5-methyl-THF.In further embodiments, the administered liposomes contain [6S]-5-methyl-THF. In other embodiments, the administered liposomes contain [6R,S]-5-methyl-THF. In some embodiments, the administered liposomes contain polyglutamylated 5-formyl-THF. In further embodiments, the administered liposomes contain polyglutamylated [6S]-5-formyl-THF. In other embodiments, the administered liposomes contain polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposomes contain γPTHF containing an L-type γ-glutamyl group. In some embodiments, the liposomes contain γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposomes contain γPTHF containing a D-type γ-glutamyl group. In some embodiments, the liposomes contain γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the liposomes contain γ-tetraglutamylated tetrahydrofolic acid. In some embodiments, the liposomes contain γ-pentaglutamylated tetrahydrofolic acid. In some embodiments, the liposomes contain γ-hexaglutamylated tetrahydrofolic acid.

[0085] In some embodiments, the administered liposomal composition comprises pegylated liposomes (e.g., TPLp-γPTHF). In some embodiments, the administered liposomal composition comprises non-pegylated liposomes. In some embodiments, the liposomes of the administered liposomal composition comprise γPTHF containing 4, 5, 2 - 10, 4 - 6, or more than 5 γ-glutamyl groups. In some embodiments, the administered liposomes comprise γPTHF containing 4 γ-glutamyl groups. In some embodiments, the administered liposomes comprise γPTHF containing 5 γ-glutamyl groups. In some embodiments, the administered liposomes comprise γPTHF containing 6 γ-glutamyl groups. In some embodiments, the administered liposomes comprise γPTHF selected from the following: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5-methyl-THF (e.g., polyglutamylated [6S]-5-methyl-THF); (e) polyglutamylated tetrahydrofolic acid THF (e.g., polyglutamylated [6S]-tetrahydrofolic acid THF); (f) polyglutamylated 5,10-methylene-THF (e.g., polyglutamylated [6R]-5,10-methylene-THF); and (g) polyglutamylated 5-formimino-THF (e.g., polyglutamylated [6S]-5-formimino-THF). In some embodiments, the administered liposomes comprise polyglutamylated 5,10-methylene-THF. In a further embodiment, the administered liposomes comprise polyglutamylated [6R]-5,10-methylene-THF. In other embodiments, the administered liposomes comprise polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the administered liposomes comprise polyglutamylated 5-methyl-THF. In a further embodiment, the administered liposomes comprise [6S]-5-methyl-THF.In other embodiments, the liposomes administered contain [6R,S]-5-methyl-THF. In some embodiments, the liposomes administered contain polyglutamylated 5-formyl-THF. In further embodiments, the liposomes administered contain polyglutamylated [6S]-5-formyl-THF. In other embodiments, the liposomes administered contain polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposomes of the liposome composition administered contain γPTHF containing D-type γ-glutamyl groups. In some embodiments, the liposomes of the liposome composition administered contain γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the liposomes of the liposome composition administered contain γPTHF containing L-type γ-glutamyl groups. In some embodiments, the liposomes of the liposome composition administered contain γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposomes of the liposome composition administered contain γPTHF containing both L- and D-type γ-glutamyl groups. In some embodiments, the liposomes of the liposome composition administered contain γPTHF containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the liposomes of the liposome composition administered contain γ-tetraglutamylated tetrahydrofolic acid. In some embodiments, the liposomes of the liposome composition administered contain γ-pentaglutamylated tetrahydrofolic acid. In other embodiments, the liposomes of the liposome composition administered contain γ-hexaglutamylated tetrahydrofolic acid. In some embodiments, the liposome composition is administered to treat cancer selected from the group consisting of: lung cancer (e.g., non-small cell), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, myeloma, leukemia, and lymphoma.In some embodiments, the liposome composition is administered to treat cancer selected from the group consisting of: colorectal cancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In certain embodiments, the liposome composition is administered to treat colorectal cancer.

[0086] In additional embodiments, the present disclosure provides a method for treating cancer, comprising administering to a subject having or at risk of having cancer that expresses a folate receptor on its cell surface an effective amount of a liposomal composition, the liposomal composition comprising liposomes comprising (a) gamma-polyglutamylated tetrahydrofolate (γPTHF) and (b) a targeting moiety having specific binding affinity for the folate receptor. In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α) and folate receptor beta (FR-β). In some embodiments, the liposomal composition to be administered comprises pegylated liposomes (e.g., TPLp-γPTHF). In some embodiments, the liposomal composition to be administered comprises non-pegylated liposomes. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPTHF containing 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the liposomes to be administered comprise γPTHF containing 4 γ-glutamyl groups. In some embodiments, the liposomes to be administered comprise γPTHF containing 5 γ-glutamyl groups. In some embodiments, the liposomes to be administered comprise γPTHF containing 6 γ-glutamyl groups.In some embodiments, the liposomes to be administered comprise a γPTHF selected from the following: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5-methyl-THF (e.g., polyglutamylated [6S]-5-methyl-THF); (e) polyglutamylated tetrahydrofolic acid THF (e.g., polyglutamylated [6S]-tetrahydrofolic acid THF); (f) polyglutamylated 5,10-methylene-THF (e.g., polyglutamylated [6R]-5,10-methylene-THF); and (g) polyglutamylated 5-formimino-THF (e.g., polyglutamylated [6S]-5-formimino-THF). In some embodiments, the liposomes to be administered comprise polyglutamylated 5,10-methylene-THF. In further embodiments, the liposomes to be administered comprise polyglutamylated [6R]-5,10-methylene-THF. In other embodiments, the liposomes to be administered comprise polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the liposomes to be administered comprise polyglutamylated 5-methyl-THF. In further embodiments, the liposomes to be administered comprise [6S]-5-methyl-THF. In other embodiments, the liposomes to be administered comprise [6R,S]-5-methyl-THF. In some embodiments, the liposomes to be administered comprise polyglutamylated 5-formyl-THF. In further embodiments, the liposomes to be administered comprise polyglutamylated [6S]-5-formyl-THF. In other embodiments, the liposomes to be administered comprise polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposomes of the liposome composition to be administered comprise a γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups.In some embodiments, the liposomes of the administered liposomal composition comprise γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPTHF containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5 or more than 5 D-type γ-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γ-pentaglutamyl oxidized tetrahydrofolic acid. In other embodiments, the liposomes of the administered liposomal composition comprise γ-hexaglutamyl oxidized tetrahydrofolic acid. In some embodiments, the liposomal composition is administered to treat cancer selected from the group consisting of: for example, non-hematological tumors including lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and, for example, hematological tumors such as leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias or cachexia. In some embodiments, the liposomal composition is administered to treat cancer selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, and villous adenoma, aleukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis, bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments the liposomal composition is administered to treat cancer selected from the group consisting of: colorectal cancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma.

[0087] In additional embodiments, the present disclosure provides a method for maintenance therapy of cancer, comprising administering to a subject who is undergoing or has undergone cancer therapy a liposomal composition comprising an effective amount of liposomes (Lp-γPTHF) containing γ-polyglutamyl oxidized tetrahydrofolic acid. In some embodiments, the liposomal composition to be administered is PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF or TPLp-γPTHF. In some embodiments, the liposomal composition to be administered comprises pegylated liposomes (e.g., PLp-γPTHF, NTPLp-γPTHF, or TPLp-γPTHF). In some embodiments, the liposomal composition to be administered comprises targeted liposomes (e.g., TLp-γPTHF or TPLp-γPTHF). In some embodiments, the liposomal composition to be administered comprises liposomes that are pegylated and contain a targeting moiety (e.g., TPLp-γPTHF). In some embodiments, the liposomes of the liposomal composition to be administered comprise γ-polyglutamyl oxidized tetrahydrofolic acid containing 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the liposomes to be administered comprise γPTHF containing 4 γ-glutamyl groups. In some embodiments, the liposomes to be administered comprise γPTHF containing 5 γ-glutamyl groups. In some embodiments, the liposomes to be administered comprise γPTHF containing 6 γ-glutamyl groups.In some embodiments, the liposomes to be administered contain γPTHF selected from the following: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5-methyl-THF (e.g., polyglutamylated [6S]-5-methyl-THF); (e) polyglutamylated tetrahydrofolic acid THF (e.g., polyglutamylated [6S]-tetrahydrofolic acid THF); (f) polyglutamylated 5,10-methylene-THF (e.g., polyglutamylated [6R]-5,10-methylene-THF); and (g) polyglutamylated 5-formimino-THF (e.g., polyglutamylated [6S]-5-formimino-THF). In some embodiments, the liposomes to be administered contain polyglutamylated 5,10-methylene-THF. In a further embodiment, the liposomes to be administered contain polyglutamylated [6R]-5,10-methylene-THF. In other embodiments, the liposomes to be administered contain polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the liposomes to be administered contain polyglutamylated 5-methyl-THF. In a further embodiment, the liposomes to be administered contain [6S]-5-methyl-THF. In other embodiments, the liposomes to be administered contain [6R,S]-5-methyl-THF. In some embodiments, the liposomes to be administered contain polyglutamylated 5-formyl-THF. In a further embodiment, the liposomes to be administered contain polyglutamylated [6S]-5-formyl-THF. In other embodiments, the liposomes to be administered contain polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposomes of the liposome composition to be administered contain γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups.In some embodiments, the liposomes of the administered liposomal composition comprise γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPTHF containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5 or more than 5 D-type γ-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γ-tetraglutamine oxidized tetrahydrofolic acid. In some embodiments, the liposomes of the administered liposomal composition comprise γ-pentaglutamine oxidized tetrahydrofolic acid. In other embodiments, the liposomes of the administered liposomal composition comprise γ-hexaglutamine oxidized tetrahydrofolic acid.

[0088] In additional embodiments, the present disclosure provides a method for treating an immune system disorder, comprising administering to a subject having or at risk of having a disorder of the immune system, a liposomal composition (e.g., Lp-γPTHF, PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF or TPLp-γPTHF) comprising liposomes containing an effective amount of γ-polyglutamyl oxidized tetrahydrofolate. In some embodiments, the liposomal composition is administered to treat an autoimmune disease. In further embodiments, the liposomal composition is administered to treat rheumatoid arthritis. In another embodiment, the liposomal composition is administered to treat inflammation. In some embodiments, the liposomal composition administered comprises pegylated liposomes (e.g., PLp-γPTHF, NTPLp-γPTHF, or TPLp-γPTHF). In some embodiments, the liposomal composition administered comprises targeted liposomes containing a targeting moiety having specific affinity for a surface antigen on a target cell of interest (e.g., an immune cell) (e.g., TLp-γPTHF or TPLp-γPTHF). In further embodiments, the liposomal composition administered comprises pegylated liposomes containing a targeting moiety (e.g., TPLp-γPTHF). In some embodiments, the liposomes of the liposomal composition administered comprise γ-pentaglutamyl oxidized tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the liposomes administered comprise γPTHF containing 4 γ-glutamyl groups. In some embodiments, the liposomes administered comprise γPTHF containing 5 γ-glutamyl groups. In some embodiments, the liposomes administered comprise γPTHF containing 6 γ-glutamyl groups.In some embodiments, the liposomes to be administered contain γPTHF selected from the following: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5-methyl-THF (e.g., polyglutamylated [6S]-5-methyl-THF); (e) polyglutamylated tetrahydrofolic acid THF (e.g., polyglutamylated [6S]-tetrahydrofolic acid THF); (f) polyglutamylated 5,10-methylene-THF (e.g., polyglutamylated [6R]-5,10-methylene-THF); and (g) polyglutamylated 5-formimino-THF (e.g., polyglutamylated [6S]-5-formimino-THF). In some embodiments, the liposomes to be administered contain polyglutamylated 5,10-methylene-THF. In a further embodiment, the liposomes to be administered contain polyglutamylated [6R]-5,10-methylene-THF. In other embodiments, the liposomes to be administered contain polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the liposomes to be administered contain polyglutamylated 5-methyl-THF. In a further embodiment, the liposomes to be administered contain [6S]-5-methyl-THF. In other embodiments, the liposomes to be administered contain [6R,S]-5-methyl-THF. In some embodiments, the liposomes to be administered contain polyglutamylated 5-formyl-THF. In a further embodiment, the liposomes to be administered contain polyglutamylated [6S]-5-formyl-THF. In other embodiments, the liposomes to be administered contain polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposomes of the liposome composition to be administered contain γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups.In some embodiments, the liposomes of the administered liposomal composition comprise γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPTHF containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5 or more than 5 D-type γ-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γ-tetraglutamine oxidized tetrahydrofolic acid. In some embodiments, the liposomes of the administered liposomal composition comprise γ-pentaglutamine oxidized tetrahydrofolic acid. In other embodiments, the liposomes of the administered liposomal composition comprise γ-hexaglutamine oxidized tetrahydrofolic acid.

[0089] The present disclosure also provides a method for delivering γ-polyglutamylated tetrahydrofolate to tumors and / or cancer cells, which includes administering to a subject having a tumor a composition comprising γ-polyglutamylated tetrahydrofolate (L-γPTHF) and a targeting moiety having specific binding affinity for an epitope of a surface antigen on tumor cells or cancer cells. In some embodiments, the targeting moiety to be administered is conjugated to a delivery vehicle. In some embodiments, the delivery vehicle is an antibody or an antigen-binding fragment of an antibody. In further embodiments, the delivery vehicle is a liposome. In some embodiments, the antibody, antigen-binding antibody fragment, or liposome is pegylated. In some embodiments, the composition to be administered comprises γ-polyglutamylated tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the composition to be administered comprises γ-tetraglutamylated tetrahydrofolate. In some embodiments, the composition to be administered comprises γ-pentaglutamylated tetrahydrofolate. In other embodiments, the composition to be administered comprises γ-hexaglutamylated tetrahydrofolate. In some embodiments, the composition to be administered comprises a γPTHF selected from the following: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5-methyl-THF (e.g., polyglutamylated [6S]-5-methyl-THF); (e) polyglutamylated tetrahydrofolate THF (e.g., polyglutamylated [6S]-tetrahydrofolate THF); (f) polyglutamylated 5,10-methylene-THF (e.g., polyglutamylated [6R]-5,10-methylene-THF); and (g) polyglutamylated 5-formimino-THF (e.g., polyglutamylated [6S]-5-formimino-THF). In some embodiments, the composition to be administered comprises polyglutamylated 5,10-methylene-THF.In further embodiments, the administered composition comprises polyglutamylated [6R]-5,10-methylene-THF. In other embodiments, the administered composition comprises polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the administered composition comprises polyglutamylated 5-methyl-THF. In further embodiments, the administered composition comprises [6S]-5-methyl-THF. In other embodiments, the administered composition comprises [6R,S]-5-methyl-THF. In some embodiments, the administered composition comprises polyglutamylated 5-formyl-THF. In further embodiments, the administered composition comprises polyglutamylated [6S]-5-formyl-THF. In other embodiments, the administered composition comprises polyglutamylated [6R,S]-5-formyl-THF.

[0090] In additional embodiments, the present disclosure provides a method of preparing a liposomal composition comprising a liposomal γ-polyglutamylated tetrahydrofolic acid (γPTHF) composition, the method comprising: forming a mixture comprising liposomal components and γ-polyglutamylated tetrahydrofolic acid in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes containing polyglutamylated tetrahydrofolic acid. In some embodiments, the γ-polyglutamylated tetrahydrofolic acid contains 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the γPTHF composition contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the γPTHF composition contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the γPTHF composition contains 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5 or more than 5 D-type γ-glutamyl groups. In some embodiments, the γPTHF composition comprises γ-pentaglutamylated tetrahydrofolic acid. In some embodiments, the γPTHF composition comprises γ-tetraglutamylated tetrahydrofolic acid. In other embodiments, the γPTHF composition comprises γ-hexaglutamylated tetrahydrofolic acid.In some embodiments, the composition comprises γPTHF selected from the following: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5-methyl-THF (e.g., polyglutamylated [6S]-5-methyl-THF); (e) polyglutamylated tetrahydrofolic acid THF (e.g., polyglutamylated [6S]-tetrahydrofolic acid THF); (f) polyglutamylated 5,10-methylene-THF (e.g., polyglutamylated [6R]-5,10-methylene-THF); and (g) polyglutamylated 5-formimino-THF (e.g., polyglutamylated [6S]-5-formimino-THF). In some embodiments, the composition comprises polyglutamylated 5,10-methylene-THF. In further embodiments, the composition comprises polyglutamylated [6R]-5,10-methylene-THF. In other embodiments, the composition comprises polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises polyglutamylated 5-methyl-THF. In further embodiments, the composition comprises [6S]-5-methyl-THF. In other embodiments, the composition comprises [6R,S]-5-methyl-THF. In some embodiments, the composition comprises polyglutamylated 5-formyl-THF. In further embodiments, the composition comprises polyglutamylated [6S]-5-formyl-THF. In other embodiments, the administered composition comprises polyglutamylated [6R,S]-5-formyl-THF.

[0091] In one embodiment, the present disclosure provides a kit comprising a γ-polyglutamylated tetrahydrofolic acid composition and / or a γPTHF delivery vehicle such as a liposome containing γPTHF or a γPTHF immunocomplex (e.g., ADC) described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0092]

FIG. 1A-1L

FIG. 2

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FIG. 9

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FIG. 21A-F

BRIEF DESCRIPTION OF THE DRAWINGS

[0093] The present disclosure generally relates to γ-polyglutamylated tetrahydrofolate compositions. The compositions provide an advance over prior treatments for proliferative disorders such as cancer. Methods of making, delivering, and using γ-polyglutamylated tetrahydrofolate compositions are also provided. The γ-polyglutamylated compositions have uses including, but not limited to, treating (e.g., treating and / or preventing) proliferative disorders such as cancer, immune system disorders such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria. The γ-polyglutamylated compositions also have uses in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effectiveness of the therapeutic agent(s), or as a “chemoprotectant” (e.g., in combination with a folate antagonist such as methotrexate) to reduce the toxic side effects associated with the therapeutic agent(s).

[0094] I. Definitions Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0095] When embodiments are described in this specification in conjunction with the term "comprising", it is to be understood that other similar embodiments described with the terms "containing", "consisting of", and / or "consisting essentially of" are provided as well. However, when used as a transitional phrase in the claims, each should be interpreted separately and in an appropriate legal and factual context (e.g., in the claims, the transitional phrase "comprising" is considered a more open-ended phrase, the transitional phrase "consisting of" is considered more exclusive, and "consisting essentially of" is considered intermediate between these).

[0096] As used in this specification, the singular forms "a", "an", and "the" include plural referents unless specifically stated otherwise or it is otherwise clear from the context that only a single referent is intended.

[0097] The term "and / or" as used in expressions such as "A and / or B" is intended in this specification to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in expressions such as "A, B and / or C" encompasses each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0098] Headings and subheadings are used for convenience and / or only for compliance with official rules, and neither limit the subject matter nor are they referred to in connection with the interpretation of the description of the subject matter. Features described under one heading or one subheading of the subject disclosure may be combined with features described under other headings or subheadings in various embodiments. Further, not all features under a single heading or subheading are necessarily used together in some embodiments.

[0099] The terms "tetrahydrofolic acid" and "THF" are used interchangeably and include salts, acids and / or free base forms of tetrahydrofolic acid (e.g., disodium tetrahydrofolate). Unless expressly stated otherwise or unambiguously clear from the context, "THF(s)" and "tetrahydrofolic acid(s)" include natural and non-natural THF forms, including 1-carbon-substituted TNF derivatives. In particular, unless expressly stated otherwise or unambiguously clear from the context, "THF(s)" and "tetrahydrofolic acid(s)" include a diastereomeric composition having an [6R] configuration at the C-6 atom of the tetrahydropterin component of THF, a diastereomeric composition having an [6S] configuration at the C-6 atom, and / or a mixture of [6,R,S] diastereomers (e.g., 1:1). Unless expressly stated otherwise or unambiguously clear from the context, "THF(s)" and "tetrahydrofolic acid(s)" include the following: (a) 5-formyl-THF (e.g., [6S], [6R,S], or [6R]-5-formyl-THF); (b) 5-formyl-THF (e.g., [6S], [6R,S], or [6R]-5-formyl-THF); (c) 5,10-methenyl-THF (e.g., [6R], [6R,S], or [6S],-5,10-methenyl-THF); (d) 5-methyl-THF (e.g., [6S], [6R,S], or [6R],-5-methyl-THF); (e) tetrahydrofolic acid THF ((2S)-2-{[4-({[2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}amino)phenyl]formamido}pentanedioic acid) ((e.g., [6S], [6R,S], and [6R,]-((2S)-2-{[4-({[2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}amino)phenyl]formamido}pentanedioic acid)); (f) 5,10-methylene-THF (e.g., [6R], [6R,S], or [SR],-5,10-methylene-THF); and (g) 5-formimino-THF (e.g., [6S], [6R,S], or [6R],-5-formimino-THF).In some embodiments, the present disclosure provides a composition containing a THF diastereomer selected from the following: (a) [6S]-5-formyl-THF; (b) [6R]-5-formyl-THF; (c) [6R]-5,10-methenyl-THF; (d) [6S]-5-methyl-THF; (e) [6S]-tetrahydrofolic acid; (f) [6R]-5,10-methylene-THF; and (g) [6S]-5-formimino-THF. In some embodiments, the present disclosure provides a composition containing a mixture of THF diastereomers (e.g., a mixture of diastereoisomers [6R,S]-5-methyl-THF (1:1) and / or a mixture of diastereoisomers [6R,S]-5-CHO-THF (1:1)). The composition containing the THF salt is Na. + , Mg 2+ , K + , NH4 + , and / or Ca 2+ and may further contain any of a variety of cations such as. In certain embodiments, the salt is a pharmaceutically acceptable salt. In additional certain embodiments, the THF salt contains Na + . Tetrahydrofolic acid contains one L-γ-glutamyl group and is considered to be monoglutamylated for the purposes of the present disclosure.

[0100] The term "tetrahydrofolic acid THF" specifically refers to a THF composition having the structure 2-{[4-({[(6S)-2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}amino)phenyl]formamide}pentanedioic acid. "Tetrahydrofolic acid THF" is also referred to herein as a type of tetrathydrolate (THF).

[0101] The terms "polyglutamylated-tetrahydrofolic acid", "polyglutamylated-THF", "THF-PG", "PTHF" and their repetitions are used herein with the same meaning and refer to a tetrahydrofolic acid composition containing at least one glutamyl group in addition to the glutamyl group of tetrahydrofolic acid (i.e., THF-PG n, where n ≧ 1). References in this specification to the number of glutamyl groups in γPTHF(THF-PG) take into account the glutamyl groups of tetrahydrofolic acid. For example, a THF-PG composition containing five glutamyl residues in addition to the glutamyl group of THF is referred to herein as hexaglutamylated tetrahydrofolic acid or tetrahydrofolic acid hexaglutamate. The polyglutamate chain contains an N-terminal glutamyl group and one or more C-terminal glutamyl groups. The N-terminal glutamyl group of the polyglutamate chain does not bind to another glutamyl group via its amino group, but binds to one or more glutamyl groups via its carboxylic acid group. In some embodiments, the N-terminal glutamyl group of polyglutamylated tetrahydrofolic acid is the glutamyl group of tetrahydrofolic acid. The C-terminal glutamyl group(s) of the polyglutamate chain bind to another glutamyl group via their amino groups, but do not bind to another glutamyl group via their carboxylic acid groups.

[0102] In some embodiments, polyglutamylated-tetrahydrofolate is a member selected from the following: (a) polyglutamylated 5-formyl-THF; (b) polyglutamylated 10-formyl-THF; (c) polyglutamylated 5,10-methenyl-THF; (d) polyglutamylated 5-methyl-THF; (e) polyglutamylated tetrahydrofolate ((2S)-2-{[4-({[2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}amino)phenyl]formamide}pentanedioic acid) (e.g., [6S], [6R,S], and [6R]-((2S)-2-{[4-({[2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}amino)phenyl]formamide}pentanedioic acid)); (f) polyglutamylated 5,10-methylene-THF; and (g) polyglutamylated 5-formimino-THF. In further embodiments, polyglutamylated-tetrahydrofolate is a member selected from the following: (a) polyglutamylated [6S]-5-formyl-THF; (b) polyglutamylated [6R]-10-formyl-THF; (c) polyglutamylated [6R]-5,10-methenyl-THF; (d) polyglutamylated [6S]-5-methyl-THF; (e) polyglutamylated [6S]-tetrahydrofolate; (f) polyglutamylated [6R]-5,10-methylene-THF; and (g) polyglutamic acid [6S]-5-formimino-THF. In some embodiments, polyglutamylated-tetrahydrofolate is [6R]-5,10-methylene-THF. In some embodiments, polyglutamylated-tetrahydrofolate is [6S]-5-methyl-THF. In some embodiments, polyglutamylated-tetrahydrofolate is [6S]-5-formyl-THF. In other embodiments, polyglutamylated-tetrahydrofolate is a [6R,S]-5,10-methylene-THF diastereomer mixture, a [6R,S]-5-methyl-THF diastereomer mixture, or a [6R,S]-5-formyl-THF diastereomer mixture (e.g., 1:1 w / w).

[0103] The terms "gamma glutamyl group", "γ-glutamyl group", and "γ-bond", when referring to the bond of a glutamyl group, refer to a glutamyl group containing a γ-carboxyl group bond. The γ-bond may be between a glutamyl group and the glutamyl group of tetrahydrofolic acid, or between a glutamyl group and a second glutamyl group not present in tetrahydrofolic acid (e.g., a glutamyl group within a polyglutamic acid chain attached to tetrahydrofolic acid). In some embodiments, the γ-bond is an amide bond between the γ-carboxyl group of one glutamyl group and a second glutamyl group. In some embodiments, the γ-bond refers to the amide bond of the glutamyl group in tetrahydrofolic acid. In some embodiments, the γ-bond is an amide bond between the γ-carboxyl group of one glutamyl group and a second glutamyl group. A reference to a γ-bond includes a γ-bond of a glutamyl group within tetrahydrofolic acid unless expressly stated otherwise or unambiguously clear from the context that such is not intended. In some embodiments, the γ-glutamyl group is of the L-type. In some embodiments, the γ-glutamyl group is of the D-type. As described herein, during tetrahydrofolic acid therapy, tetrahydrofolic acid enters cells and is polyglutaminated by the enzyme holylpoly-γ-glutamate synthetase (FPGS), which sequentially adds L-glutamyl groups to the γ-carboxyl group of glutamate within the tetrahydrofolic acid L-glutamyl group of tetrahydrofolic acid. As a result, D-γ-polyglutaminated tetrahydrofolic acid compositions are not formed intracellularly during tetrahydrofolic acid therapy.

[0104] The terms "γ-polyglutaminated tetrahydrofolic acid", "γ-polyglutamylated tetrahydrofolic acid", "γPTHF", "γ-polyglutamylated-tetrahydrofolic acid", "polyglutamylated-THF", "γTHF-PG", and their repeats are used herein with the same meaning and refer to a tetrahydrofolic acid composition (e.g., THF-PG) that includes at least one γ-glutamyl group having a γ-carboxyl group bond in addition to the γ-glutamyl group of tetrahydrofolic acid n, where n ≧ 1 refers to the γ-glutamyl group). In this specification, references to the number of glutamyl groups in γPTHF (γTHF-PG) take into account the glutamyl groups of tetrahydrofolic acid. For example, a γTHF-PG composition containing five γ-glutamyl groups in addition to the glutamyl group of THF may be referred to herein as γ-hexaglutamylated tetrahydrofolic acid or γ-tetrahydrofolic acid hexaglutamate.

[0105] The terms "α-glutamyl group", "α-glutamyl group", and "α-bond" refer to a glutamyl group containing an α-carboxyl group bond, as they relate to the bond of the glutamyl group.

[0106] As used herein, the term "isolated" means a composition in a form not found in nature. Isolated γ-polyglutamylated compositions include those that are purified to the extent that they are no longer in the form in which they are naturally found. In some embodiments, the isolated γ-polyglutamylated tetrahydrofolic acid is substantially pure. Isolated compositions are free or substantially free of substances that are naturally incorporated, such as other cellular components such as proteins and nucleic acids that may be found in nature or in the environment in which they are produced (e.g., cell culture). γ-Polyglutamylated compositions can be formulated with diluents or adjuvants and further isolated for practical purposes - for example, when used in a diagnostic or therapeutic agent, the γ-polyglutamylated composition is usually mixed with a pharmaceutically acceptable carrier or diluent. In some embodiments, the isolated γ-polyglutamylated composition (e.g., a delivery vehicle such as γ-polyglutamic acid and liposomes containing γ-polyglutamic acid) contains less than 1% or less than 0.1% of unwanted DNA or protein content. In some embodiments, the γ-polyglutamic acid composition (e.g., a delivery vehicle such as γ-polyglutamic acid and liposomes containing γ-polyglutamic acid) is "isolated".

[0107] As used herein, the term "targeting moiety" means a molecule that confers enhanced affinity for a selected target, such as a cell, cell type, tissue, organ, region of the body, or compartment, e.g., a compartment of a cell, tissue or organ. The targeting moiety can include a wide variety of entities. The targeting moiety includes natural molecules, or recombinant or synthetic molecules. In some embodiments, the targeting moiety is an antibody antigen-binding antibody fragment, a bispecific antibody or other antibody-based molecule or compound. In some embodiments, the targeting moiety is an aptamer, an avimer, a receptor-binding ligand, a nucleic acid, a biotin-avidin binding pair, a peptide, a protein, a carbohydrate, a lipid, a vitamin, a toxin, a microbial component, a hormone, a receptor ligand or any derivative thereof. Other targeting moieties are known in the art and are encompassed by the present disclosure.

[0108] The terms "specific affinity", "specifically bind", and "enhanced affinity" mean that a targeting moiety, such as an antibody or an antigen-binding antibody fragment, reacts or binds to an epitope, protein, or target molecule with a higher frequency, more rapidly, for a longer period, with a greater affinity, or in some combination of these, than to another substance containing a protein unrelated to the antigen containing the target epitope. Due to sequence identity between homologous proteins in different species, in some embodiments, a particular affinity involves a binding substance that recognizes an epitope on a protein and / or target molecule in two or more species. Similarly, due to homology within a particular region of the polypeptide sequences of different proteins, the terms "specific affinity" or "specifically bind" can include a binding substance that recognizes an epitope present on two or more proteins and / or target molecules. In certain embodiments, it is understood that a targeting moiety that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific affinity" does not necessarily (although it can) require exclusive binding, e.g., binding to only one epitope on one target. Thus, in certain embodiments, a targeting moiety can specifically bind to epitopes present on two or more targets. In certain embodiments, multiple targets can be bound by the same targeting moiety that specifically binds to epitopes present on the multiple targets.

[0109] The term "epitope" means a portion of an antigen that can be recognized and specifically bound by a targeting moiety (i.e., a binding portion), such as an antibody. When the antigen is a polypeptide, the epitope can be formed from both contiguous and non-contiguous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed from contiguous amino acids are usually retained upon protein denaturation, whereas epitopes formed by tertiary folding are usually lost upon protein denaturation. An epitope typically contains at least 3 amino acids, more generally at least 5 or 8 - 10 amino acids, in a unique spatial higher-order structure.

[0110] Expressions such as "binding affinity for a target", "binding to a target", "enhanced affinity" that are known in the art, and similar expressions, refer to properties of a targeting moiety that can be directly measured by determining an affinity constant, e.g., the amount of targeting moiety that binds and dissociates at a given antigen concentration. Without limitation, intermolecular interactions can be characterized using other methods such as competitive analysis, equilibrium analysis, and microcalorimetry, and real-time interaction analysis based on surface plasmon resonance interactions (e.g., using a Biacore® instrument). These methods are well known to those skilled in the art and are described, for example, in Neri et al., Tibtech 14:465-470 (1996), and Jansson et al., J. Biol. Chem. 272:8189-8197 (1997).

[0111] The term "delivery vehicle" generally means any composition that acts to assist, facilitate, or ease the entry of γ-polyglutamyl oxidized tetrahydrofolate into cells. Such delivery vehicles are known in the art and include, without limitation, liposomes, lipospheres, polymers (e.g., polymer conjugates), peptides, proteins such as antibodies (e.g., immunoconjugates such as antibody-drug conjugates (ADCs), antigen-binding antibody fragments, and derivatives thereof), cell components, cyclic oligosaccharides (e.g., cyclodextrins), micelles, microparticles (e.g., microspheres), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), hydrogels, lipoprotein particles, viral sequences, viral materials, or lipid or liposome formulations, and combinations thereof. The delivery vehicle can be bound directly or indirectly to the targeting moiety. In some embodiments, the targeting moiety is selected from a polymer, protein, peptide, monoclonal antibody, or fatty acid lipid.

[0112] "Subject" means a vertebrate mammal, including, but not limited to, humans, dogs, cats, horses, goats, and primates, such as monkeys. Thus, the present invention can also be used to treat diseases or conditions in non-human subjects. For example, cancer is one of the leading causes of death in companion animals (e.g., cats and dogs). In some embodiments of the present invention, the subject is human. In the present disclosure, the terms "subject" and "patient" are used interchangeably and have the same meaning. Generally, the maximum dose, i.e., the maximum safe dose in accordance with sound medical judgment, is preferably used.

[0113] As used herein, "effective amount" means an amount of a drug sufficient to produce a medically desired result. The effective amount can vary depending on the desired outcome, the particular condition to be treated or prevented, the age and health of the subject being treated, the severity of the condition, the duration of the treatment, the nature of concurrent or concomitant therapies (if any), the specific route of administration, and similar factors within the knowledge and professional opinion of the health care provider. The "effective amount" can be determined experimentally and routinely in relation to the indicated purpose. In the case of cancer, an effective amount of a drug reduces the number of cancer cells; reduces the size of the tumor; inhibits the invasion of cancer cells into surrounding organs (i.e., slows down to some extent and preferably stops); inhibits the metastasis of the tumor (i.e., slows down to some extent and preferably stops); inhibits the growth of the tumor to some extent; and / or alleviates one or more of the symptoms associated with the disorder to some extent. Depending on the extent to which the drug can prevent and / or kill the growth of existing cancer cells, the drug can be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by evaluating survival time, progression-free survival (PFS) period, response rate (RR), response duration, and / or quality of life.

[0114] The terms "hyperproliferative disorder", "proliferative disease", and "proliferative disorder" are used interchangeably herein and relate to unwanted or abnormal cell growth, such as neoplastic or hyperplastic growth, regardless of in vitro or in vivo context. In some embodiments, a proliferative disease is a cancer or tumor disease (including benign or cancerous) and / or any tumor metastasis, regardless of the location of the cancer, tumor, and / or tumor metastasis. In some embodiments, a proliferative disease is a benign or malignant tumor. In some embodiments, a proliferative disease is a non-cancerous disease. In some embodiments, a proliferative disease is a hyperproliferative condition such as hyperplasia, fibrosis (particularly pulmonary, but also other types such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis, and smooth muscle proliferation in blood vessels such as stenosis or restenosis after angioplasty.

[0115] "Cancer", "tumor", or "malignant tumor" are used as synonymous terms and refer to any of a number of cell types or diseases characterized by uncontrolled, abnormal growth of cells and the ability to spread (metastasize) locally or to other parts of the body via the bloodstream and lymphatic system, and / or any of the characteristic structures and / or molecular features known to be associated with these cell types or diseases. As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. "Cancerous tumor", or "malignant cells", are understood to be cells that have specific structural characteristics, lack differentiation, and are capable of invasion and metastasis. Cancers that can be treated with the γPTHF compositions provided herein include, but are not limited to, for example, non-blood system tumors such as lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and blood system tumors such as, for example, leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias or malignancies. In some embodiments, the cancer is selected from the group consisting of: colorectal cancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma.

[0116] Other types of cancers and tumors that can be treated with the γPTHF compositions are described herein or are known in the art. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder", and "tumor" are not mutually exclusive when referred to herein.

[0117] The terms "treating," "treatment," or "treat" mean both (a) therapeutic means to cure, slow, mitigate symptoms of, and / or halt the progression of a diagnosed medical condition or disorder and (b) prophylactic or preventative means to prevent and / or delay the occurrence of a targeted disease or condition. Thus, a subject in need of treatment includes a subject already having the cancer, disorder or disease, a subject at risk of becoming a cancer or condition, and a subject in which an infection or condition is to be prevented. A subject is identified as "at risk of having" cancer, an infectious disease, an immune system disorder, a proliferative disorder, or another disease or disorder mentioned herein using well-known medical and diagnostic techniques. In certain embodiments, a subject is "being treated successfully" by the methods provided herein if the subject exhibits, for example, an overall, partial, or temporary remission or removal of symptoms associated with a disease or condition (e.g., cancer, inflammation and rheumatoid arthritis). In certain embodiments, the term "treating" or "treatment" or "treat" means an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which may not be distinguishable by the patient. In other embodiments, the term "treating" or "treatment" or "treat" means inhibiting the progression of a proliferative disorder, for example, physically by stabilization of distinguishable symptoms or physiologically by stabilization of physical parameters, or both. In other embodiments, the term "treating" or "treatment" or "treat" means a decrease or stabilization in size, tumor cell proliferation or survival, or cancer cell number. The γPTHF composition can be used alone or in combination with additional therapeutic agents for treatment.

[0118] "Subject", "patient", and "animal" are used interchangeably and mean mammalian subjects such as human patients and non-human primates, as well as laboratory animals such as rabbits, rats, mice and other animals. Animals include all vertebrates, for example, mammals and non-mammals such as chickens, amphibians, and reptiles. As used herein, "mammal" includes, but is not limited to, humans and non-human primates such as chimpanzees and other apes and monkey species; livestock animals such as cows, sheep, pigs, goats, and horses; pet mammals such as dogs and cats; laboratory animals such as rodents such as mice, rats, guinea pigs, and other members of the mammalian class known in the art. In certain embodiments, the subject is human.

[0119] As used herein, "treatment of a proliferative disorder" includes maintaining or reducing the tumor size of a subject with a proliferative disorder, inducing (partial or complete) tumor shrinkage, suppressing tumor growth, and / or extending lifespan. In one embodiment, the proliferative disorder is a solid tumor. Such tumors include, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma. In one embodiment, the proliferative disorder is a hematological tumor. Such hematological tumors include, for example, leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dyscrasias or cachexia.

[0120] As used herein, the term "autoimmune disease" is defined as a disorder resulting from an autoimmune reaction. Autoimmune diseases are the result of an inappropriate and excessive reaction against self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type I), dystrophic epidermolysis bullosa, orchitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis and inflammation, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis are particularly mentioned.

[0121] As used herein, the term "therapeutic agent" means an agent or its derivative that can interact with hyperproliferative cells such as cancer cells or immune cells, thereby reducing the proliferative state of the cells and / or killing the cells. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, cytotoxic agents, platinum-based agents (e.g., cisplatin, carboplatin, oxaliplatin), taxanes (e.g., Taxol®), etoposide, alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., tetrahydrofolic acid (THF)), 5-fluorouracil, gemcitabine, or their derivatives), antitumor antibiotics (e.g., mitomycin, doxorubicin), plant-derived antitumor agents (e.g., vincristine, vindesine, taxol). Such agents further include, but are not limited to, the anticancer agents trimethoprim, temozolomide, tetrahydrofolic acid, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), bis-chloronitrosourea (BCNU) and camptothecin, or any of their therapeutic derivatives. Further examples of therapeutic agents that may be suitable for use in the methods of the present disclosure include, but are not limited to, anti-restenosis agents, growth promoting or anti-proliferative agents, anti-inflammatory agents, anti-neoplastic agents, anti-mitotic agents, antiplatelet agents, anticoagulants, antifibrin agents, antithrombin agents, cell growth inhibitory agents, antibiotics and other anti-infective agents, anti-enzyme agents, antimetabolites, angiogenesis agents, cytoprotective agents, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists and / or cardioprotective agents. "Therapeutic agent" also means salts, acids, and free base forms of the above agents.

[0122] As used herein, the term "chemotherapeutic agent," when used in the context of cancer therapy, means any agent that causes the death of cancer cells or inhibits the growth or spread of cancer cells. Examples of such chemotherapeutic agents include alkylating agents, antibiotics, antimetabolites, plant-derived drugs, and hormones. In some embodiments, the disclosed γ-polyglutamylated tetrahydrofolate compositions are used in combination with chemotherapeutic drugs. In some embodiments, the chemotherapeutic drug is 5-fluorouracil. In some embodiments, the chemotherapeutic agent is cisplatin. In some embodiments, the chemotherapeutic agent is carboplatin. In some embodiments, the chemotherapeutic agent is oxaliplatin. In other embodiments, the chemotherapeutic agent is gemcitabine. In other embodiments, the chemotherapeutic agent is doxorubicin. In certain embodiments, the chemotherapeutic drug is a pyrimidine analog (e.g., a fluoropyrimidine such as 5-fluorouracil (5-FU)).

[0123] As used herein, the term "antimetabolite" means a therapeutic agent that inhibits the utilization of a metabolite or its prodrug. Examples of antimetabolites include 5-FU and 5-FU metabolites and / or prodrugs, such as 5-FUMP, 5-FUDP, 5-FdUMP, capecitabine, tegafur 5-fluorodeoxyuridine monophosphate; as well as cytarabine, and cytarabine prodrugs, such as nelarabine, 5-azacitidine, gemcitabine, mercaptopurine, thioguanine, azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, and cladribine. Antimetabolites useful for practicing the disclosed methods include nucleoside analogs that include purine or pyrimidine analogs. In some embodiments, the γ-polyglutamylated tetrahydrofolate composition is used in combination with an antimetabolite selected from the group consisting of fluoropyrimidines, 5-fluorouracil, 5-fluoro-1-(oxolan-2-yl)pyrimidine-2,4-dione, 5-fluoro-2'-deoxycytidine, cytarabine, gemcitabine, troxacitabine, decitabine, azacitidine, pseudoisocytidine, zebularine, ancitabine, fazarabine, 6-azacitidine, capecitabine, N4-octadecylcytarabine, elaidic acid cytarabine, fludarabine, cladribine, clofarabine, nelarabine, foladesine, and pentostatin, or derivatives thereof. In one example, the nucleoside analog is a substrate of a nucleoside deaminase that is adenosine deaminase or cytidine deaminase. In some examples, the nucleoside analog is selected from fludarabine, cytarabine, gemcitabine, decitabine, and azacitidine or derivatives thereof.In some embodiments, the nucleoside analog is selected from the following: N3-alkylated analogs of 5-fluorouracil, 5-fluorouracil derivatives having a 1,4-oxaheteroepane moiety, 5-fluorouracil and nucleoside analogs, cis- and trans-5-fluoro-5,6-dihydro-6-alkoxyuracil, cyclopentane 5-fluorouracil analogs, A-OT-fluorouracil, N4-trimethoxybenzoyl-5′-deoxy-5-fluorocytidine and 5′-deoxy-5-fluorouridine, 1-hexylcarbamoyl-5-fluorouracil, B-3839, uracil-1-(2-tetrahydrofuryl)-5-fluorouracil, 1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-fluorouracil, doxifluridine, 5′-deoxy-5-fluorouridine, 1-acetyl-3-O-toluoyl-5-fluorouracil, 5-fluorouracil-m-formylbenzene-sulfonate (JP55059173), N′-(2-franidyl)-5-fluorouracil (JP53149985) and 1-(2-tetrahydrofuryl)-5-fluorouracil or derivatives thereof. In certain embodiments, the antimetabolite is a pyrimidine analog or a pyrimidine analog prodrug (e.g., a fluoropyrimidine). In some embodiments, the antimetabolite is 5-fluorouracil.

[0124] As used herein, "taxane" is an anti-cancer agent that interferes with or disrupts microtubule stability, formation and / or function. Taxane agents include paclitaxel and docetaxel and their derivatives, which function in the same mode of action on microtubules as the taxanes from which they are derived. In certain embodiments, the taxane is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid or derivative of paclitaxel or docetaxel. In certain embodiments, the taxane is paclitaxel (Taxol®), docetaxel (Taxotere®), albumin-bound paclitaxel (nab-paclitaxel; Abraxane®), DHA-paclitaxel, or PG-paclitaxel.

[0125] The term "pharmaceutically acceptable carrier" means a component other than the active ingredient in a pharmaceutical formulation that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, carriers, excipients, stabilizers, diluents, or preservatives. Pharmaceutically acceptable carriers include, for example, one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans or other subjects.

[0126] The present disclosure generally relates to γ-polyglutamylated tetrahydrofolate (γPTHF) compositions and methods of making and using the compositions for treating diseases including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria. The γ-polyglutamylated compositions also have use in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effectiveness of the therapeutic agent(s), or as a "chemoprotectant" (e.g., in combination with folate antagonists such as methotrexate) to reduce the toxic side effects associated with the therapeutic agent(s).

[0127] In some embodiments, the present disclosure provides the following: [1] A composition comprising γPTHF; [2]The composition of [1], wherein γ-polyglutamyl oxidized tetrahydrofolic acid is selected from the group consisting of: (a) Polyglutamyl oxidized 5-formyl-THF (e.g., polyglutamyl oxidized [6S]-5-formyl-THF); (b) Polyglutamyl oxidized 10-formyl-THF (e.g., polyglutamyl oxidized [6R]-10-formyl-THF); (c) Polyglutamyl oxidized 5,10-methenyl-THF (e.g., polyglutamyl oxidized [6R]-5,10-methenyl-THF); (d) Polyglutamyl oxidized 5-methyl-THF (e.g., polyglutamyl oxidized [6S]-5-methyl-THF); (e) Polyglutamyl oxidized tetrahydrofolic acid (e.g., polyglutamyl oxidized [6S]-tetrahydrofolic acid); (f) Polyglutamyl oxidized 5,10-methylene-THF (e.g., polyglutamyl oxidized [6R]-5,10-methylene-THF); and (g) Polyglutamyl oxidized 5-formimino-THF (e.g., polyglutamyl oxidized [6S]-5-formimino-THF); [3]γPTHF is a composition of [1] or [2] that contains glutamyl groups having γ-carboxyl group linkages of 4, 5, 2-10, 4-6, or more than 5; [4]γPTHF is a composition according to any one of [1]-[3], which is γ-tetraglutamyl oxidized tetrahydrofolic acid; [5]γPTHF is a composition according to any one of [1]-[3], which is γ-pentaglutamyl oxidized tetrahydrofolic acid; [6]γPTHF is a composition according to any one of [1]-[3], which is γ-hexaglutamyl oxidized tetrahydrofolic acid; [7] (a) γPTHF contains two or more L-type glutamyl groups having γ-carboxyl group linkages, (b) each of the glutamyl groups of γPTHF is of the L-type and has a γ-carboxyl group linkage, (c) at least one of the glutamyl groups of γPTHF is of the D-type and has a γ-carboxyl group linkage, (d) Each glutamyl group of γPTHF other than the glutamyl group of tetrahydrofolic acid is of the D-type and has a γ-carboxyl group bond, or (e) γPTHF is a composition according to any one of [1]-[6], comprising two or more L-type glutamyl groups and at least one D-type glutamyl group having a γ-carboxyl group bond; [8](a) Each glutamyl group is of the L-type and has a γ-carboxyl group bond, or (b) each glutamyl group other than the glutamyl group of tetrahydrofolic acid is of the D-type and each glutamyl (gluytamyl) group has a γ-carboxyl group bond, a composition according to [4]; [9](a) Each glutamyl group is of the L-type and has a γ-carboxyl group bond, or (b) each glutamyl group other than the glutamyl group of tetrahydrofolic acid is of the D-type and each glutamyl (gluytamyl) group has a γ-carboxyl group bond, a composition according to [5];

[10] (a) Each glutamyl group is of the L-type and has a γ-carboxyl group bond, or (b) each glutamyl group other than the glutamyl group of tetrahydrofolic acid is of the D-type and each glutamyl (gluytamyl) group has a γ-carboxyl group bond, a composition according to [6];

[11] γPTHF is polyglutaminoxidisable by FGPS under physiological conditions and / or polyglutaminoxylated THF has a lower hepatocyte uptake rate (<30%) than THF, a composition according to any one of [1]-

[10] ;

[12] A liposomal composition (Lp-γPTHF) containing γPTHF according to any one of [1]-

[11] ;

[13] γPTHF is an Lp-γPTHF composition according to

[12] , comprising two or more L-type glutamyl groups;

[14] An Lp-γPTHF composition according to

[12] or

[13] , wherein each glutamyl group of γPTHF is of the L-type;

[15] An Lp-γPTHF composition according to

[12] or

[13] , wherein at least one of the glutamyl groups of γPTHF is of the D-type;

[16] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[15] , comprising γPTHF containing 1-10 glutamyl groups having γ-carboxyl group bonds;

[17] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[16] , comprising γPTHF containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups;

[18] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[17] , comprising γ-tetraglutamine oxidized tetrahydrofolic acid;

[19] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[17] , comprising γ-pentaglutamine oxidized tetrahydrofolic acid;

[20] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[17] , comprising γ-hexaglutamine oxidized tetrahydrofolic acid;

[21] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[20] , which is not pegylated (PγLp-γPTHF);

[22] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[20] , which is pegylated (PγLp-γPTHF);

[23] The liposome comprises at least 1% weight / weight (w / w) of γPTHF, or at least 1% of the starting material of γ-polyglutamine oxidized THF is encapsulated (trapped) within the Lp-γPTHF during the process of preparing the Lp-γPTHF, and is an Lp-γPTHF composition according to any one of

[12] -

[22] ;

[24] The liposome has a diameter in the range of 20 nm to 500 nm, and is an Lp-γPTHF composition according to any one of

[12] -

[23] ;

[25] The liposome has a diameter in the range of 20 nm to 200 nm, and is an Lp-γPTHF composition according to any one of

[12] -

[24] ;

[26] The liposome has a diameter in the range of 80 nm to 120 nm, and is an Lp-γPTHF composition according to any one of

[12] -

[25] ;

[27] The liposome is formed from liposome components, and is an Lp-γPTHF composition according to any one of

[12] -

[26] ;

[28] The liposome component includes at least one of an anionic lipid and a neutral lipid, the Lp-γPTHF composition according to

[27] ;

[29] The liposome component includes at least one selected from the group consisting of the following, the Lp-γPTHF composition according to

[27] or

[28] : DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide;

[30] The liposome component includes at least one selected from the group consisting of the following, the Lp-γPTHF composition according to any one of

[27] -

[29] : DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC;

[31] The Lp-γPTHF composition according to any one of

[27] -

[30] , wherein one or more liposome components further include a steric stabilizer;

[32] The steric stabilizer is at least one selected from the group consisting of the following, the Lp-γPTHF composition according to

[31] : polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymer; oligoglycerol, polyethylene glycol and polypropylene oxide-containing copolymer, poloxamer 188, and polyvinyl alcohol;

[33] The steric stabilizer is PEG, and PEG has a number average molecular weight (Mn) of 200 to 5000 daltons, the Lp-γPTHF composition according to

[32] ;

[34] The liposome is anionic or neutral, the Lp-γPTHF composition according to any one of

[12] -

[33] ;

[35] The liposome has a zeta potential that is zero or less, the Lp-γPTHF composition according to any one of

[12] -

[33] ;

[36] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[33] , having a zeta potential of 0 to -150 mV;

[37] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[33] , having a zeta potential of -30 to -50 mV;

[38] The liposome is a cationic Lp-γPTHF composition according to any one of

[12] -

[33] ;

[39] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[38] , having an internal space containing γPTHF and an aqueous, pharmaceutically acceptable carrier;

[40] The pharmaceutically acceptable carrier is an Lp-γPTHF composition according to

[39] , containing an isotonic agent such as dextrose, mannitol, glycerin, potassium chloride, sodium chloride, etc. at a concentration exceeding 1%;

[41] The aqueous, pharmaceutically acceptable carrier is trehalose, an Lp-γPTHF composition according to

[39] ;

[42] The pharmaceutically acceptable carrier is an Lp-γPTHF composition according to

[41] , containing 1% to 50% trehalose;

[43] The pharmaceutically acceptable carrier is an Lp-γPTHF composition according to any one of

[39] -

[42] , containing a 1% to 50% dextrose solution;

[44] The internal space of the liposome is an Lp-γPTHF composition according to any one of

[39] -

[43] , containing 5% dextrose suspended in HEPES buffer;

[45] The pharmaceutically acceptable carrier is an Lp-γPTHF composition according to any one of

[39] -

[44] , containing a buffer such as HEPES buffered saline (HBS) or the like at a concentration of 1 to 200 mM and a pH of 2 to 8;

[46] The pharmaceutically acceptable carrier is an Lp-γPTHF composition according to any one of

[39] -

[45] , containing sodium acetate and calcium acetate at a total concentration of 50 mM to 500 mM;

[47] The internal space of the liposome has a pH of 5 - 8 or 6 - 7, or any range therebetween, an Lp-γPTHF composition according to any one of

[12] -

[46] ;

[48] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[47] , containing less than 500,000 or less than 200,000 γPTHF molecules;

[49] The liposome is an Lp-γPTHF composition according to any one of

[12] -

[48] , containing 10 to 100,000 γPTHF molecules, or any range in between;

[50] An Lp-γPTHF composition according to any one of

[12] -

[49] , further comprising a targeting moiety, wherein the targeting moiety has specific affinity for a surface antigen on a target cell of interest;

[51] The targeting moiety is attached to one or both of the outer surface of PEG and the liposome, and optionally, the targeting moiety is attached to one or both of the outer surface of PEG and the liposome by a covalent bond, an Lp-γPTHF composition according to

[50] ;

[52] The targeting moiety is a polypeptide, an Lp-γPTHF composition of

[50] or

[51] ;

[53] The targeting moiety is an antibody or an antigen-binding fragment of an antibody, an Lp-γPTHF composition according to any one of

[50] -

[52] ;

[54] The targeting moiety binds to the surface antigen with an equilibrium dissociation constant (Kd) in the range of 0.5x10 -10 ~10x10 -6 as measured by BIACORE® analysis, an Lp-γPTHF composition according to any one of

[50] -

[53] ;

[55] The targeting moiety specifically binds one or more folate receptors selected from the group consisting of: folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ), an Lp-γPTHF composition according to any one of

[50] -

[54] ;

[56] The targeting moiety comprises one or more selected from the group consisting of: antibody, humanized antibody, antigen-binding fragment of an antibody, single-chain antibody, single-domain antibody, bispecific antibody, synthetic antibody, pegylated antibody, and multimeric antibody, an Lp-γPTHF composition according to any one of

[50] -

[55] ;

[57] Each pegylated liposome is an Lp-γPTHF composition according to any one of

[50] -

[56] , containing 1 to 1000 or 30 - 200 targeting moieties;

[58] An Lp-γPTHF composition according to any one of

[39] -

[57] , further comprising one or more of an immunostimulant, a detectable marker, and maleimide, wherein the immunostimulant, detectable marker, or maleimide is attached to the outer surface of the PEG or liposome;

[59] The immunostimulant of the Lp-γPTHF composition of

[58] is at least one selected from the group consisting of: protein immunostimulants; nucleic acid immunostimulants; chemical immunostimulants; haptens; and adjuvants;

[60] The immunostimulant of the Lp-γPTHF composition of

[58] or

[59] is at least one selected from the group consisting of: fluorescein; fluorescein isothiocyanate (FITC); DNP; β-glucan; β-1,3-glucan; β-1,6-glucan; resorcinol (e.g., D n-6DPA or D n-3DPA such as resorcinol D, resorcinol E, or T-series resorcinol); and toll-like receptor (TLR) modulators such as oxidized low density lipoprotein (e.g., OXPAC, PGPC), and erythran lipids (e.g., E5564);

[61] The immunostimulant and the detectable marker of the Lp-γPTHF composition according to any one of

[58] -

[60] are the same;

[62] An Lp-γPTHF composition according to any one of

[58] -

[61] , further comprising a hapten;

[63] The hapten of the Lp-γPTHF composition of

[62] comprises one or more of fluorescein or β1,6-glucan;

[64] An Lp-γPTHF composition according to any one of

[12] -

[63] , further comprising at least one cryoprotective substance selected from the group consisting of mannitol; trehalose; sorbitol; and sucrose;

[65] A targeted composition comprising a composition according to any one of [1]-

[64] ;

[66] An untargeted composition comprising a composition according to any one of [1]-

[49] ; An Lp-γPTHF composition according to any one of

[12] -

[66] , further comprising carboplatin and / or pembrolizumab; A pharmaceutical composition comprising a liposomal γPTHF composition according to any one of

[68] -

[12] ; A pharmaceutical composition comprising a γPTHF composition according to any one of [1]-[7]; A composition according to any one of [1]-

[69] for use in the treatment of a disease; Use of a composition according to any one of [1]-

[70] in the manufacture of a medicament for the treatment of a disease and / or in combination therapy with one or more therapeutic agents such as chemotherapeutic agents (e.g., 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or to reduce the toxic side effects associated with the therapeutic agent(s) as a "chemoprotective agent" (e.g., in combination with folate antagonists such as methotrexate); A method for treating (e.g., treating or preventing) a disease or chemical-induced toxicity in a subject in need of such treatment or prevention, the method comprising administering to the subject a composition according to any one of [1]-

[70] ; A method for treating (e.g., treating or preventing) a disease or chemical-induced toxicity in a subject in need of such treatment or prevention, the method comprising administering to the subject a liposomal γPTHF composition according to any one of

[12] -

[69] ; A method for killing proliferative cells, comprising contacting the proliferative cells with a composition according to any one of [1]-

[69] ; A method for killing proliferative cells, comprising contacting the proliferative cells with a liposomal γPTHF composition according to any one of

[12] -

[69] ; The method of

[74] or

[75] , wherein the proliferative cells are cancer cells, mammalian cells, and / or human cells; A method for treating cancer, comprising delivering to a subject having or at risk of having cancer an effective amount of a composition according to any one of [1]-

[69] ;

[78] A method for treating (e.g., treating or preventing) cancer, comprising administering to a subject having cancer or at risk of having cancer an effective amount of any of the liposomal γPTHF compositions of

[12] -

[68] ;

[79] The composition is administered for treating or preventing cancer, and the cancer is selected from the group consisting of: the method of

[77] or

[78] , for example, non-blood tumors including lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and blood tumors such as leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias;

[80] The composition is administered for treating or preventing cancer, and the cancer is a member selected from the group consisting of: the method of

[77] or

[78] , lung cancer, breast cancer, colon cancer, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, ovarian cancer, and cervical cancer;

[81] The composition is administered for treating or preventing cancer, and the cancer is a member selected from the group consisting of: the method of

[77] or

[78] , colorectal cancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma;

[82] The composition is administered for treating or preventing colorectal cancer, the method of

[77] or

[78] ;

[83] A method for treating (e.g., treating or preventing) cancer, comprising administering to a subject having or at risk of having cancer cells expressing folate receptor on its surface that are bound by a targeting moiety an effective amount of any of the Lp-γPTHF compositions of

[50] -

[66] ;

[84] Maintenance therapy, comprising administering to a subject receiving or having received cancer therapy an effective amount of any of the compositions of [1]-

[69] ;

[85] Maintenance therapy, comprising administering to a subject receiving or having received cancer therapy an effective amount of any of the liposomal γPTHF compositions of

[12] -

[69] ;

[86] A method for treating an immune system disorder, comprising administering to a subject having or at risk of having an immune system disorder an effective amount of any of the compositions of [1]-

[69] , optionally, the immune system disorder is selected from the following: inflammation (e.g., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, systemic lupus erythematosus, and Takayasu disease, as well as psoriasis;

[87] A method for treating an immune system disorder, comprising administering to a subject having or at risk of having an immune system disorder an effective amount of any of the liposomal γPTHF compositions of [8]-

[69] , optionally, the immune system disorder is selected from the following: inflammation (e.g., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, systemic lupus erythematosus, and Takayasu disease, as well as psoriasis;

[88] A method for treatment as follows: (a) A method for treating leukopenia, comprising administering to a subject having or at risk of having leukopenia an effective amount of a composition according to any of [1]-

[59] ; (b) A method for treating an infectious disease, comprising administering to a subject having or at risk of having an infectious disease an effective amount of a composition according to any of [1]-

[69] ; (c) A method for treating a cardiovascular disease or a metabolic disease, comprising administering to a subject having or at risk of having an infectious disease, a cardiovascular disease, or another disease an effective amount of a composition according to any of [1]-

[59] , wherein the disease is a member selected from the following: atherosclerosis, cardiovascular disease (CVD), coronary artery disease, myocardial infarction, stroke, metabolic syndrome, gestational trophoblastic disease, and ectopic pregnancy; (d) A method for treating an autoimmune disease, comprising administering to a subject having or at risk of having an autoimmune disease an effective amount of a composition according to any of [1]-

[59] ; (e) A method for treating rheumatoid arthritis, comprising administering to a subject having or at risk of having rheumatoid arthritis an effective amount of a composition according to any one of [1]-

[59] ; (f) A method for treating an inflammatory condition, comprising administering to a subject having or at risk of having inflammation an effective amount of a composition according to any one of [1]-

[59] , wherein optionally the inflammation is acute, chronic, and / or systemic inflammation; or (g) A method for treating a skin disease, comprising administering to a subject having or at risk of having a skin disease an effective amount of a composition according to any one of [1]-

[59] , wherein optionally the skin disease is psoriasis;

[89] A method for treating an infectious disease, comprising administering to a subject having or at risk of having an infectious disease an effective amount of a liposomal γPTHF composition according to any one of

[12] -

[69] ;

[90] A method for delivering γPTHF to a tumor expressing a folate receptor on its surface, the method comprising: administering to a subject having a tumor an Lp-γPTHF composition according to any one of [1]-

[69] in an amount effective to deliver a therapeutically effective amount of γPTHF to the tumor;

[91] A method for preparing a γ-polyglutamylated tetrahydrofolic acid composition comprising a liposomal γ-polyglutamylated tetrahydrofolic acid composition according to any one of

[12] -

[69] , the method comprising: forming a mixture comprising liposomal components and a γ-polyglutamylated folic acid antimetabolite in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes containing γ-polyglutamylated tetrahydrofolic acid;

[92] A method for preparing a γ-polyglutamylated tetrahydrofolic acid composition comprising a liposomal γ-polyglutamylated tetrahydrofolic acid composition according to any one of

[12] -

[69] , the method comprising: forming a mixture comprising liposomal components and γ-polyglutamylated tetrahydrofolic acid in solution; and treating the mixture to form liposomes containing γ-polyglutamylated tetrahydrofolic acid;

[93] Treating the mixture includes homogenizing the mixture in solution to form liposomes, the method of

[92] ,

[94] A method for preparing a composition according to any one of

[50] -

[69] , comprising the following steps: forming a mixture comprising liposome components and γ-polyglutamylated tetrahydrofolic acid in solution; homogenizing the mixture in solution to form liposomes; treating the mixture to form liposomes that capture and / or encapsulate γ-polyglutamylated tetrahydrofolic acid; and providing a targeting moiety on the surface of the liposomes, the targeting moiety having specific affinity for at least one of folate receptor α (FR-α), folate receptor β (FR-β), and folate receptor δ (FR-δ);

[95] A method for preparing a composition according to any one of

[50] -

[69] , comprising the following steps: forming a mixture comprising liposome components and γ-polyglutamylated tetrahydrofolic acid in solution; treating the mixture to form liposomes that capture and / or encapsulate γ-polyglutamylated tetrahydrofolic acid; and providing a targeting moiety on the surface of the liposomes, the targeting moiety having specific affinity for at least one of folate receptor α (FR-α), folate receptor β (FR-β), and folate receptor δ (FR-δ);

[96] The treatment step includes homogenizing the mixture in solution to form liposomes, the method of

[95] ,

[97] The treatment step includes one or more of the following steps of the method according to

[92] : thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw technique, reverse phase evaporation, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, freeze-dried double emulsion, 3D printing, membrane contactor method, and stirring; and / or

[98] The treatment step includes one or more steps of changing the size of the liposomes by one or more of extrusion, high pressure microfluidization, and / or sonication, the method according to any one of

[95] -

[97] ; and / or A method according to any one of

[91] to

[98] , wherein at least 1% of the starting material of γ-polyglutamyl oxidized tetrahydrofolic acid is encapsulated or entrapped in liposomes.

[0128] II. γ-Polyglutamyl oxidized tetrahydrofolic acid (γPTHF) Generally, the present disclosure relates to γPTHF (γPTHF) compositions. The γPTHF compositions contain at least one glutamyl group having a γ-carboxyl group bond. These are structurally different from the L-γ-polyglutamyl oxidized form of tetrahydrofolic acid (Lγ1PTHF) produced by the enzyme folylpolyγglutamate synthetase (FPGS) in cells during tetrahydrofolic acid therapy.

[0129] In some embodiments, the γPTHF composition contains 2 - 20, 2 - 15, 2 - 10, 2 - 5, or more than 5 glutamyl groups (including the glutamyl groups in tetrahydrofolic acid). In some embodiments, each of the glutamyl groups in γPTHF other than the glutamyl groups of tetrahydrofolic acid has a γ-bond. In some embodiments, two or more of the glutamyl groups in γPTHF have a γ-bond (g1amma linkage). In some embodiments, each of the glutamyl groups in γPTHF is of the L-type. In some embodiments, each of the glutamyl groups in γPTHF other than the glutamyl groups of tetrahydrofolic acid is of the D-type. In some embodiments, γPTHF contains two or more L-type glutamyl groups and one or more D-type glutamyl groups.

[0130] In some embodiments, γPTHF is diglutamylated. That is, γPTHF contains one γ-glutamyl group in addition to the glutamyl groups of tetrahydrofolic acid (γTHF-PG1). In some embodiments, each of the glutamyl groups of γ-diglutamylated tetrahydrofolic acid is of the L-type. In other embodiments, γ-diglutamylated THF contains a D-type glutamyl group.

[0131] In some embodiments, γPTHF is triglutaminylated. That is, γPTHF contains two γ-glutamyl groups in addition to the glutamyl group of tetrahydrofolic acid (γTHF-PG2). In some embodiments, each of the glutamyl groups of γ-triglutaminylated tetrahydrofolic acid is of the L-type. In other embodiments, γ-triglutaminylated THF contains a D-type glutamyl group. In further embodiments, each of the glutamyl groups of γ-triglutaminylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, γ-triglutaminylated THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0132] In some embodiments, γPTHF is tetraglutaminylated, and thus contains three γ-glutamyl groups in addition to the γ-glutamyl group in tetrahydrofolic acid (γTHF-PG3). In some embodiments, γ-tetraglutaminylated THF contains two or more L-type γ-glutamyl groups. In further embodiments, each of the γ-glutamyl groups of γ-tetraglutaminylated tetrahydrofolic acid is of the L-type. In other embodiments, γ-tetraglutaminylated THF contains a D-type γ-glutamyl group. In some embodiments, γ-tetraglutaminylated THF contains two D-type γ-glutamyl groups. In some embodiments, each of the glutamyl groups of γ-tetraglutaminylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, tetraglutaminylated THF contains a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0133] In some embodiments, γPTHF is pentaglutamylated (γTHF-PG4) and contains a chain of four γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ pentaglutamylated THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ pentaglutamylated tetrahydrofolic acid is of the L-type. In other embodiments, γ pentaglutamylated THF contains a D-type glutamyl group. In some embodiments, γ tetraglutamylated THF contains two or three D-type γ-glutamyl groups. In further embodiments, each of the γ-glutamyl groups of γ pentaglutamylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, pentaglutamylated THF contains a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0134] In some embodiments, γPTHF is hexaglutamylated (γTHF-PG5) and contains a chain of five γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ hexaglutamylated THF contains two or more L-type γ-glutamyl groups. In further embodiments, each of the glutamyl groups of γ hexaglutamylated tetrahydrofolic acid is of the L-type. In other embodiments, γ hexaglutamylated THF contains a D-type γ-glutamyl group. In some embodiments, γ tetraglutamylated THF contains two, three, four, or five D-type γ-glutamyl groups. In further embodiments, each of the glutamyl groups of γ hexaglutamylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, hexaglutamylated THF contains a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0135] In some embodiments, γPTHF is heptaglutamylated (γTHF-PG6) and thus contains a chain of six γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ-heptaglutamylated THF contains two or more L-type γ-glutamyl groups. In further embodiments, each of the γ-glutamyl groups of γ-heptaglutamylated tetrahydrofolic acid is of the L-type. In other embodiments, γ-heptaglutamylated THF contains a D-type γ-glutamyl group. In some embodiments, γ-tetraglutamylated THF contains two, three, four, five, or six D-type γ-glutamyl groups. In further embodiments, each of the γ-glutamyl groups of γ-heptaglutamylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, heptaglutamylated THF contains a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0136] In some embodiments, γPTHF is octaglutamylated (γTHF-PG7) and thus contains a chain of seven γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ-octaglutamylated THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ-octaglutamylated tetrahydrofolic acid is of the L-type. In other embodiments, γ-octaglutamylated THF contains a D-type glutamyl group. In some embodiments, γ-octaglutamylated THF contains two, three, four, five, six, or seven D-type γ-glutamyl groups. In further embodiments, each of the glutamyl groups of γ-octaglutamylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, octaglutamylated THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0137] In some embodiments, γPTHF is nonaglutamylated (γTHF-PG8) and contains a chain of eight γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ nonaglutamylated THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ nonaglutamylated tetrahydrofolic acid is of the L-type. In other embodiments, γ nonaglutamylated THF contains a D-type glutamyl group. In further embodiments, each of the glutamyl groups of γ nonaglutamylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, nonaglutamylated THF contains a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0138] In some embodiments, γPTHF is decaglutamylated (γTHF-PG9) and contains a chain of nine γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ decaglutamylated THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ decaglutamylated tetrahydrofolic acid is of the L-type. In other embodiments, γ decaglutamylated THF contains a D-type glutamyl group. In further embodiments, each of the glutamyl groups of γ decaglutamylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, decaglutamylated THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0139] In some embodiments, γPTHF is undecaglutamylated (γTHF-PG 10) It contains a chain of 10 γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ-undecaglutamyl oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ-undecaglutamyl oxidized tetrahydrofolic acid is of the L-type. In other embodiments, γ-undecaglutamyl oxidized THF contains a D-glutamyl group. In further embodiments, each of the glutamyl groups of γ-undecaglutamyl oxidized tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, undecaglutamyl oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0140] In some embodiments, γPTHF is dodecaglutamyl oxidized (γTHF-PG 11 ) It contains a chain of 11 γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ-dodecaglutamyl oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ-dodecaglutamyl oxidized tetrahydrofolic acid is of the L-type. In other embodiments, γ-dodecaglutamyl oxidized THF contains a D-glutamyl group. In further embodiments, each of the glutamyl groups of γ-dodecaglutamyl oxidized tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, dodecaglutamyl oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0141] In some embodiments, γPTHF is tridecaglutamyl oxidized (γTHF-PG 12) It contains a chain of 12 γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ-triskadecaglutaminyl oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ-triskadecaglutaminyl oxidized tetrahydrofolic acid is of the L-type. In other embodiments, γ-triskadecaglutaminyl oxidized THF contains a D-type glutamyl group. In further embodiments, each of the glutamyl groups of γ-triskadecaglutaminyl oxidized tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, triskadecaglutaminyl oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0142] In some embodiments, γPTHF is tetradecaglutaminylated (γTHF-PG 13 ) It contains a chain of 13 γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ-tetradecaglutaminyl oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ-tetradecaglutaminyl oxidized tetrahydrofolic acid is of the L-type. In other embodiments, γ-tetradecaglutaminyl oxidized THF contains a D-type glutamyl group. In further embodiments, each of the glutamyl groups of γ-tetradecaglutaminyl oxidized tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, tetradecaglutaminyl oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0143] In some embodiments, γPTHF is pentadecaglutaminylated (γTHF-PG 14) It contains a chain of 14 γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ-pentadeca-glutamylated THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ-pentadeca-glutamylated tetrahydrofolic acid is of the L-type. In other embodiments, γ-pentadeca-glutamylated THF contains a D-type glutamyl group. In further embodiments, each of the glutamyl groups of γ-pentadeca-glutamylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, pentadeca-glutamylated THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0144] In some embodiments, γPTHF is hexadeca-glutamylated (γTHF-PG 15 ) It contains a chain of 15 γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ-hexadeca-glutamylated THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ-hexadeca-glutamylated tetrahydrofolic acid is of the L-type. In other embodiments, γ-hexadeca-glutamylated THF contains a D-type glutamyl group. In further embodiments, each of the glutamyl groups of γ-hexadeca-glutamylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, hexadeca-glutamylated THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0145] In other embodiments, γPTHF is heptadeca-glutamylated (γTHF-PG 16) It contains a chain of 16 γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ-heptadeca-glutamylated THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ-heptadeca-glutamylated tetrahydrofolic acid is of the L-type. In other embodiments, γ-heptadeca-glutamylated THF contains a D-glutamyl group. In further embodiments, each of the glutamyl groups of γ-heptadeca-glutamylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, heptadeca-glutamylated THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0146] In some embodiments, γPTHF is octadeca-glutamylated (γTHF-PG 17 ) It contains a chain of 17 γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ-octadeca-glutamylated THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ-octadeca-glutamylated tetrahydrofolic acid is of the L-type. In other embodiments, γ-octadeca-glutamylated THF contains a D-glutamyl group. In further embodiments, each of the glutamyl groups of γ-octadeca-glutamylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, octadeca-glutamylated THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0147] In some embodiments, γPTHF is enea-deca-glutamylated (γTHF-PG 18) It contains a chain of 18 γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ-eneradeca glutamylated THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ-eneradeca glutamylated tetrahydrofolic acid is of the L-type. In other embodiments, γ-eneradeca glutamylated THF contains a D-glutamyl group. In further embodiments, each of the glutamyl groups of γ-eneradeca glutamylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, eneradeca glutamylated THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0148] In some embodiments, γPTHF is eicosaglutamylated (γTHF-PG 19 ) It contains a chain of 19 γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ-eicosaglutamylated THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ-eicosaglutamylated tetrahydrofolic acid is of the L-type. In other embodiments, γ-eicosaglutamylated THF contains a D-glutamyl group. In further embodiments, each of the glutamyl groups of γ-eicosaglutamylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, eicosaglutamylated THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0149] In some embodiments, γPTHF is eicosakaienaglutamylated (γTHF-PG 20) It contains a chain of 20 γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, γ-eicosakaienaglutamine oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each of the glutamyl groups of γ-eicosakaienaglutamine oxidized tetrahydrofolic acid is of the L-type. In other embodiments, γ-eicosakaienaglutamine oxidized THF contains a D-type glutamyl group. In further embodiments, each of the glutamyl groups of γ-eicosakaienaglutamine oxidized tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In additional embodiments, eicosakaienaglutamine oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0150] In some embodiments, γPTHF contains a chain of 4-7 glutamyl groups attached to tetrahydrofolic acid (i.e., γTHF-PGn, where n = 4-7), and each of the 4-7 attached glutamyl groups has a γ-bond. In some embodiments, each of the 4-7 attached glutamyl groups is of the L-type. In other embodiments, each of the 4-7 attached glutamyl groups is of the D-type. In other embodiments, the 4-7 attached glutamyl groups are of the L-type and the D-type.

[0151] In one embodiment, γPTHF is tetraglutamine oxidized, and each of the three glutamyl groups in the polyglutamic acid chain attached to tetrahydrofolic acid contains a γ-bond. In some embodiments, each of the four glutamyl groups is of the L-type. In some embodiments, each of the glutamyl groups in γ-tetraglutamine oxidized tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is of the D-type. In other embodiments, at least two of the glutamyl groups in γ-tetraglutamine oxidized tetrahydrofolic acid are of the L-type and at least one glutamyl group is of the D-type.

[0152] In one embodiment, γPTHF is pentaglutamylated, and each of the four glutamyl groups in the polyglutamic acid chain attached to tetrahydrofolic acid contains a γ bond. In some embodiments, each of the four glutamyl groups is of the L-type. In some embodiments, each of the glutamyl groups in γ-pentaglutamylated tetrahydrofolic acid other than the glutamyl groups of tetrahydrofolic acid is of the D-type. In other embodiments, at least two of the glutamyl groups in γ-pentaglutamylated tetrahydrofolic acid are of the L-type and at least one glutamyl group is of the D-type.

[0153] In one embodiment, γPTHF is hexaglutamylated. In some embodiments, each of the five glutamyl groups is of the L-type. In some embodiments, each of the glutamyl groups in γ-hexaglutamylated tetrahydrofolic acid other than the glutamyl groups of tetrahydrofolic acid is of the D-type. In other embodiments, at least two of the glutamyl groups in γ-hexaglutamylated tetrahydrofolic acid are of the L-type and at least one glutamyl group is of the D-type.

[0154] In another embodiment, γPTHF is heptaglutamylated. In some embodiments, each of the six glutamyl groups is of the L-type. In some embodiments, each of the glutamyl groups in γ-heptaglutamylated tetrahydrofolic acid other than the glutamyl groups of tetrahydrofolic acid is of the D-type. In other embodiments, at least two of the glutamyl groups in γ-heptaglutamylated tetrahydrofolic acid are of the L-type and at least one glutamyl group is of the D-type.

[0155] In some embodiments, γPTHF contains a total of 1-15, 1-10, 2-15, 2-10, 3-15, 3-10, 3-6, 3-5, 4-10, 4-7, or 4-6 glutamyl groups containing glutamyl groups in tetrahydrofolic acid, or any range therebetween. In some embodiments, each of the glutamyl groups in γPTHF other than the glutamyl groups of tetrahydrofolic acid has a γ-bond. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in γPTHF have a γ-bond. In some embodiments, γPTHF contains L-type and D-type γ-glutamyl groups. In some embodiments, each of the glutamyl groups in the polyglutamic acid structure of polyglutaminated tetrahydrofolic acid is of the L-type. In some embodiments, each of the glutamyl groups in γPTHF other than the glutamyl groups of tetrahydrofolic acid is of the D-type. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the glutamyl groups in γPTHF are of the L-type. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in γPTHF are of the D-type.

[0156] In additional embodiments, γPTHF contains 20-100, 20-75, 20-50, 20-40, 20-30, 20-25, or more than 100 γ-glutamyl groups, or any range therebetween. In some embodiments, each of the glutamyl groups of γPTHF is of the L-type. In other embodiments, each of the glutamyl groups of γPTHF other than the glutamyl groups of tetrahydrofolic acid is of the D-type. In other embodiments, at least two of the glutamyl groups in γPTHF are of the L-type and at least one of the glutamyl groups in γPTHF is of the D-type.

[0157] In additional embodiments, the compositions provided include γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 - 10, or 1 - 20 glutamyl groups (having γ bonds). In some embodiments, the γPTHF contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 - 10, or 1 - 20 L - type glutamyl groups. In some embodiments, the γPTHF contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 - 10, or 1 - 20 D - type glutamyl groups. In some embodiments, the γPTHF contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 - 10, or 1 - 20 L - type glutamyl groups and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1 - 10 or 1 - 20 D - type glutamyl groups.

[0158] In some embodiments, the γPTHF compositions provided herein can have one or more additional glutamyl groups added, i.e., the compositions can serve as a substrate for FPGS (folylpolyglutamate synthetase). Reagents and assays for measuring the ability of γPTHF compositions to act as substrates for FPGS (e.g., human FPGS, or rat liver FPGS) are readily available and can be performed as routine business.

[0159] In some embodiments, the naked γPPMX compositions disclosed herein (e.g., γPTHF not bound to a delivery vehicle) are taken up by liver cells at a rate that is significantly reduced compared to the uptake rate of tetrahydrofolic acid under the same physiological conditions. In some embodiments, the liver cell uptake rate of the naked γPTHF composition is less than 30%, 20%, 15%, or 10% compared to the rate of tetrahydrofolic acid. In further embodiments, the efflux (transport) rate of the γPTHF compositions disclosed herein from liver cells occurs at a significantly slower rate (less than 30%, 20%, 15%, or 10%) compared to tetrahydrofolic acid.

[0160] In some embodiments, the γPTHF composition does not contain fluorine atoms. In some embodiments, the γPTHF composition does not contain 4-fluoroglutamyl groups.

[0161] γ-Polygamma-glutamylated tetrahydrofolate (γPTHF) compositions and their uses are further disclosed in U.S. Patent Applications Nos. 62 / 374,458, 62 / 583,432, 62 / 630,820, and 62 / 630,821; 62 / 630,824, 62 / 630,825, 15 / 675,695, and 15 / 675,701; and International Patent Applications Nos. PCT / US2017 / 046666, and PCT / US2017 / 046667, the contents of each of which are hereby incorporated by reference in their entirety.

[0162] A. Polyglutamylated Tetrahydrofolate Analogs and Derivatives The present disclosure also encompasses γPTHF derivatives and analogs. The compositions and methods disclosed herein are contemplated for application to any and all known derivatives or analogs of polyglutamylated tetrahydrofolate. In some embodiments, the polyglutamylated tetrahydrofolate analog or derivative composition prepared and used by the disclosed compositions and methods is shown in FIGS. 1I-1J. In some embodiments, the analog corresponds to a modified form of tetrahydrofolate, in which case the glutamyl group of tetrahydrofolate is not linked to the remainder of the tetrahydrofolate molecule via a γ-peptide bond. In some embodiments, the analog is a variant of tetrahydrofolate, in which case the glutamyl group of tetrahydrofolate is of the D-form. In some embodiments, the polyglutamylated form of tetrahydrofolate, or polyglutamylated tetrahydrofolate analog or derivative, is not fluorinated.

[0163] B. THF-PG Synthesis The tetrahydrofolic acid polyglutamic acid composition provided in this specification can be obtained by the following synthesis methods using available reagents and synthetic intermediates. The addition of glutamyl residues to the glutamyl residues of tetrahydrofolic acid can be achieved using synthetic procedures known in the art. In some embodiments, the glutamyl residues are added sequentially to the glutamyl residues of tetrahydrofolic acid. In additional embodiments, the polyglutamic acid is added to the glutamyl residues (reside) of tetrahydrofolic acid using the "click chemistry" method or other bioconjugate chemistries known to those skilled in the art. Alternatively, a peptide of glutamyl residues of the desired length can be prepared and added to a precursor of tetrahydrofolic acid that does not have a glutamyl residue. The peptide can be generated using synthetic procedures known in the art. In some embodiments, the first glutamyl residue is attached to the wang resin, and additional glutamyl residues are added sequentially using F-moc chemistry by solid-phase peptide synthesis. After adding the final glutamyl residue, the tetrahydrofolic acid precursor (precurser) is coupled to the peptide, and the molecule is cleaved from the resin.

[0164] The addition of a glutamyl residue to the glutamyl residue of tetrahydrofolic acid can be achieved using synthetic procedures known in the art. In some embodiments, the glutamyl residue is added sequentially to the glutamyl residue of tetrahydrofolic acid. In additional embodiments, the polyglutamic acid is added to the glutamyl residue of tetrahydrofolic acid using "click chemistry" methods or other bioconjugate chemistries known to those of skill in the art. Alternatively, a peptide of the desired length of glutamyl residues can be made and added to a precursor of tetrahydrofolic acid that does not have a glutamyl residue. The peptide can be generated using synthetic procedures known in the art. In some embodiments, the first glutamyl residue is attached to a Wang resin and additional glutamyl residues are added sequentially using F-moc chemistry by solid-phase peptide synthesis. After the final glutamyl residue is added, the tetrahydrofolic acid precursor is coupled to the peptide and the molecule is cleaved from the resin.

[0165] C. Tetrahydrofolic Acid-PG Complex Surprisingly, the inventors have found that polyglutamylated folic acid antagonists that share similar structural and chemical characteristics to tetrahydrofolic acid (γPTHF) can form complexes with other compositions, including therapeutic agents that contain cytotoxic compounds such as platinum-based compounds. Accordingly, in some embodiments, the present disclosure provides complexes of γPTHF (e.g., γPTHF as disclosed herein) with a therapeutic agent or a salt or acid thereof.

[0166] In some embodiments, the γPTHF / composite comprises γPTHF and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound such as a chemotherapeutic agent. In further embodiments, the γPTHF / composite contains a platinum-based drug such as a platinum-based chemotherapeutic agent (e.g., cisplatin, carboplatin, and oxaliplatin). In other embodiments, the αPTHF / composite comprises a taxane-based chemotherapeutic agent (e.g., paclitaxel and docetaxel). In other embodiments, the γPTHF / composite comprises cyclodextrin. In further embodiments, the γPTHF / composite is encapsulated within liposomes.

[0167] In some embodiments, the present disclosure provides a composition comprising γPTHF and a complex of a therapeutic agent or a salt or acid thereof. In further embodiments, the γPTHF / therapeutic agent complex comprises one or more γPTHFs containing 2 - 150, 2 - 100, 2 - 75, 2 - 50, 2 - 24, 2 - 30, 2 - 20, 2 - 19, 2 - 15, 2 - 10, or 2 - 5 glutamyl groups. In some embodiments, the γPTHF / therapeutic agent complex comprises one or more γPTHFs containing 3 - 10, 3 - 9, 3 - 8, or 3 - 7 glutamyl groups, or any range therebetween. In other embodiments, the γPTHF / therapeutic agent complex comprises one or more γPTHFs containing 4 - 10, 4 - 9, 4 - 8, 4 - 7, 4 - 6, or 4 - 5 glutamyl groups, or any range therebetween. In one particular embodiment, the complex comprises one or more γPTHFs containing 3 - 10 glutamyl groups. In further embodiments, the γPTHF / therapeutic agent complex comprises one or more γPTHFs containing 3 - 7 glutamyl groups. In another embodiment, the γPTHF / therapeutic agent complex comprises one or more γPTHFs containing 5 glutamyl groups. In another embodiment, the γPTHF / therapeutic agent complex comprises one or more γPTHFs containing 6 glutamyl groups. In some embodiments, the therapeutic agent is a cytotoxic compound or a salt or acid thereof. In further embodiments, the therapeutic agent is a chemotherapeutic agent or a salt or acid thereof. In another embodiment, the therapeutic agent is a platinum-based drug. In another embodiment, the therapeutic agent is a taxane-based drug. In additional embodiments, the molar ratio of γPTHF / therapeutic agent in the complex is in the range of 1 - 10:1. In some embodiments, the molar ratio of γPTHF / therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In other embodiments, the molar ratio of γPTHF / therapeutic agent in the complex is in the range of 1:1 - 20, 1:1 - 10, or 1:2 - 8, or any range therebetween.In some embodiments, the molar ratio of γPPTHF / to the therapeutic agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In some embodiments, the γPTHF / therapeutic agent complex is encapsulated in liposomes (e.g., as described herein, or otherwise, as known in the art).

[0168] In other embodiments, the γPTHF complex comprises γPTHF and cyclodextrin. In some embodiments, the molar ratio of γPTHF (e.g., γPTHF salt) / cyclodextrin in the complex is in the range of 1 - 20:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / cyclodextrin in the complex is in the range of 1 - 10:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / cyclodextrin in the complex is in the range of 2 - 8:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / cyclodextrin in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In other embodiments, the molar ratio of γPTHF / cyclodextrin in the complex is in the range of 1:1 - 20, 1:1 - 10, or 1:2 - 8, or any range therebetween. In some embodiments, the molar ratio of γPTHF / cyclodextrin is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In some embodiments, the γPTHF / cyclodextrin complex is encapsulated in liposomes (e.g., as described herein, or otherwise, as known in the art).

[0169] In some embodiments, the present disclosure provides a composition comprising a γPTHF / platinum-based chemotherapeutic agent complex. In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of: cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the γPTHF / platinum-based chemotherapeutic agent complex comprises an analog of cisplatin, carboplatin, oxaliplatin, or salts or acids thereof. In some embodiments, the molar ratio of γPTHF / platinum-based agent in the complex ranges from 1-20:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / platinum-based agent in the complex ranges from 1-10:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / platinum-based agent in the complex ranges from 2-8:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / platinum-based agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPTHF / platinum-based agent in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPTHF / platinum-based agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, the γPTHF / / platinum-based agent complex is encapsulated in liposomes (e.g., as described herein, or otherwise, as known in the art).

[0170] In additional embodiments, the γPTHF / platinum-based chemotherapeutic complex comprises cisplatin, carboplatin, an oxaliplatin analog, or a salt or acid thereof. In some embodiments, the molar ratio of γPTHF / platinum-based analog in the complex ranges from 1-20:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / platinum-based analog in the complex ranges from 1-10:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / platinum-based agent in the complex ranges from 2-8:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / platinum-based analog in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPTHF / platinum-based analog in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPTHF / platinum-based analog is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, the γPTHF / / platinum-based analog complex is encapsulated in liposomes (e.g., as described herein or otherwise known in the art).

[0171] In a further embodiment, the present disclosure provides a complex containing γPTHF and cisplatin or a salt or acid thereof. In some embodiments, the molar ratio of γPTHF / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1-20:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1-10:1, or any range therebetween. In a further embodiment, the molar ratio of γPTHF / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 2-8:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / cisplatin (or a salt or acid of cisplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPTHF / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPTHF / cisplatin (or a salt or acid of cisplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In an additional embodiment, the γPTHF / / cisplatin (or a salt or acid of cisplatin) complex is encapsulated in liposomes (e.g., as described herein or otherwise known in the art).

[0172] In another embodiment, the present disclosure provides a complex containing γPTHF and carboplatin or a salt or acid thereof. In some embodiments, the molar ratio of γPTHF / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1-20:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1-10:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 2-8:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / carboplatin (or a salt or acid of carboplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPTHF / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPTHF / carboplatin (or a salt or acid of carboplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, the γPTHF / carboplatin (or a salt or acid of carboplatin) complex is encapsulated in liposomes (e.g., as described herein or otherwise known in the art).

[0173] In another embodiment, the present disclosure provides a complex containing γPTHF and oxaliplatin, or salts or acids thereof. In some embodiments, the molar ratio of γPTHF / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1-20:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1-10:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 2-8:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / oxaliplatin (or a salt or acid of oxaliplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPTHF / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPTHF / oxaliplatin (or a salt or acid of oxaliplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, the γPTHF / oxaliplatin (or a salt or acid of oxaliplatin) complex is encapsulated in liposomes (e.g., as described herein, or otherwise, as known in the art).

[0174] In additional embodiments, the present disclosure provides a complex comprising γPTHF and a platinum-based chemotherapeutic agent (platinum) selected from the group consisting of nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platinol, cisplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin (traplatin), enroplatin, JM216, NK121, CI973, DWA2114R, NDDP, and dedaplatin, or salts or acids thereof. In other embodiments, the γPTHF / platinum-based chemotherapeutic agent complex comprises a nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platinol, cisplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin (traplatin), enroplatin, JM216, NK121, CI973, DWA2114R, NDDP, or an analog of dedaplatin, or salts or acids thereof. In some embodiments, the molar ratio of γPTHF / platinum (or platinum salt or acid) in the complex ranges from 1-20:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / platinum (or platinum salt or acid) in the complex ranges from 1-10:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / platinum (or platinum salt or acid) in the complex ranges from 2-8:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / platinum (or platinum salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPTHF / platinum (or platinum salt or acid) in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween.In some embodiments, the molar ratio of γPTHF / platinum (or platinum salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In additional embodiments, the γPTHF / platinum (or its salt or acid or analog) complex is encapsulated within liposomes (e.g., as described herein or otherwise known in the art).

[0175] In some embodiments, the present disclosure provides a composition comprising a γPTHF / taxane chemotherapeutic agent (taxane) complex. In some embodiments, the taxane chemotherapeutic agent is selected from the group consisting of: paclitaxel (PTX), docetaxel (DTX), larotaxel (LTX), and cabazitaxel (CTX), or salts or acids thereof. In some embodiments, the molar ratio of γPTHF / taxane agent in the complex ranges from 1-20:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / taxane (or salt or acid of taxane) in the complex ranges from 1-10:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / taxane (or salt or acid of taxane) in the complex ranges from 2-8:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / taxane (or salt or acid of taxane) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPTHF / taxane (or salt or acid of taxane) in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γγPTHF / taxane (or salt or acid of taxane) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, the γPTHF / taxane agent complex is encapsulated in liposomes (e.g., as described herein or otherwise known in the art).

[0176] In additional embodiments, the present disclosure provides a complex comprising γPTHF and paclitaxel (PTX), or a salt or acid thereof. In other embodiments, the γPTHF / taxane-based chemotherapeutic complex comprises an analogue of paclitaxel (PTX), or a salt or acid thereof. In some embodiments, the molar ratio of γPTHF / paclitaxel (or a salt or acid of paclitaxel) in the complex ranges from 1-20:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / paclitaxel (or a salt or acid of paclitaxel) in the complex ranges from 1-10:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / paclitaxel (or a salt or acid of paclitaxel) in the complex ranges from 2-8:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / paclitaxel (or a salt or acid of paclitaxel) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPTHF / paclitaxel (or a salt or acid of paclitaxel) in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPTHF / paclitaxel (or a salt or acid of paclitaxel) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, the γPTHF / paclitaxel (or a salt or acid of paclitaxel) complex is encapsulated in liposomes (e.g., as described herein or otherwise known in the art).

[0177] In additional embodiments, the present disclosure provides a complex comprising γPTHF and docetaxel (DTX), or a salt or acid thereof. In other embodiments, the γPTHF / taxane-based chemotherapeutic complex comprises an analog of docetaxel (DTX), or a salt or acid thereof. In some embodiments, the molar ratio of γPTHF / docetaxel (or a salt or acid of docetaxel) in the complex ranges from 1-20:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / docetaxel (or a salt or acid of docetaxel) in the complex ranges from 1-10:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / docetaxel (or a salt or acid of docetaxel) in the complex ranges from 2-8:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / docetaxel (or a salt or acid of docetaxel) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPTHF / docetaxel (or a salt or acid of docetaxel) in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPTHF / docetaxel (or a salt or acid of docetaxel) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, the γPTHF / docetaxel (or a salt or acid of docetaxel) complex is encapsulated in liposomes (e.g., as described herein, or otherwise, as known in the art).

[0178] In additional embodiments, the present disclosure provides a complex comprising γPTHF and larotaxel (LTX), or a salt or acid thereof. In other embodiments, the γPTHF / taxane-based chemotherapeutic complex comprises an analog of larotaxel (LTX), or a salt or acid thereof. In some embodiments, the molar ratio of γPTHF / larotaxel (or a salt or acid of larotaxel) in the complex ranges from 1-20:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / larotaxel (or a salt or acid of larotaxel) in the complex ranges from 1-10:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / larotaxel (or a salt or acid of larotaxel) in the complex ranges from 2-8:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / larotaxel (or a salt or acid of larotaxel) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPTHF / larotaxel (or a salt or acid of larotaxel) in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPTHF / larotaxel (or a salt or acid of larotaxel) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, the γPTHF / larotaxel (or a salt or acid of larotaxel) complex is encapsulated in liposomes (e.g., as described herein, or otherwise, as known in the art).

[0179] In additional embodiments, the present disclosure provides a complex comprising γPTHF and cabazitaxel (CTX), or a salt or acid thereof. In other embodiments, the γPTHF / taxane-based chemotherapeutic complex comprises an analog of cabazitaxel (CTX), or a salt or acid thereof. In some embodiments, the molar ratio of γPTHF / cabazitaxel (or a salt or acid of cabazitaxel) in the complex ranges from 1-20:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / cabazitaxel (or a salt or acid of cabazitaxel) in the complex ranges from 1-10:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / cabazitaxel (or a salt or acid of cabazitaxel) in the complex ranges from 2-8:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / cabazitaxel (or a salt or acid of cabazitaxel) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPTHF / cabazitaxel (or a salt or acid of cabazitaxel) in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPTHF / cabazitaxel (or a salt or acid of cabazitaxel) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, the γPTHF / cabazitaxel (or a salt or acid of cabazitaxel) complex is encapsulated in liposomes (e.g., as described herein, or otherwise as known in the art).

[0180] In additional embodiments, the present disclosure provides a complex comprising γPTHF and another antimetabolite, or a salt or acid thereof. An antimetabolite is a chemical substance that is similar to a metabolite required for normal biochemical reactions but has a structure with sufficient differences to interfere with one or more normal functions of cells, such as cell division. In some embodiments, the present disclosure provides a complex comprising γPTHF and tetrahydrofolic acid (THF), or a salt or acid thereof. In some embodiments, the present disclosure provides a complex comprising γPTHF and an antimetabolite selected from the group consisting of: gemcitabine, fluorouracil, capecitabine, antifoilate (e.g., tetrahydrofolic acid, tetrahydrofolic acid), tegafur, cytosine arabinoside, thioguanine, 5-azacitidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, and any pharmaceutically acceptable salt or acid (single or plural) thereof, or derivative. In some embodiments, the molar ratio of γPTHF / antimetabolite (or salt or acid of the antimetabolite) in the complex ranges from 1-20:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / antimetabolite (or salt or acid of the antimetabolite) in the complex ranges from 1-10:1, or any range therebetween. In further embodiments, the molar ratio of γPTHF / antimetabolite (or salt or acid of the antimetabolite) in the complex ranges from 2-8:1, or any range therebetween. In some embodiments, the molar ratio of γPTHF / antimetabolite (or salt or acid of the antimetabolite) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPTHF / antimetabolite (or salt or acid of the antimetabolite) in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween.In some embodiments, the molar ratio of γPTHF / antimetabolite (or salt or acid of an antimetabolite) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In additional embodiments, the γPTHF / antimetabolite (or salt or acid of an antimetabolite) complex is encapsulated in liposomes (e.g., as described herein or otherwise known in the art).

[0181] In additional embodiments, the present disclosure provides a complex of γPTHF (e.g., γPTHF as disclosed herein) and cyclodextrin (CD). Cyclodextrin (CD) is a group of cyclic oligosaccharides that have been shown to improve the physicochemical properties of many drugs through complex formation. CD is a cyclic oligosaccharide composed of several D-glucose units linked by α-(1,4) bonds. This cyclic structure provides a hydrophobic inner cavity and gives CD a truncated cone shape. Many hydroxyl groups are located on the ends of the ring, which makes CD both lipophilic and water-soluble. As a result, CD can form complexes with a wide variety of hydrophobic agents, thereby changing the physicochemical properties of these complexed agents.

[0182] The term "cyclodextrin" or "CD" generally means a parent or derivatized cyclic oligosaccharide that can form a complex with tetrahydrofolic acid-PG, including a variable number of (α-1,4) linked D-glucopyranoside units, unless otherwise specified. Each cyclodextrin ring glucopyranoside subunit has secondary hydroxyl groups at the 2 and 3 positions and a primary hydroxyl group at the 6 position. The terms "parent", "undenivatized", or "inactive" cyclodextrin refer to the basic formula C6H 12Cyclodextrin having an O6 and glucose structure and no additional chemical substituents (e.g., α-cyclodextrin consisting of six D-glucopyranoside units, β-cyclodextrin consisting of seven D-glucopyranoside units, and γ-cyclodextrin consisting of eight D-glucopyranoside units). The physical and chemical properties of the parent cyclodextrin can be modified by derivatizing the hydroxyl groups with other functional groups. Any substance located in the inner phase of the cyclodextrin is said to be "complexed" with the cyclodextrin or to form a complex (inclusion complex) with the cyclodextrin.

[0183] As used herein, there are no special restrictions on the cyclodextrin component of the γPTHF / cyclodextrin complex as long as the cyclodextrin can form a complex with γPTHF. In certain embodiments, the cyclodextrin is derivatized to have ionizable (e.g., weakly basic and / or weakly acidic) functional groups to facilitate complex formation with γPTHF and / or liposomal encapsulates.

[0184] Modification of the hydroxyl groups of cyclodextrin, such as hydroxyl groups directed from the inner phase of cyclodextrin using ionizable chemical groups, is known to facilitate the addition of cyclodextrin and a therapeutic agent complexed with cyclodextrin. In some embodiments, the cyclodextrin of the γPTHF / cyclodextrin complex has hydroxyl groups substituted with at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ionizable chemical groups. The term "charged cyclodextrin" means a cyclodextrin having a hydroxyl group substituted with one or more of its charged moieties. Such moieties can include a charged group per se or an organic moiety substituted with one or more charged moieties (e.g., a C1-C6 alkyl or C1-C6 alkyl ether moiety).

[0185] In some embodiments, the "ionizable" or "charged" portion of the CD derivative is weakly ionizable. The weakly ionizable portion is a weakly basic or weakly acidic portion. The weakly basic functional group (W) has a pKa by CH3-W in the range of about 6.0 to 9.0, 6.5 to 8.5, 7.0 to 8.0, 7.5 to 8.0, and any range therebetween (including the endpoints). Similarly, the weakly acidic functional group (X) has a logarithmically recorded dissociation constant (pKa) by CH3-X in the range of about 3.0 to 7.0, 4.0 to 6.5, 4.5 to 6.5, 5.0 to 6.0, 5.0 to 5.5, and any range therebetween (including the endpoints). Representative anionic portions include, but are not limited to, carboxylate, carboxymethyl, succinyl, sulfonyl, phosphate, sulfoalkyl ether, sulfate carbonate, thiocarbonate, dithiocarbonate, phosphate, phosphonate, sulfonate, nitrate, and borate groups. Representative cationic portions include, but are not limited to, amino, guanidine, and quaternary ammonium groups.

[0186] In another embodiment, the derivatized cyclodextrin is a "polyanion" or a "polycat ion". A polyanion is a derivatized cyclodextrin having two or more negatively charged groups, resulting in a net negative ionic charge of three or more units. A polycation is a derivatized cyclodextrin having two or more positively charged groups, resulting in a net positive ionic charge of three or more units.

[0187] In another embodiment, the derivatized cyclodextrin is a "chargeable amphiphile". "Chargeable" means that the amphiphile has a pK in the range of pH 4 to 8 or 4 to 8.5. The chargeable amphiphile can thus be a weak acid or base. "Amphoteric" herein means a derivatized cyclodextrin having ionizable groups with both anionic and cationic characteristics, (a) at least one, optionally both, of the cationic and anionic amphiphiles being chargeable and having at least one charge group with a pK between 4 and 8 to 8.5, (b) the cationic charge being dominant at pH 4, and (c) the anionic charge being dominant at pH 8 to 8.5.

[0188] In some embodiments, the "ionizable" or "charged" derivatized cyclodextrins are generally polyionic, amphiphilic, or otherwise weakly ionizable (e.g., having a pKa of about 4.0 - 8.5, 4.5 - 8.0, 5.0 - 7.5, 5.5 - 7.5, 6.0 - 6.5, and any range (including both ends) therebetween).

[0189] Any one, some, or all of the hydroxyl groups of any one, some, or all of the α-D-glucopyranoside units of any cyclodextrin can be modified to an ionizable chemical group as described herein. Since each cyclodextrin hydroxyl group has different chemical reactivity, the reaction with the modification moiety can produce a non-crystalline mixture of positional and optical isomers. Alternatively, with specific chemistry, it can be reacted to form a homogeneous product of pre-modified α-D-glucopyranoside units.

[0190] Aggregate substitution occurring in cyclodextrin derivatives in a mixture is described by a term called degree of substitution. For example, 6-ethylenediamino-β-cyclodextrin having a degree of substitution of 7 would be composed of the distribution of isomers of 6-ethylenediamino-β-cyclodextrin where the number of ethylenediamino groups per 6-ethylenediamino-β-cyclodextrin molecule is 7. The degree of substitution of a cyclodextrin derivative mixture can be routinely measured using mass spectrometry or nuclear magnetic resonance spectroscopy.

[0191] In one embodiment, at least one hydroxyl moiety oriented in the opposite direction from within the cyclodextrin is replaced by an ionizable chemical group. For example, at least one of the γ-D-glucopyranoside units of C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and all three of C2-C3-C6 hydroxyls is replaced by an ionizable chemical group. Any such combination of hydroxyls can be combined with any of the degrees of substitution described herein, in addition to being able to similarly combine at least 2, 3, 4, 5, 6, 7, 8, 9, the maximum of all 10, 11, all of the α-D-glucopyranoside units in the modified cyclodextrin. One such derivative is sulfalkyl ether cyclodextrin (SAE-CD). The sulfobutyl ether derivative of β-cyclodextrin (SBE-β-CD) has been shown to have significantly improved water solubility compared to the parent cyclodextrin.

[0192] Additional cyclodextrin derivatives that can complex with a therapeutic agent in the disclosed liposome compositions include sugammadex or Org-25969, in which case the 6-hydroxy group on γ-CD is substituted with a carboxythioacetate ether bond and hydroxybutenyl-β-CD. Alternative forms of cyclodextrin include 2,6-di-O-methyl-β-CD (DIMEB), 2-hydroxylpropyl-3-cyclodextrin (HP-β-CD), randomly methylated-β-cyclodextrin (RAMEB), sulfobutyl ether β-cyclodextrin (SBE-β-CD), and sulfobutyl ether-γ-cyclodextrin (SBEγCD), sulfobutylated-β-cyclodextrin sodium salt, (2-hydroxypropyl)-γ-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, 2,6-di-O-methyl)-β-cyclodextrin (DIMEB-50 heptakis), 2,3,6-tri-O-methyl)-β-cyclodextrin (TRIMEB heptakis), methyl-β-cyclodextrin, octakis(6-deoxy-6-iodo)-γ-cyclodextrin, and octakis(6-deoxy-6-bromo)-γ-cyclodextrin.

[0193] In some embodiments, the cyclodextrin has high solubility in water to facilitate capture of a greater amount of cyclodextrin in the inner phase of the liposome. In some embodiments, the aqueous solubility of the cyclodextrin is at least 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, or more. In some embodiments, the water solubility of the cyclodextrin is in the range of 10 - 150 mg / mL, 20 - 100 mg / mL, 20 - 75 mg / mL, and within any range therebetween (including both ends).

[0194] In some embodiments, a large binding constant between cyclodextrin and γPTHF and / or other therapeutic agents complexed with cyclodextrin is preferred and can be obtained by selecting the number of glucose units in the cyclodextrin based on the size of the therapeutic agent (see, e.g., Albers et al., Crit. Rev. Therap. Drug Carrier Syst. 12:311-337 (1995); Stella et al., Toxicol. Pathol. 36:30-42 (2008)). When the binding constant is pH-dependent, cyclodextrin can be selected such that the binding constant is large at the pH of the inner phase of the liposome. As a result, the solubility (apparent solubility) of the therapeutic agent in the presence of cyclodextrin can be further improved. In some embodiments, the binding constant between cyclodextrin and the therapeutic agent is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. In some embodiments, the binding constant between cyclodextrin and the therapeutic agent is in the range of 100-1,200, 200-1,000, 300-750, and any range therebetween.

[0195] In some embodiments, the cyclodextrin of the γPTHF / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is not derivatized.

[0196] In some embodiments, the cyclodextrin of the γPTHF / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is derivatized. In a further embodiment, the cyclodextrin derivative of the complex has the structure of Formula I:

Chemical Formula

[0197] In some embodiments, the cyclodextrin derivative of the γPTHF / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is of formula II:

Chemical formula

[0198] In some embodiments, the cyclodextrin derivatives of the γPTHF / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex are the cyclodextrins disclosed in U.S. Patent Nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, 6,869,939; and International Publication No. 02005 / 117911. The content of each of these patent documents is hereby incorporated by reference into this specification preferentially.

[0199] In some embodiments, the cyclodextrin derivatives of the γPTHF / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex are sulfalkyl ether cyclodextrins. In some embodiments, the cyclodextrin derivative of the complex is a sulfobutyl ether-3-cyclodextrin such as CAPTISOL® (CyDex Pharma.Inc., Lenexa, Kansas). Methods for preparing sulfobutyl ether-3-cyclodextrin and other sulfalkyl ether cyclodextrins are known in the art.

[0200] In some embodiments, the cyclodextrin derivative of the γPTHF / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is of Formula III: [Chemical formula] a compound wherein R is (a) (H) 21-X or (-(CH2)4-SO3Na) X and x = 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0; (b) (H) 21-X or (-(CH2CH(OH)CH3) X and x = 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0; (c) (H) 21-X or (sulfonaliphatic ether) X and x = 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0; or (d) (H) 21-X or (-(CH2)4-SO3Na) X and x = 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0.

[0201] In additional embodiments, the γPTHF / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is encapsulated in liposomes (e.g., as described herein or otherwise known in the art).

[0202] III. γPTHF Delivery Vehicle In other embodiments, the present disclosure provides γPTHF delivery systems and their use for delivering γPTHF payloads to cells (single or plural) in vitro or in vivo. In some embodiments, γPTHF is complexed with or incorporated into a delivery vehicle. Such delivery vehicles are known in the art and include, but are not limited to, liposomes, lipospheres, polymers, peptides, proteins, antibodies (e.g., ADCs such as antibody-γPTHF conjugates), cell components, cyclic oligosaccharides (e.g., cyclodextrins), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), lipoprotein particles, and combinations thereof. In certain embodiments, the delivery vehicle is a liposome. In other certain embodiments, the delivery vehicle is an antibody or antigen-binding antibody fragment.

[0203] A. Liposomes In some embodiments, the present disclosure provides a liposomal composition comprising liposomes encapsulating (i.e., filled with) γPTHF (e.g., γPTHF disclosed herein). In some embodiments, the liposomes in the liposomal composition comprise γPTHF containing 4, 5, 2 - 10, 4 - 6, or more than 5 glutamyl groups (including the glutamyl groups in tetrahydrofolic acid). In some embodiments, the γPTHF in Lp - γPTHF comprises two or more L - type glutamyl groups. In other embodiments, the γPTHF in Lp - γPTHF comprises a D - type glutamyl group. In further embodiments, the γPTHF in Lp - γPTHF comprises a D - type glutamyl group and two or more L - type glutamyl groups. In additional embodiments, the γPTHF in Lp - γPTHF comprises two or more glutamyl groups having a γ - carboxyl bond. In some embodiments, the liposomal composition comprises liposomes containing γ - pentaglutamyl oxidized THF. In further embodiments, the liposomes comprise L - γ - pentaglutamyl oxidized THF, D - γ - pentaglutamyl oxidized THF, or L - and D - γ - pentaglutamyl oxidized THF. In some embodiments, the liposomal composition comprises liposomes containing γ - hexaglutamyl oxidized THF (Lp - γPTHF). In further embodiments, the liposomes comprise L - γ - hexaglutamyl oxidized THF, D - γ - hexaglutamyl oxidized THF, or L - and D - γ - hexaglutamyl oxidized THF. In some embodiments, the liposomal composition comprises liposomes that are anionic or neutral. In some embodiments, the liposomal composition comprises liposomes that are cationic. In some embodiments, the Lp - γPTHF composition is not pegylated. In some embodiments, the Lp - γPTHF composition is not targeted (NTLp - γPTHF). In other embodiments, the Lp - γPTHF composition is targeted (TLp - γPTHF). In some embodiments, the liposomal composition comprises liposomes having a diameter in the range of 20 nm to 500 nm, or any range therebetween. In some embodiments, the liposomal composition comprises liposomes having a diameter in the range of 20 nm to 400 nm, or any range therebetween.In some embodiments, the liposome composition comprises liposomes having a diameter of 20 nm to 300 nm, or any range therebetween. In some embodiments, the liposome composition comprises liposomes having a diameter of 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposome composition comprises liposomes having a diameter of 20 nm to 150 nm, or any range therebetween. In further embodiments, the liposome composition comprises liposomes having a diameter of 80 nm to 120 nm, or any range therebetween. In additional embodiments, 30 - 70%, 30 - 60%, or 30 - 50% w / w, or any range therebetween of γPTHF is encapsulated (trapped) in Lp-γPTHF during the process of preparing the liposomes. In some embodiments, the Lp-αPTHF composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or greater than 75% w / w of γ-polyglutamylated THF. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or greater than 75% w / w of γPTHF is encapsulated in Lp-γPTHF during the process of preparing the liposomes.

[0204] In some embodiments, the liposomes provided further comprise an immunostimulant, a detectable marker, or both, disposed on the outer surface of the liposome. The immunostimulant or detectable marker can be ionically or covalently bound to the outer surface of the liposome, optionally including binding to the steric stabilizer component of the liposome.

[0205] The term "immunostimulatory agent" is also known as "immunostimulant" and "immunostimulator", and refers to a substance that stimulates immunity (including existing immune responses) by inducing the activation or increased activity of any component of the immune system. These immunostimulatory agents include one or more of haptens, adjuvants, protein immunostimulatory agents, nucleic acid immunostimulatory agents, and chemical immunostimulatory agents. Many adjuvants contain substances designed to stimulate immune responses, such as lipid A, proteins derived from Bordetella pertussis or Mycobacterium tuberculosis. Specific adjuvants include, for example, Freund's incomplete adjuvant and complete adjuvant (Difco Laboratories, Detroit, Mich.); Merck adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, PA.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron, or zinc; insoluble suspensions of acylated tyrosine; acylated saccharides; polysaccharides derivatized cationically or anionicly; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A; IFNα, IFNγ, FLT3 ligand; and immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3) are commercially available. Cytokines such as GM-CSF, interleukin 2, 7, 12, and 15, and other similar growth factors can also be used as adjuvants. In a preferred embodiment, the immunostimulant can be at least one selected from the group consisting of fluorescein, DNP, beta-glucan, β-1,3-glucan, β-1,6-glucan. In a further preferred embodiment, the immunostimulant is a toll-like receptor (TLR) regulator. In a further embodiment, the toll-like receptor (TLR) regulator is one or more of oxidized low density lipoprotein (e.g., OXPAC, PGPC), eritoran lipid (e.g., E5564), and resolvin.In some embodiments, the liposome contains fluorescein isothiocyanate (FITC), which, based on our experiments, surprisingly functions as both an immunostimulant and a detectable marker.

[0206] In some embodiments, the liposome contains a detectable marker. Detectable markers can include, for example, any suitable means known in the art, such as, at least, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelating agents, enzymes, dyes, inks, magnetic compounds, biocatalysts, or pigments that are detectable by magnetic resonance imaging (MRI), optical imaging, fluorescence / bioluminescence imaging, and / or nuclear imaging techniques.

[0207] In some embodiments, the immunostimulant and / or detectable marker is bound to the outer surface by co-incubation with the liposome. For example, the immunostimulant and / or detectable marker can be bound to the liposome membrane by hydrophobic interactions or ionic bonds such as avidin / biotin binding or metal chelate binding (e.g., Ni-NTA). Alternatively, the immunostimulant or detectable marker can be covalently bound to the outer surface of the liposome, for example, by covalently binding to a liposome component or to a steric stabilizer that is PEG.

[0208] In some embodiments, the liposome further contains an agent that increases the uptake of the liposome into the intracellular compartment of the target cell containing the cytosol.

[0209] In some embodiments, the liposome comprises a mitochondrial targeting agent. In some embodiments, the liposome comprises triphenylphosphonium (TPP). Methods and mechanisms for surface functionalization of liposomes using TPP are known in the art (e.g., binding TPP to a lipid anchor via a peg spacer group and modifying TPP with a stearyl group (stearyltriphenylphosphonium (STPP))). In some embodiments, the liposome comprises high density octaarginine. In some embodiments, the liposome comprises sphingomyelin and / or sphingomyelin metabolites. Sphingomyelin metabolites used to formulate the liposomes of the present invention can include, for example, ceramide, sphingosine or sphingosine 1-phosphate. In some embodiments, the liposome comprises rhodamine 123. In some embodiments, the liposome comprises a mitochondrial permeability peptide. In some embodiments, the liposome comprises a mitochondrial permeabilizing agent selected from the group consisting of: mitofusin peptide, a mitochondrial targeting signal peptide, and antennapedia helix III homeodomain cell membrane permeable peptide (ANT) (e.g., RQIKIWFQNRRMKWKKRKKRRQRRR (SEQ ID NO: 1), RKKRRXRRRGC, wherein X is any natural or non-natural amino acid (SEQ ID NO: 2), CCGCCAAGAAGCG (SEQ ID NO: 3), GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTGAGCGGCGTGGCGCGGGGGCGTCAT (SEQ ID NO: 4), ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTCATCCGTCAGCTC (SEQ ID NO: 5), or ACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTG (SEQ ID NO: 6)), or a mitochondrial permeable fragment thereof.

[0210] In some embodiments, the liposomes in the provided liposomal composition include a mitochondrial permeabilizing agent selected from guanidine-rich peptides, tetraguanidium, triguanidium, diguanidium, monoguanidium, guanidine-rich polycarbamate, beta-oligoarginine, proline-rich dendrimers, and phosphonium salts (e.g., methyltriphenylphosphonium and / or tetraphenylphosphonium).

[0211] In some embodiments, the liposomes in the provided liposomal composition include sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposomes include sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposomes include DOPE, sphingomyelin, stearyl octaarginine sphingomyelin and stearyl octaarginine. In some embodiments, the liposomes include DOPE, sphingomyelin, stearyl octaarginine sphingomyelin and stearyl octaarginine in a molar ratio of 9:2:1. In some embodiments, the liposomes include the MITO-Porter® system or a variant thereof.

[0212] In some embodiments, the liposomes in the provided liposome composition include agents such as membrane permeabilizing agents that facilitate the delivery of the liposomes across the cell membrane and confer the ability to bypass the harsh environment of the endocytic pathway and lysosomes to the liposomes. Membrane permeabilizing agents are known in the art and are routinely used and applicable to the manufacture and use of the provided liposome compositions. In some embodiments, the membrane penetration agent / lysosome bypass agent is chloroquine. In some embodiments, the membrane permeabilizing agent is a cell permeable peptide.In some embodiments, the liposomes in the provided liposome composition comprise a membrane permeabilizing agent selected from the following group: RKKRRQRRR (SEQ ID NO: 7), GRKKRRQRRRTPQ (SEQ ID NO: 8), YGRKKRRQRRR (SEQ ID NO: 9), AAVALLPAVLLALLA (SEQ ID NO: 10), MGLGLHLLVLAAALQ (SEQ ID NO: 11), GALFLGFLGAAGSTM (SEQ ID NO: 12), AGYLLGKINLKALAALAKKIL (SEQ ID NO: 13), RVIRVWFQNKRCKDKK (SEQ ID NO: 14), RQIKIWFQNRRMKWKK (SEQ ID NO: 15), GLFEAIAGFIENGWEGMIDG (SEQ ID NO: 16), GWTLNSAGYLLGKIN (SEQ ID NO: 17), RSQSRSRYYRQRQRS (SEQ ID NO: 18), LAIPEQEY (SEQ ID NO: 19), LGIAEQEY (SEQ ID NO: 20), LGIPAQEY (SEQ ID NO: 21), LGIPEAEY (SEQ ID NO: 22), LGIPEQAY (SEQ ID NO: 23), LGIAEAEY (SEQ ID NO: 24), LGIPEAAY (SEQ ID NO: 25), LGIAEQAY (SEQ ID NO: 26), LGIAEAAY (SEQ ID NO: 27), LLIILRRRIRKQAHAHSK (SEQ ID NO: 28), LKALAALAKKIL (SEQ ID NO: 29), KLALKLALKALKAALKLA (SEQ ID NO: 30), KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 31), DHQLNPAF (SEQ ID NO: 32), DPKGDPKG (SEQ ID NO: 33), VTVTVTVTVTGKGDPKPD (SEQ ID NO: 34), RQIKIWFQNRRMKWKK (SEQ ID NO: 35), GRKKRRQRRRPPQ (SEQ ID NO: 36), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 37), GRKKRRQRRR (SEQ ID NO: 38), RRRRRRR (SEQ ID NO: 39), RRRRRRRR (SEQ ID NO: 40), RRRRRRRRR (SEQ ID NO: 41), RRRRRRRRRR (SEQ ID NO: 42), RRRRRRRRRRR (SEQ ID NO: 43), and YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG(R)n (wherein n = 2 to 15 R in L- and / or D-form) (SEQ ID NO: 44), or cell permeable fragments thereof.

[0213] As discussed above, liposomes can include steric stabilizers that can extend their lifetimes in circulation. For these embodiments incorporating steric stabilizers, the steric stabilizer can be at least one member selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymers; oligoglycerol, polyethylene glycol and polypropylene oxide-containing copolymers, poloxamer 188, and polyvinyl alcohol. In some embodiments, the steric stabilizer or group of steric stabilizers is PEG. In one embodiment, the steric stabilizer is PEG. In a further embodiment, the PEG has a number average molecular weight (Mn) of 200 to 5000 daltons. These PEGs can have any structure such as linear, branched, star or comb-shaped structures and are commercially available.

[0214] In some embodiments, the liposome composition comprises pegylated liposomes (PLp-γPTHF). In some embodiments, the liposomes in the pegylated liposome composition comprise γPTHF containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the γPTHF in Lp-γPTHF comprises two or more L-type glutamyl groups. In other embodiments, the γPTHF in Lp-γPTHF comprises a D-type glutamyl group. In further embodiments, the γPTHF in Lp-γPTHF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the liposome composition comprises pegylated liposomes comprising γ-pentaglutamyl oxidized THF. In further embodiments, the liposomes comprise L-γ-pentaglutamyl oxidized THF, D-γ-pentaglutamyl oxidized THF, or L- and D-γ-pentaglutamyl oxidized THF. In some embodiments, the liposome composition comprises pegylated liposomes comprising γ-hexaglutamyl oxidized THF. In further embodiments, the liposomes comprise L-γ-hexaglutamyl oxidized THF, D-γ-hexaglutamyl oxidized THF, or L- and D-γ-hexaglutamyl oxidized THF. In some embodiments, the liposome composition comprises pegylated liposomes that are anionic or neutral. In some embodiments, the liposome composition comprises pegylated liposomes that are cationic. In some embodiments, the PLp-γPTHF composition is not targeted (NTPLp-γPTHF). In other embodiments, the PLp-γPTHF composition is targeted (TPLp-γPTHF). In some embodiments, the liposome composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of γPTHF. In some embodiments, the liposome composition comprises pegylated liposomes encapsulating (trapping) at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of γPTHF, which is encapsulated in PLp-γPTHF during the process of preparing the liposomes.In some embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 20 nm to 500 nm. In some embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 20 nm to 400 nm. In some embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 20 nm to 300 nm. In some embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 20 nm to 200 nm. In further embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 80 nm to 120 nm.

[0215] In some embodiments, more than 70%, 80% or 90% of the polyglutamylated tetrahydrofolic acid in the provided liposome composition is pentaglutamylated. In some embodiments, more than 70%, 80% or 90% of the polyglutamylated tetrahydrofolic acid in the provided composition is hexaglutamylated. In some embodiments, more than 70%, 80% or 90% of the polyglutamylated tetrahydrofolic acid in the composition has 4 - 10, 4 - 6, or more than 5 γ - glutamyl groups.

[0216] In some embodiments, the γPTHF composition (e.g., delivery vehicles such as liposomes containing polyglutamic acid and polyglutamic acid) is in an aqueous solution. In some embodiments, the γPTHF composition is in the liposome composition and is administered at a dose of 0.005 to 5000 mg of γPTHF per square meter (m 2 ) of body surface area or any range therebetween. In further embodiments, the γPTHF composition is in the liposome composition and is administered at a dose of 0.1 to 1000 mg of γPTHF per square meter of body surface area, or any range therebetween.

[0217] (1) Liposome composition The lipids and other components of the liposomes contained in the liposome composition can be any lipid, combination and ratio of lipids, or combination of lipids and other liposome components and their respective ratios known in the art. However, it will be understood by those skilled in the art that liposomal encapsulation of any particular drug, such as γ-polyglutamylated THF discussed herein, which is not limited, may involve substantially routine experimentation to obtain a useful and functional liposome formulation. Generally, the liposomes provided can have any liposome structure, for example, a structure having an inner space isolated from the outer medium by one or more lipid bilayers, or any microcapsule structure having a semipermeable membrane with a lipophilic central portion where the membrane isolates the interior. The lipid bilayer can be any amphiphilic molecule of any structure characterized by a hydrophilic moiety (hydrophilic moiety hydrophilic moiety) and a hydrophobic moiety (hydrophobic moiety). Usually, the amphiphilic molecules in the bilayer are arranged in a two-dimensional sheet where the hydrophobic moieties face the inside of the sheet while the hydrophilic moieties face the outside. The amphiphilic molecules forming the liposomes provided can be any known or hereafter discovered amphiphilic molecules (e.g., synthetic or naturally occurring lipids or biocompatible lipids). Liposomes can be formed by amphiphilic polymers and surfactants, such as polymersomes and niosomes. In the present disclosure, without limitation, these liposome-forming materials are also referred to as "lipids".

[0218] The liposome composition formulations provided herein can be in liquid or dry forms such as dry powder or dry cake. The dry powder or dry cake can be subjected to primary drying, for example, under lyophilization conditions, or can be subjected to only primary drying or both primary and secondary drying. In the dry form, the powder or cake can have, for example, 1% to 6% moisture, such as 2% to 5% moisture or 2% to 4% moisture. An example of the drying method is lyophilization (also called freeze-drying or cryodessication). Any of the compositions and methods of the present disclosure can include liposomes, lyophilized liposomes, or liposomes reconstituted from lyophilized liposomes. In some embodiments, the disclosed compositions and methods include one or more lyoprotectants or cryoprotective substances. These protectants are typically saccharides (monosaccharides, disaccharides, and polysaccharides), polyhydric alcohols, and their derivatives, polyhydroxy compounds such as glycerol or polyethylene glycol, trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol, or aminoglycoside. In further embodiments, the lyoprotectant or cryoprotective substance comprises up to 10% or up to 20% of the solution outside, inside, or both outside and inside the liposome.

[0219] In some embodiments, the liposomes contain steric stabilizers that extend their lifespan in circulation. One or more steric stabilizers, such as hydrophilic polymers (polyethylene glycol (PEG)), glycolipids (monosialoganglioside (GM1)), and others, occupy the space directly adjacent to the liposome surface and exclude other polymers from this space. As a result, access and binding of plasma opsonins to the liposome surface are hindered, thus suppressing the interaction of such liposomes with macrophages or any other clearance mechanism, and extending the lifespan of the liposomes in circulation. In some embodiments, the steric stabilizer or group of steric stabilizers is PEG or a combination containing PEG. In further embodiments, the steric stabilizer is PEG or a combination containing PEG with a number average molecular weight (Mn) in the range of 200 to 5000 daltons. These PEGs can have any structure, such as linear, branched, star, or comb-shaped structures, and are commercially available.

[0220] The diameter of the disclosed liposomes is not particularly limited. In some embodiments, the liposomes have a diameter in the range of, for example, 30 - 150 nm (nanometers). In other embodiments, the liposomes have a diameter in the range of 40 - 70 nm.

[0221] The properties of the liposomes are affected by the nature of the lipids used to produce the liposomes. A wide variety of lipids have been used to produce liposomes. These include cationic, anionic, and neutral lipids. In some embodiments, the liposomes containing γPTHF are anionic or neutral. In other embodiments, the provided liposomes are cationic. The determination of the charge (e.g., anionic, neutral, or cationic) can be determined by a routine operation by measuring the zeta potential of the liposomes. The zeta potential of the liposomes can be positive, zero, or negative. In some embodiments, the zeta potential of the liposomes is below zero. In some embodiments, the zeta potential of the liposomes is in the range of 0 to -150 mV. In another embodiment, the zeta potential of the liposomes is in the range of -30 to -50 mV.

[0222] In some embodiments, cationic lipids are used to prepare cationic liposomes, which are commonly used as gene delivery agents. The positive charges on the cationic liposomes enable interaction with the negative charges on the cell surface. After the cationic liposomes bind to the cells, the liposomes are transported into the cells by endocytosis.

[0223] In some preferred embodiments, neutral to anionic liposomes are used. In a preferred embodiment, anionic liposomes are used. For example, by using a mixture of neutral lipids such as HSPC and anionic lipids such as PEG-DSPE, anionic liposomes are formed, which have a low potential for non-specific binding to normal cells. Specific binding to tumor cells can be achieved using tumor targeting antibodies such as folate receptor antibodies including, for example, folate receptor alpha antibody, folate receptor beta antibody, and / or folate receptor delta antibody.

[0224] As an example, at least one (or several) lipid is an amphiphilic lipid defined as having hydrophilic and hydrophobic moieties (usually a hydrophilic head and a hydrophobic tail). The hydrophobic moiety usually faces the hydrophobic phase (e.g., within the bilayer), while the hydrophilic moiety usually faces the aqueous phase (e.g., outside the bilayer). The hydrophilic moiety can include polar or charged groups such as carbohydrates, phosphates, carboxylic acids, sulfates, aminos, sulfhydryls, nitros, hydroxyls, and other similar groups. The hydrophobic moiety can include nonpolar groups including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted by one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphiphilic compounds include, but are not limited to, phospholipids, amino lipids, and sphingolipids.

[0225] Typically, for example, the lipid is a phospholipid. Phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and the like. It should be understood that other lipid membrane components such as cholesterol, sphingomyelin, and cardiolipin can also be used.

[0226] The lipids including liposomes provided herein can be anionic and neutral (including zwitterio...

Claims

**Claim 1** A liposome composition comprising liposomes encapsulating γ-polyglutamylated tetrahydrofolic acid and one or more non-γ-polyglutamylated γ-polyglutaminatable folic acid antagonists or non-γ-polyglutaminatable folic acid antagonists, wherein the γ-polyglutamylated tetrahydrofolic acid contains 2 to 15 glutamyl groups having γ-carboxyl group linkages, and the γ-polyglutamylated tetrahydrofolic acid is (a) γ-polyglutamylated 5-formyl-THF; (b) γ-polyglutamylated 10-formyl-THF; (c) γ-polyglutamylated 5,10-methenyl-THF; (d) γ-polyglutamylated 5-methyl-THF; (e) γ-polyglutamylated 5,10-methylene-THF; and (f) selected from the group consisting of γ-polyglutamylated 5-formimino-THF, where (i) at least two of the glutamyl groups of the γ-polyglutamylated tetrahydrofolic acid are of the L-type, or (ii) each of the glutamyl groups of the γ-polyglutamylated tetrahydrofolic acid is of the L-type, or (iii) at least one of the glutamyl groups of the γ-polyglutamylated tetrahydrofolic acid is of the D-type, or (iv) each of the glutamyl groups of the γ-polyglutamylated tetrahydrofolic acid other than the glutamyl group of pemetrexed is of the D-type, or, (v) at least two of the glutamyl groups of the γ-polyglutamylated tetrahydrofolic acid are of the L-type and at least one glutamyl group is of the D-type, and the liposomes have a diameter of 50 to 150 nm, a liposome composition.

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