Alpha polyglutamated tetrahydrofolate and uses thereof

Abstract

To provide methods for improving the effect of tetrahydrofolate in treating disease and polyglutamated alpha tetrahydrofolate compositions with improved therapeutic effect.SOLUTION: Disclosed is a liposome composition comprising liposomes encapsulating alpha polyglutamated tetrahydrofolate, where at least one glutamyl group of the alpha polyglutamated tetrahydrofolate has two or more alpha carboxyl group linkage.SELECTED DRAWING: None

Description

Background Art

[0001] The present disclosure generally relates to compositions of polyglutamylated alpha-tetrahydrofolate, including delivery vehicles such as liposomes containing the polyglutamylated alpha-tetrahydrofolate compositions, and methods of manufacturing and using such 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 alpha-tetrahydrofolate compositions have uses as adjunctive therapies with one or more therapeutic agents such as chemotherapeutic agents (e.g., 5-fluorouracil) that enhance the effects of the therapeutic agent(s), or as “chemoprotective agents” that reduce the toxic side effects associated with the therapeutic agent(s) (e.g., in combination with folate antagonists such as methotrexate).

[0002] Folate is an essential cofactor that mediates the transfer of one-carbon units involved in nucleotide biosynthesis and DNA repair, remethylation of homocysteine (Hcy), and methylation of DNA, proteins, and lipids. The only circulating form of folate in the blood is monoglutamate, and monoglutamate folate is the only form of folate that is transported across cell membranes. Similarly, monoglutamate forms of tetrahydrofolate are also transported across cell membranes. Once taken up into cells, intracellular tetrahydrofolate is polyglutamylated by the enzyme folylpoly-gamma-glutamate synthase (FPGS). Tetrahydrofolate polyglutamylation by FPGS serves at least two major therapeutic purposes: (1) significantly increasing the affinity of tetrahydrofolate for DHFR; and (2) facilitating the accumulation of polyglutamylated tetrahydrofolate, which, unlike tetrahydrofolate (monoglutamate), is not readily transported out of cells by cellular efflux pumps.

[0003] The provided polyglutamylated alpha-tetrahydrofolate compositions provide a strategy for improving the therapeutic efficacy of tetrahydrofolate.

Summary of the Invention

[0004] The present disclosure generally relates to compositions of polyglutamylated alpha - tetrahydrofolate (THF), and methods of making and using such compositions for treating diseases including hyperproliferative diseases such as cancer, immune system disorders such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria. The polyglutamylated alpha - tetrahydrofolate compositions have uses in combination therapies with one or more therapeutic agents (e.g., chemotherapeutic agents such as 5 - fluorouracil) that enhance the effect of the therapeutic agent(s), or as "chemoprotective agents" (e.g., in combination with folate antagonists such as methotrexate) that 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 polyglutamylated alpha - tetrahydrofolate. [2] The polyglutamylated alpha - tetrahydrofolate is (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 (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) The composition according to item [1], selected from the group consisting of. [3] The composition according to item [1] or [2], wherein the polyglutamylated alpha-tetrahydrofolic acid contains 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups having an alpha-carboxyl group bond. [4] The composition according to any one of items [1] to [3], wherein the polyglutamylated alpha-tetrahydrofolic acid is tetraglutamylated alpha-tetrahydrofolic acid (for example, [6R]-5,10-methenyl-THF, [6S]-5-formyl-THF, and / or [6R]-10-formyl-THF). [5] The composition according to any one of items [1] to [3], wherein the polyglutamylated alpha-tetrahydrofolic acid is pentaglutamylated alpha-tetrahydrofolic acid (for example, [6R]-5,10-methenyl-THF, [6S]-5-formyl-THF, and / or [6R]-10-formyl-THF). [6] The composition according to any one of items [1] to [3], wherein the polyglutamylated alpha-tetrahydrofolic acid is hexaglutamylated alpha-tetrahydrofolic acid (for example, [6R]-5,10-methenyl-THF, [6S]-5-formyl-THF, and / or [6R]-10-formyl-THF). [7] (a) Two or more glutamyl groups have an alpha-carboxyl group bond, (b) Each glutamyl group other than the glutamyl group of tetrahydrofolic acid has an alpha-carboxyl group bond; or (c) Two or more glutamyl groups have a gamma-carboxyl group bond, The composition according to any one of items [1] to [6]. [8] The composition according to any one of items [1] to [7], wherein at least one glutamyl group has both an alpha-carboxyl group bond and a gamma-carboxyl group bond. [9] (a) At least two of the glutamyl groups of the polyglutamylated alpha-tetrahydrofolic acid are of the L type, (b) Each of the glutamyl groups of the polyglutamylated alpha-tetrahydrofolic acid is of the L type, (c) at least one of the glutamyl groups of polyglutamylated alpha-tetrahydrofolic acid is of the D type, (d) the glutamyl groups of polyglutamylated alpha-tetrahydrofolic acid other than the glutamyl groups of tetrahydrofolic acid are each of the D type, or (e) at least two of the glutamyl groups of polyglutamylated alpha-tetrahydrofolic acid are of the L type and at least one of the glutamyl groups is of the D type, The composition according to any one of items [1] to [8].

[10] The composition according to any one of items [1] to [9], wherein the polyglutamic acid is linear.

[11] The composition according to any one of items [1] to [9], wherein the polyglutamic acid is branched.

[12] A liposome composition (Lp-αPTHF) comprising the polyglutamylated alpha-tetrahydrofolic acid according to any one of items [1] to

[11] ;

[13] The LαPP composition according to item

[12] , wherein the polyglutamylated alpha-tetrahydrofolic acid contains an L-type glutamyl group having an alpha-carboxyl group bond;

[14] The Lp-αPTHF composition according to item

[12] or

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

[15] The Lp-αPTHF composition according to item

[12] or

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

[16] The Lp-αPTHF composition according to any one of items

[12] to

[15] , wherein the liposome contains polyglutamylated alpha-tetrahydrofolic acid containing 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups;

[17] The Lp-αPTHF composition according to any one of items

[12] to

[16] , wherein at least one of the glutamyl groups of the polyglutamylated alpha-tetrahydrofolic acid has a gamma-carboxyl group bond;

[18] The composition according to any one of items

[12] to

[17] , wherein at least one glutamyl group has both an alpha carboxyl group bond and a gamma carboxyl group bond;

[19] The composition according to any one of items

[12] to

[18] , comprising 2, 3, 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups having both an alpha carboxyl group bond and a gamma carboxyl group bond;

[20] The Lp-αPTHF composition according to any one of items

[12] to

[19] , wherein the liposome contains a polyglutamylated alpha tetrahydrofolic acid containing tetraglutamylated alpha tetrahydrofolic acid, pentaglutamylated alpha tetrahydrofolic acid, or hexaglutamylated alpha tetrahydrofolic acid;

[21] The Lp-αPTHF composition according to any one of items

[12] to

[19] , wherein the liposome contains a polyglutamylated alpha tetrahydrofolic acid containing tetraglutamylated alpha tetrahydrofolic acid, pentaglutamylated alpha tetrahydrofolic acid, or hexaglutamylated alpha tetrahydrofolic acid;

[22] The Lp-αPTHF composition according to any one of items

[12] to

[21] , wherein the polyglutamic acid is linear or branched;

[23] The Lp-αPTHF composition according to any one of items

[12] to

[22] , wherein the liposome is pegylated (PαLp-αPTHF);

[24] The Lp-αPTHF composition according to any one of items

[12] to

[23] , wherein the liposome contains at least 1% by weight (w / w) of polyglutamylated alpha tetrahydrofolic acid, or at least 1% of the starting material of polyglutamylated alpha THF is encapsulated (enclosed) in αPTHF in the process of preparing Lp-αPTHF;

[25] The Lp-αPTHF composition according to any one of items

[12] to

[24] , wherein the liposome has a diameter in the range of 20 nm to 500 nm or 20 nm to 200 nm;

[26] The Lp-αPTHF composition according to any one of items

[12] to

[25] , wherein the liposome has a diameter in the range of 80 nm to 120 nm;

[27] A liposome is an Lp-αPTHF composition according to any one of items

[12] to

[26] , formed from liposome components;

[28] The liposome composition according to item

[27] , wherein the liposome component contains at least one anionic lipid and neutral lipid;

[29] The liposome composition according to item

[27] or

[28] , wherein the liposome component contains at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide;

[30] The liposome composition according to any one of items

[27] to

[29] , wherein the liposome component contains at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC;

[31] The liposome composition according to any one of items

[27] to

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

[32] The liposome composition according to item

[31] , wherein the steric stabilizer is at least one 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 polymer; oligoglycerol, a copolymer containing polyethylene glycol and polypropylene oxide, poloxamer 188, and polyvinyl alcohol;

[33] The liposome composition according to item

[32] , wherein the steric stabilizer is PEG and PEG has a number average molecular weight (Mn) of 200 to 5000 daltons;

[34] The Lp-αPTHF composition according to any one of items

[12] to

[33] , wherein the liposome is anionic or neutral;

[35] The Lp-αPTHF composition according to any one of items

[12] to

[33] , wherein the liposome has a zeta potential of zero or less;

[36] The Lp-αPTHF composition according to any one of items

[12] to

[33] , wherein the liposome has a zeta potential of 0 to -150 mV;

[37] The Lp-αPTHF composition according to any one of items

[12] to

[33] , wherein the liposome has a zeta potential of -30 to -50 mV;

[38] The Lp-αPTHF composition according to any one of items

[12] to

[33] , wherein the liposome is cationic;

[39] The Lp-αPTHF composition according to any one of items

[12] to

[38] , wherein the liposome has an internal space containing polyglutamylated alpha-tetrahydrofolic acid and an aqueous pharmaceutically acceptable carrier;

[40] The Lp-αPTHF composition according to item

[39] , wherein the pharmaceutically acceptable carrier contains an isotonic agent such as dextrose, mannitol, glycerol, potassium chloride, sodium chloride, etc. at a concentration of more than 1%;

[41] The Lp-αPTHF composition according to item

[39] , wherein the aqueous pharmaceutically acceptable carrier is trehalose;

[42] The Lp-αPTHF composition according to item

[41] , wherein the pharmaceutically acceptable carrier contains 5 wt% to 20 wt% of trehalose;

[43] The Lp-αPTHF composition according to any one of items

[39] to

[42] , wherein the pharmaceutically acceptable carrier contains 1 wt% to 15 wt% of dextrose;

[44] The Lp-αPTHF composition according to any one of items

[39] to

[43] , wherein the internal space of the liposome contains 5% dextrose suspended in HEPES buffer;

[45] The Lp-αPTHF composition according to any one of items

[39] to

[44] , wherein the pharmaceutically acceptable carrier contains a buffer solution 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] An Lp-αPTHF composition according to any one of items

[39] to

[45] , wherein the pharmaceutically acceptable carrier comprises sodium acetate and calcium acetate at a total concentration of 50 mM to 500 mM;

[47] An Lp-αPTHF composition according to any one of items

[12] to

[46] , wherein the internal space of the liposome has a pH of 5 to 8 or 6 to 7, or any range in between;

[48] An Lp-αPTHF composition according to any one of items

[12] to

[47] , wherein the liposome contains less than 500,000 molecules or less than 200,000 molecules of polyglutamylated alpha-tetrahydrofolic acid;

[49] An Lp-αPTHF composition according to any one of items

[12] to

[48] , wherein the liposome contains 10 to 100,000 molecules or any range in between of polyglutamylated alpha-tetrahydrofolic acid molecules;

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

[12] to

[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-αPTHF composition according to item

[50] , wherein the targeting moiety is bound to one or both of the PEG and the outer side of the liposome, and optionally, the targeting moiety is covalently bound to one or both of the PEG and the outer side of the liposome;

[52] An Lp-αPTHF composition according to item

[50] or

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

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

[50] to

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

[54] The targeting moiety has an equilibrium dissociation constant (Kd) in the range of 0.5×10 -10 ~10×10 -6 and binds to the surface antigen as measured using BIACORE® analysis. An Lp-αPTHF composition according to any one of items

[50] to

[53] ;

[55] The Lp-αPTHF composition according to any one of items

[50] to

[55] , wherein the targeting moiety specifically binds to 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);

[56] The Lp-αPTHF composition according to any one of items

[50] to

[56] , wherein the targeting moiety comprises one or more selected from the group consisting 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;

[57] The Lp-αPTHF composition according to any one of items

[50] to

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

[58] The Lp-αPTHF composition according to any one of items

[39] to

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

[59] The Lp-αPTHF composition according to item

[58] , wherein the immunostimulant is at least one selected from the group consisting of a protein immunostimulant, a nucleic acid immunostimulant, a chemical immunostimulant, a hapten, and an adjuvant;

[60] The immunostimulant is at least one selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan; resorcin (e.g., D n-6DPA or D n-3DPA , etc. of resorcin D, resorcin E, or T-series resorcin), and toll-like receptor (TLR) modulators such as oxidized low-density lipoprotein (e.g., OXPAC, PGPC) and erythran lipids (e.g., E5564), the Lp-αPTHF composition according to item

[58] or

[59] ;

[61] The Lp-αPTHF composition according to any one of items

[58] to

[60] , wherein the immunostimulant and the detectable marker are the same;

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

[58] to

[61] , further comprising a hapten;

[63] The Lp-αPTHF composition according to item

[62] , wherein the hapten comprises one or more of fluorescein or beta-1,6-glucan;

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

[12] to

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

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

[64] ;

[66] An untargeted composition comprising the composition according to any one of items [1] to

[49] ;

[67] An Lp-αPTHF composition according to any one of items

[12] to

[66] , further comprising carboplatin and / or pembrolizumab;

[68] A pharmaceutical composition comprising the liposomal polyglutamylated alpha-tetrahydrofolic acid composition according to any one of items

[12] to

[67] ;

[69] A pharmaceutical composition comprising polyglutamylated alpha-tetrahydrofolic acid according to any one of items [1] to [7];

[70] The composition according to any one of items [1] to

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

[71] For the manufacture of a drug for use in combination therapy with one or more therapeutic agents (such as a chemotherapeutic agent (e.g., 5-fluorouracil)) for the treatment of a disease and / or for enhancing the effect of a therapeutic agent (s), or for use as a "chemoprotective agent" for reducing the toxic side effects associated with a therapeutic agent (s) (e.g., in combination with a folic acid antagonist such as methotrexate), the use of the composition according to any one of items [1] to

[70] ;

[72] A method of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject a composition according to any one of items [1] to

[70] ;

[73] A method for treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject the liposomal polyglutamate oxidized alpha-tetrahydrofolate composition according to any one of items

[12] to

[69] ;

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

[69] ;

[75] A method for killing hyperproliferative cells, the method comprising contacting the hyperproliferative cells with the liposomal polyglutamate oxidized alpha-tetrahydrofolate composition according to any one of items

[12] to

[69] ;

[76] The method according to item

[74] or

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

[77] A method for treating cancer, the method comprising administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having cancer;

[78] A method for treating cancer, the method comprising administering an effective amount of the liposomal polyglutamate oxidized alpha-tetrahydrofolate composition according to any one of items

[12] to

[68] to a subject having or at risk of having cancer;

[79] A method for treating or preventing cancer, wherein the cancer is a non-hematological tumor including, 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; and, for example, a hematological tumor such as leukemia, lymphoma and other B cell malignancies, multiple myeloma and other plasma cell dyscrasias, the method according to item

[77] or

[78] ;

[80] A method for treating or preventing cancer, wherein the cancer is a member selected from the group consisting of lung cancer, breast cancer, colon cancer, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, ovarian cancer, and cervical cancer, the method according to item

[77] or

[78] ;

[81] A method according to item

[77] or

[78] for treating or preventing cancer, wherein the cancer is a member selected from the group consisting of colorectal cancer, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer;

[82] A method according to item

[77] or

[78] for treating or preventing cancer, wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma;

[83] A method for treating cancer, comprising administering an effective amount of the Lp-αPTHF composition according to any one of items

[50] to

[66] to a subject having or at risk of having cancer cells expressing folate receptor to which the targeting moiety binds;

[84] A maintenance therapy for a subject who has received or has received cancer treatment, comprising administering an effective amount of the composition according to any one of items [1] to

[69] to a subject who has received or has received cancer treatment;

[85] A maintenance therapy for a subject who has received or has received cancer treatment, comprising administering an effective amount of the liposomal polyglutamine oxidized alpha tetrahydrofolate composition according to any one of items

[12] to

[69] to a subject who has received or has received cancer treatment;

[86] A method for treating an immune system disorder, comprising administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having an immune system disorder, and optionally, the immune system disorder is selected from 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 an immune system disorder, comprising administering an effective amount of the liposomal polyglutamine oxidized tetrahydrofolate composition according to any one of items [8] to

[69] to a subject having or at risk of having an immune system disorder, and optionally, the immune system disorder is selected from 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] The following treatment methods: (a) A method for treating leukopenia, comprising administering an effective amount of the composition according to any one of items [1] to

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

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

[69] 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 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 an effective amount of the composition according to any one of items [1] to

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

[69] to a subject having or at risk of having rheumatoid arthritis; (f) A method for treating an inflammatory condition, comprising administering an effective amount of the composition according to any one of items [1] to

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

[69] to a subject having or at risk of having a skin disease;

[89] A method for treating an infectious disease, comprising administering an effective amount of the liposomal polyglutamine oxidized alpha-tetrahydrofolic acid composition according to any one of items

[12] to

[69] to a subject having or at risk of having an infectious disease;

[90] A method for delivering polyglutamylated alpha-tetrahydrofolic acid to tumors expressing folate receptors on their surfaces, comprising administering to a subject having a tumor an Lp-αPTHF composition according to any one of items [1] to

[69] in an amount such that a therapeutically effective amount of polyglutamylated alpha-tetrahydrofolic acid is delivered to the tumor;

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

[12] to

[69] , comprising forming a mixture comprising a liposomal component and a polyglutamylated alpha-folate antagonist in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes comprising polyglutamylated alpha-tetrahydrofolic acid;

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

[12] to

[69] , comprising forming a mixture comprising a liposomal component and polyglutamylated alpha-tetrahydrofolic acid in solution; homogenizing the mixture in solution to form liposomes; treating the mixture to form liposomes in which polyglutamylated alpha-tetrahydrofolic acid is encapsulated and / or enveloped; and imparting a targeting moiety to the surface of the liposomes, wherein the targeting moiety has specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ);

[93] The method according to item

[92] , wherein the treating step comprises one or more steps selected from thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw method, reverse phase evaporation method, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, freeze-drying double emulsion, 3D printing, membrane contactor method, and agitation; and / or

[94] The method according to item

[92] , wherein the treating step comprises one or more steps of changing the size of the liposomes by one or more steps selected from extrusion, high pressure microfluidization, and / or sonication.

[0006] In some embodiments, the present disclosure provides a polyglutamylated alpha - tetrahydrofolic acid (αPTHF) composition in which at least two of the glutamyl residues of polyglutamylated alpha - tetrahydrofolic acid have an alpha - carboxyl group bond. In some embodiments, αPTHF contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 6 or more glutamyl groups (including the glutamyl groups in tetrahydrofolic acid). In some embodiments, αPTHF contains 2 or more L - type glutamyl groups. In other embodiments, αPTHF contains a D - type glutamyl group. In further embodiments, αPTHF contains a D - type glutamyl group and 2 or more L - type glutamyl groups. In further embodiments, αPTHF contains 2 or more glutamyl groups having an alpha - bond. In some embodiments, at least one glutamyl group has both an alpha - bond and a gamma - bond.

[0007] In one embodiment, the αPTHF composition contains a chain of 3 glutamyl groups bonded to the glutamyl group of tetrahydrofolic acid (i.e., tetraglutamylated tetrahydrofolic acid). In some embodiments, tetraglutamylated THF contains 2 or more L - type glutamyl groups. In other embodiments, tetraglutamylated THF contains a D - type glutamyl group. In further embodiments, tetraglutamylated THF contains a D - type glutamyl group and 2 or more L - type glutamyl groups. In further embodiments, tetraglutamylated THF contains 2 or more glutamyl groups having a gamma - bond.

[0008] In one embodiment, the αPTHF composition comprises a chain of four glutamyl groups (e.g., α-pentaglutamyl oxidized tetrahydrofolate) bound to the gamma-glutamyl group of tetrahydrofolate. In some embodiments, the pentaglutamyl oxidized alpha THF comprises two or more L-type glutamyl groups. In other embodiments, the pentaglutamyl oxidized alpha THF comprises a D-type glutamyl group. In further embodiments, the pentaglutamyl oxidized alpha THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the pentaglutamyl oxidized THF comprises two or more glutamyl groups having a gamma bond.

[0009] In one embodiment, the αPTHF composition comprises a chain of five glutamyl groups (e.g., α-hexaglutamyl oxidized tetrahydrofolate) bound to the gamma-glutamyl group of tetrahydrofolate. In some embodiments, the hexaglutamyl oxidized alpha THF comprises two or more L-type glutamyl groups. In other embodiments, the hexaglutamyl oxidized alpha THF comprises a D-type glutamyl group. In further embodiments, the hexaglutamyl oxidized alpha THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the hexaglutamyl oxidized THF comprises two or more glutamyl groups having a gamma bond.

[0010] In a further embodiment, the present disclosure provides compositions comprising a delivery medium such as a liposome filled (e.g., encapsulated) with polyglutamylated alpha-tetrahydrofolate and / or otherwise associated therewith, and methods of making and using an alphaPTHF-filled / associated delivery vehicle composition (DV-αPTHF) for delivering polyglutamylated alpha-tetrahydrofolate to diseased (e.g., cancerous) and / or target cells. These compositions have use, including, but not limited to, treating (e.g., treating or preventing) diseases such as 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 polyglutamylated alpha-tetrahydrofolate in the DV-αPTHF comprises 2 to 20, 2 to 15, 2 to 10, 2 to 5, 6 or more, or 21 or more glutamyl groups (including the glutamyl groups in tetrahydrofolate). The DV-αPTHF-filled / associated delivery vehicle composition improves the efficacy and safety of delivering tetrahydrofolate to cancer cells by preferentially delivering a payload (e.g., polyglutamylated tetrahydrofolate) that is more cytotoxic than the cytotoxicity of tetrahydrofolate (THF) administered in the monoglutamic acid state.

[0011] In some embodiments, the present disclosure provides for the use of a delivery medium, such as a liposome, filled (e.g., encapsulated) with polyglutamylated alpha-tetrahydrofolate and / or otherwise associated therewith, as a "chemoprotective agent" that enhances the effect of one or more therapeutic agents (e.g., chemotherapy drugs such as 5-fluorouracil) in combination therapy with one or more therapeutic agents or reduces the toxic side effects associated with the therapeutic agent(s) (e.g., in combination with a folate antagonist such as methotrexate). In some embodiments, the polyglutamylated alpha-tetrahydrofolate in DV-αPTHF contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, 6 or more, or 21 or more glutamyl groups (including the glutamyl groups in tetrahydrofolate). The DV-αPTHF-filled / associated delivery medium composition improves the effectiveness and safety of delivering tetrahydrofolate to cancer cells by preferentially delivering a payload (e.g., polyglutamylated tetrahydrofolate) that is more cytotoxic than the cytotoxicity of tetrahydrofolate (THF) administered in the monoglutamate state.

[0012] In further embodiments, the present disclosure provides a composition comprising polyglutamylated alpha-tetrahydrofolate (αPTHF).

[0013] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha 5-formyl-THF. In some embodiments, the polyglutamylated alpha 5-formyl-THF is polyglutamylated from polyglutamylated alpha [6S]-5-formyl-THF. In some embodiments, the composition comprises polyglutamylated alpha [6R,S]-5-formyl-THF. In some embodiments, the composition comprises polyglutamylated alpha [6R]-5-formyl-THF. In some embodiments, the composition comprises polyglutamylated alpha 5-formyl-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5-formyl-THF). In some embodiments, the polyglutamylated alpha 5-formyl-THF comprises 1, 2, 3, or 4 or more glutamyl groups having an alpha bond. In some embodiments, the composition comprises polyglutamylated alpha 5-formyl-THF having 2 or more L-type glutamyl groups. In other embodiments, the composition comprises polyglutamylated alpha 5-formyl-THF having a D-type glutamyl group. In further embodiments, the composition comprises polyglutamylated alpha 5-formyl-THF having a D-type glutamyl group and 2 or more L-type glutamyl groups. In further embodiments, the polyglutamylated alpha tetrahydrofolate in Lp-αPTHF comprises 2 or more glutamyl groups having an alpha bond. In further embodiments, the polyglutamylated alpha tetrahydrofolate in Lp-αPTHF comprises 1 or more glutamyl groups having both an alpha bond and a gamma bond. In some embodiments, the polyglutamylated alpha tetrahydrofolate in Lp-αPTHF comprises 2 to 10 or any number in between of glutamyl groups having both an alpha bond and a gamma bond. In some embodiments, the polyglutamic acid chain of polyglutamylated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of polyglutamylated alpha 5-formyl-THF is branched.

[0014] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha 5-formyl-THF (i.e., tetraglutamylated 5-formyl-THF) comprising a chain of three glutamyl groups linked to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises tetraglutamylated alpha [6S]-5-formyl-THF. In some embodiments, the composition comprises tetraglutamylated alpha [6R,S]-5-formyl-THF. In some embodiments, the composition comprises tetraglutamylated alpha [6R]-5-formyl-THF. In some embodiments, tetraglutamylated alpha 5-formyl-THF comprises one, two, or three glutamyl groups having an alpha bond. In some embodiments, tetraglutamylated alpha 5-formyl-THF comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamylated alpha 5-formyl-THF comprises a D-type glutamyl group. In further embodiments, tetraglutamylated alpha 5-formyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5-formyl-THF is branched.

[0015] In some embodiments, the present disclosure provides a composition comprising pentaglutamylated alpha 5-formyl-THF (i.e., pentaglutamylated 5-formyl-THF) comprising a chain of four glutamyl groups linked to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises pentaglutamylated alpha [6S]-5-formyl-THF. In some embodiments, the composition comprises pentaglutamylated alpha [6R,S]-5-formyl-THF. In some embodiments, the composition comprises pentaglutamylated alpha [6R]-5-formyl-THF. In some embodiments, pentaglutamylated alpha 5-formyl-THF comprises one, two, three, or four glutamyl groups having an alpha linkage. In some embodiments, pentaglutamylated 5-formyl-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamylated 5-formyl-THF comprises a D-type glutamyl group. In further embodiments, pentaglutamylated 5-formyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5-formyl-THF is branched.

[0016] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha 5-formyl-THF (i.e., hexaglutamylated 5-formyl-THF) comprising a chain of five glutamyl groups linked to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises hexaglutamylated alpha [6S]-5-formyl-THF. In some embodiments, the composition comprises hexaglutamylated alpha [6R,S]-5-formyl-THF. In some embodiments, the composition comprises hexaglutamylated alpha [6R]-5-formyl-THF. In some embodiments, hexaglutamylated alpha 5-formyl-THF comprises one, two, three, four, or five glutamyl groups having an alpha linkage. In some embodiments, hexaglutamylated alpha 5-formyl-THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamylated THF comprises a D-type glutamyl group. In further embodiments, hexaglutamylated alpha 5-formyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5-formyl-THF is branched.

[0017] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha 5,10-methenyl-THF. In some embodiments, the composition comprises polyglutamylated alpha [6R]-5,10-methenyl-THF. In some embodiments, the composition comprises polyglutamylated alpha [6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprises polyglutamylated alpha [6S]-5,10-methenyl-THF. In some embodiments, the composition comprises polyglutamylated alpha 5,10-methenyl-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5,10-methenyl-THF). In some embodiments, the polyglutamylated alpha 5,10-methenyl-THF comprises 1, 2, 3, or 4 or more glutamyl groups having an alpha bond. In some embodiments, the composition comprises polyglutamylated alpha 5-formyl-THF having 2 or more L-type glutamyl groups. In other embodiments, the composition comprises polyglutamylated alpha 5,10-methenyl-THF having a D-type glutamyl group. In further embodiments, the composition comprises polyglutamylated alpha 5,10-methenyl-THF having a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of the polyglutamylated alpha 5,10-methenyl-THF is linear. In some embodiments, the polyglutamic acid chain of the polyglutamylated alpha 5,10-methenyl-THF is branched.

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

[0019] In some embodiments, the present disclosure provides a composition comprising pentaglutamylated alpha 5,10-methenyl-THF (i.e., pentaglutamylated 5,10-methenyl-THF) comprising a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises pentaglutamylated alpha [6R]-5,10-methenyl-THF. In some embodiments, the composition comprises pentaglutamylated alpha [6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprises pentaglutamylated alpha [6S]-5,10-methenyl-THF. In some embodiments, pentaglutamylated alpha 5,10-methenyl-THF comprises one, two, three, or four glutamyl groups having an alpha bond. In some embodiments, pentaglutamylated alpha 5,10-methenyl-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamylated alpha 5,10-methenyl-THF comprises a D-type glutamyl group. In further embodiments, pentaglutamylated alpha 5,10-methenyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5,10-methenyl-THF is linear. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5,10-methenyl-THF is branched.

[0020] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha 5,10-methenyl-THF (i.e., hexaglutamylated 5,10-methenyl-THF) comprising a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises hexaglutamylated alpha [6R]-5,10-methenyl-THF. In some embodiments, the composition comprises hexaglutamylated alpha [6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprises hexaglutamylated alpha [6S]-5,10-methenyl-THF. In some embodiments, hexaglutamylated alpha 5,10-methenyl-THF comprises one, two, three, four, or five glutamyl groups having an alpha bond. In some embodiments, hexaglutamylated alpha 5,10-methenyl-THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamylated alpha 5,10-methenyl-THF comprises a D-type glutamyl group. In further embodiments, hexaglutamylated alpha 5,10-methenyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5,10-methenyl-THF is linear. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5,10-methenyl-THF is branched.

[0021] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha 5-methyl-THF. In some embodiments, the composition comprises polyglutamylated alpha [6S]-5-methyl-THF. In some embodiments, the composition comprises polyglutamylated alpha [6R,S]-5-methyl-THF. In some embodiments, the composition comprises polyglutamylated alpha [6R]-5-methyl-THF. In some embodiments, the composition comprises polyglutamylated alpha 5-methyl-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5-methyl-THF). In some embodiments, polyglutamylated alpha 5-methyl-THF comprises 1, 2, 3, or 4 or more glutamyl groups having an alpha bond. In some embodiments, the composition comprises polyglutamylated alpha 5-methyl-THF having 2 or more L-type glutamyl groups. In other embodiments, the composition comprises polyglutamylated alpha 5-methyl-THF having a D-type glutamyl group. In further embodiments, the composition comprises polyglutamylated alpha 5-methyl-THF having a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of polyglutamylated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamic acid chain of polyglutamylated alpha 5-methyl-THF is branched.

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

[0023] In some embodiments, the present disclosure provides a composition comprising pentaglutamylated alpha 5-methyl-THF (i.e., pentaglutamylated 5-methyl-THF) comprising a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises pentaglutamylated alpha [6S]-5-methyl-THF. In some embodiments, the composition comprises pentaglutamylated alpha [6R,S]-5-methyl-THF. In some embodiments, the composition comprises pentaglutamylated alpha [6R]-5-methyl-THF. In some embodiments, pentaglutamylated alpha 5-methyl-THF comprises one, two, three, or four glutamyl groups having a gamma bond. In some embodiments, pentaglutamylated alpha 5-methyl-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamylated alpha 5-methyl-THF comprises a D-type glutamyl group. In further embodiments, pentaglutamylated alpha 5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5-methyl-THF is branched.

[0024] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha 5-methyl-THF (i.e., hexaglutamylated 5-methyl-THF) comprising a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises hexaglutamylated alpha [6S]-5-methyl-THF. In some embodiments, the composition comprises hexaglutamylated alpha [6R,S]-5-methyl-THF. In some embodiments, the composition comprises hexaglutamylated alpha [6R]-5-methyl-THF. In some embodiments, hexaglutamylated alpha 5-methyl-THF comprises one, two, three, four, or five glutamyl groups having an alpha bond. In some embodiments, hexaglutamylated alpha 5-methyl-THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamylated alpha 5-methyl-THF comprises a D-type glutamyl group. In further embodiments, hexaglutamylated alpha 5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5-methyl-THF is branched.

[0025] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha-tetrahydrofolate (THF). In some embodiments, the composition comprises polyglutamylated alpha-[6S]-tetrahydrofolate (THF). In some embodiments, the composition comprises polyglutamylated alpha-[6R,S]-tetrahydrofolate (THF). In some embodiments, the composition comprises polyglutamylated alpha-[6R]-tetrahydrofolate (THF). In some embodiments, the composition comprises polyglutamylated alpha-tetrahydrofolate (THF) having from 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in tetrahydrofolate (THF)). In some embodiments, alpha-polyglutamylated tetrahydrofolate-THF comprises 1, 2, 3, or 4 or more glutamyl groups having an alpha-linkage. In some embodiments, the composition comprises polyglutamylated alpha-tetrahydrofolate (THF) having 2 or more L-type glutamyl groups. In other embodiments, the composition comprises polyglutamylated alpha-tetrahydrofolate (THF) having a D-type glutamyl group. In further embodiments, the composition comprises polyglutamylated alpha-tetrahydrofolate (THF) having a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of polyglutamylated alpha-tetrahydrofolate (THF) is linear. In some embodiments, the polyglutamate chain of polyglutamylated alpha-tetrahydrofolate (THF) is branched.

[0026] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha tetrahydrofolate (THF) (i.e., tetraglutamylated tetrahydrofolate (THF)) comprising a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolate (THF). In some embodiments, the composition comprises tetraglutamylated alpha [6S] tetrahydrofolate (THF). In some embodiments, the composition comprises tetraglutamylated alpha [6R,S]-tetrahydrofolate (THF). In some embodiments, the composition comprises tetraglutamylated alpha [6R]-tetrahydrofolate (THF). In some embodiments, tetraglutamylated alpha tetrahydrofolate-THF comprises one, two, or three glutamyl groups having an alpha bond. In some embodiments, tetraglutamylated alpha tetrahydrofolate (THF) comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamylated alpha tetrahydrofolate (THF) comprises a D-type glutamyl group. In further embodiments, tetraglutamylated alpha tetrahydrofolate (THF) comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha tetrahydrofolate (THF) is linear. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha tetrahydrofolate (THF) is branched.

[0027] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha tetrahydrofolate THF (i.e., pentaglutamylated tetrahydrofolate THF) comprising a chain of four glutamyl groups attached to the glutamyl group of THF. In some embodiments, the composition comprises pentaglutamylated alpha [6S] tetrahydrofolate THF. In some embodiments, the composition comprises pentaglutamylated alpha [6R,S]-tetrahydrofolate THF. In some embodiments, the composition comprises pentaglutamylated alpha [6R]-tetrahydrofolate THF. In some embodiments, pentaglutamylated alpha tetrahydrofolate-THF comprises one, two, three, or four glutamyl groups having an alpha linkage. In some embodiments, pentaglutamylated alpha THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamylated alpha tetrahydrofolate THF comprises a D-type glutamyl group. In further embodiments, pentaglutamylated alpha tetrahydrofolate THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha tetrahydrofolate THF is linear. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha THF is branched.

[0028] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha tetrahydrofolate (THF) (i.e., hexaglutamylated tetrahydrofolate (THF)) comprising a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolate (THF). In some embodiments, the composition comprises hexaglutamylated alpha [6S] tetrahydrofolate (THF). In some embodiments, the composition comprises hexaglutamylated alpha [6R,S] - tetrahydrofolate (THF). In some embodiments, the composition comprises hexaglutamylated alpha [6R] tetrahydrofolate (THF). In some embodiments, hexaglutamylated alpha tetrahydrofolate - THF comprises one, two, three, four, or five glutamyl groups having an alpha bond. In some embodiments, hexaglutamylated alpha tetrahydrofolate tetrahydrofolate (THF) comprises two or more L - type glutamyl groups. In other embodiments, hexaglutamylated tetrahydrofolate (THF) comprises a D - type glutamyl group. In further embodiments, hexaglutamylated alpha tetrahydrofolate (THF) comprises a D - type glutamyl group and two or more L - type glutamyl groups. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha tetrahydrofolate (THF) is linear. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha tetrahydrofolate (THF) is branched.

[0029] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha 5,10-methylene-THF. In some embodiments, the composition comprises polyglutamylated alpha [6R]-5,10-methylene-THF. In some embodiments, the composition comprises polyglutamylated alpha [6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises polyglutamylated alpha [6S]-5,10-methylene-THF. In some embodiments, the composition comprises polyglutamylated alpha 5,10-methylene-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5,10-methylene-THF). In some embodiments, polyglutamylated alpha 5,10-methylene-THF comprises 1, 2, 3, or 4 or more glutamyl groups having an alpha bond. In some embodiments, the composition comprises polyglutamylated alpha 5,10-methylene-THF having 2 or more L-type glutamyl groups. In other embodiments, the composition comprises polyglutamylated alpha 5,10-methylene-THF having a D-type glutamyl group. In further embodiments, the composition comprises polyglutamylated alpha 5,10-methylene-THF having a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of polyglutamylated alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of polyglutamylated alpha 5,10-methylene-THF is branched.

[0030] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha 5,10-methylene-THF (i.e., tetraglutamylated 5,10-methylene-THF) comprising a chain of three glutamyl groups linked to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises tetraglutamylated alpha [6R]-5,10-methylene-THF. In some embodiments, the composition comprises tetraglutamylated alpha [6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises tetraglutamylated alpha [6S]-5,10-methylene-THF. In some embodiments, tetraglutamylated alpha 5,10-methylene-THF comprises one, two, or three glutamyl groups having an alpha linkage. In some embodiments, tetraglutamylated alpha 5,10-methylene-THF comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamylated alpha 5,10-methylene-THF comprises a D-type glutamyl group. In further embodiments, tetraglutamylated alpha 5,10-methylene-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5,10-methylene-THF is branched.

[0031] In some embodiments, the present disclosure provides a composition comprising pentaglutamylated alpha 5,10-methylene-THF (i.e., pentaglutamylated 10-methylene-THF) comprising a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises pentaglutamylated alpha [6R] 5,10-methylene-THF. In some embodiments, the composition comprises pentaglutamylated alpha [6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises pentaglutamylated alpha [6S]-5,10-methylene-THF. In some embodiments, pentaglutamylated alpha 5,10-methylene-THF comprises one, two, three, or four glutamyl groups having an alpha bond. In some embodiments, pentaglutamylated alpha 5,10-methylene-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamylated alpha 5,10-methylene-THF comprises a D-type glutamyl group. In further embodiments, pentaglutamylated alpha 5,10-methylene-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5,10-methylene-THF is branched.

[0032] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha 5,10-methylene-THF (i.e., hexaglutamylated 5,10-methylene-THF) comprising a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises hexaglutamylated alpha [6R]5,10-methylene-THF. In some embodiments, the composition comprises hexaglutamylated alpha [6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises hexaglutamylated alpha [6S]-5,10-methylene-THF. In some embodiments, alpha hexaglutamylated 5,10-methylene-THF comprises one, two, three, four, or five glutamyl groups having an alpha bond. In some embodiments, hexaglutamylated alpha 5,10-methylene-THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamylated alpha 5,10-methylene-THF comprises a D-type glutamyl group. In further embodiments, hexaglutamylated alpha 5,10-methylene-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5,10-methylene-THF is branched.

[0033] In some embodiments, the present disclosure provides a composition comprising polyglutaminyl oxidized alpha 5-formiminino-THF. In some embodiments, the composition comprises polyglutaminyl oxidized alpha [6S]-5-formiminino-THF. In some embodiments, the composition comprises polyglutaminyl oxidized alpha [6R,S]-5-formiminino-THF. In some embodiments, the composition comprises polyglutaminyl oxidized alpha [6R]-5-formiminino-THF. In some embodiments, the composition comprises polyglutaminyl oxidized alpha 5-formiminino-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl group in 5-formiminino-THF). In some embodiments, polyglutaminyl oxidized alpha 5-formiminino-THF comprises 1, 2, 3, or 4 or more glutamyl groups having an alpha bond. In some embodiments, the composition comprises polyglutaminyl oxidized alpha 5-formiminino-THF having 2 or more L-type glutamyl groups. In other embodiments, the composition comprises polyglutaminyl oxidized alpha 5-formiminino-THF having a D-type glutamyl group. In further embodiments, the composition comprises polyglutaminyl oxidized alpha 5-formiminino-THF having a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of polyglutaminyl oxidized alpha 5-formiminino-THF is linear. In some embodiments, the polyglutamic acid chain of polyglutaminyl oxidized alpha 5-formiminino-THF is branched.

[0034] In one embodiment, the composition comprises polyglutamylated alpha 5-formimino-THF (i.e., tetraglutamylated 5-formimino-THF) comprising a chain of three glutamyl groups linked to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises tetraglutamylated alpha [6S]-5-formimino-THF. In some embodiments, the composition comprises tetraglutamylated alpha [6R,S]-5-formimino-THF. In some embodiments, the composition comprises tetraglutamylated alpha [6R]5-formimino-THF. In some embodiments, tetraglutamylated alpha 5-formimino-THF comprises one, two, or three glutamyl groups having an alpha linkage. In some embodiments, tetraglutamylated alpha 5-formimino-THF comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamylated alpha 5-formimino-THF comprises a D-type glutamyl group. In further embodiments, tetraglutamylated alpha 5-formimino-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5-formimino-THF is branched.

[0035] In one embodiment, the composition comprises polyglutamylated alpha 5-formimino-THF (i.e., pentaglutamylated 5-formimino-THF) comprising a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises pentaglutamylated alpha [6S]-5-formimino-THF. In some embodiments, the composition comprises pentaglutamylated alpha [6R,S]-5-formimino-THF. In some embodiments, the composition comprises pentaglutamylated alpha [6R]5-formimino-THF. In some embodiments, pentaglutamylated alpha 5-formimino-THF comprises one, two, three, or four glutamyl groups having an alpha bond. In some embodiments, pentaglutamylated alpha 5-formimino-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamylated alpha 5-formimino-THF comprises a D-type glutamyl group. In further embodiments, pentaglutamylated alpha 5-formimino-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5-formimino-THF is branched.

[0036] In one embodiment, the composition comprises polyglutamylated alpha 5-formimino-THF (i.e., hexaglutamylated 5-formimino-THF) comprising a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the composition comprises hexaglutamylated alpha [6S]-5-formimino-THF. In some embodiments, the composition comprises hexaglutamylated alpha [6R,S]-5-formimino-THF. In some embodiments, the composition comprises hexaglutamylated alpha [6R]5-formimino-THF. In some embodiments, hexaglutamylated alpha 5-formimino-THF comprises one, two, three, four, or five glutamyl groups having an alpha bond. In some embodiments, hexaglutamylated alpha 5-formimino-THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamylated alpha 5-formimino-THF comprises a D-type glutamyl group. In further embodiments, hexaglutamylated alpha 5-formimino-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5-formimino-THF is branched.

[0037] In further embodiments, the present disclosure provides a composition comprising liposomes (Lp-αPTHF) encapsulated (filled) with polyglutamylated alpha tetrahydrofolic acid.

[0038] In some embodiments, the present disclosure provides a composition comprising liposomes encapsulated (filled) with polyglutamylated alpha 5-formyl-THF. In some embodiments, the liposomes comprise polyglutamylated alpha [6S]-5-formyl-THF. In some embodiments, the liposomes comprise polyglutamylated alpha [6R,S]-5-formyl-THF. In some embodiments, the liposomes comprise polyglutamylated alpha [6R]-5-formyl-THF. In some embodiments, the liposomes comprise polyglutamylated alpha 5-formyl-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5-formyl-THF). In some embodiments, polyglutamylated alpha 5-formyl-THF comprises 1, 2, 3, or 4 or more glutamyl groups having an alpha bond. In some embodiments, the liposomes comprise polyglutamylated alpha 5-formyl-THF having 2 or more L-type glutamyl groups. In other embodiments, the liposomes comprise polyglutamylated alpha 5-formyl-THF having a D-type glutamyl group. In further embodiments, the liposomes comprise polyglutamylated alpha 5-formyl-THF having a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of polyglutamylated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of polyglutamylated alpha 5-formyl-THF is branched.

[0039] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha 5-formyl-THF (i.e., tetraglutamylated 5-formyl-THF) comprising a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises tetraglutamylated alpha [6S]-5-formyl-THF. In some embodiments, the liposome comprises tetraglutamylated alpha [6R,S]-5-formyl-THF. In some embodiments, the liposome comprises tetraglutamylated alpha [6R]-5-formyl-THF. In some embodiments, tetraglutamylated alpha 5-formyl-THF comprises one, two, or three glutamyl groups having gamma linkages. In some embodiments, tetraglutamylated alpha 5-formyl-THF comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamylated alpha 5-formyl-THF comprises a D-type glutamyl group. In further embodiments, tetraglutamylated alpha 5-formyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5-formyl-THF is branched.

[0040] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha 5-formyl-THF (i.e., pentaglutamylated 5-formyl-THF) comprising a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises pentaglutamylated alpha [6S]-5-formyl-THF. In some embodiments, the liposome comprises pentaglutamylated alpha [6R,S]-5-formyl-THF. In some embodiments, the liposome comprises pentaglutamylated alpha [6R]-5-formyl-THF. In some embodiments, pentaglutamylated alpha 5-formyl-THF comprises one, two, three, or four glutamyl groups having an alpha bond. In some embodiments, pentaglutamylated 5-formyl-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamylated 5-formyl-THF comprises a D-type glutamyl group. In further embodiments, pentaglutamylated 5-formyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5-formyl-THF is branched.

[0041] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha 5-formyl-THF (i.e., hexaglutamylated 5-formyl-THF) comprising a chain of five glutamyl groups linked to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises hexaglutamylated alpha [6S]-5-formyl-THF. In some embodiments, the liposome comprises hexaglutamylated alpha [6R,S]-5-formyl-THF. In some embodiments, the liposome comprises hexaglutamylated alpha [6R]-5-formyl-THF. In some embodiments, hexaglutamylated alpha 5-formyl-THF comprises one, two, three, four, or five glutamyl groups having an alpha bond. In some embodiments, hexaglutamylated alpha 5-formyl-THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamylated THF comprises a D-type glutamyl group. In further embodiments, hexaglutamylated alpha 5-formyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5-formyl-THF is branched.

[0042] In some embodiments, the present disclosure provides a composition comprising liposomes encapsulated (filled) with polyglutamylated alpha 5,10-methenyl-THF. In some embodiments, the liposomes comprise polyglutamylated alpha [6R]-5,10-methenyl-THF. In some embodiments, the liposomes comprise polyglutamylated alpha [6R,S]-5,10-methenyl-THF. In some embodiments, the liposomes comprise polyglutamylated alpha [6S]-5,10-methenyl-THF. In some embodiments, the liposomes comprise polyglutamylated alpha 5,10-methenyl-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5,10-methenyl-THF). In some embodiments, the polyglutamylated alpha 5,10-methenyl-THF comprises 1, 2, 3, or 4 or more glutamyl groups having an alpha bond. In some embodiments, the liposomes comprise polyglutamylated alpha 5-formyl-THF having 2 or more L-type glutamyl groups. In other embodiments, the liposomes comprise polyglutamylated alpha 5,10-methenyl-THF having a D-type glutamyl group. In further embodiments, the liposomes comprise polyglutamylated alpha 5,10-methenyl-THF having a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of polyglutamylated alpha 5,10-methenyl-THF is linear. In some embodiments, the polyglutamic acid chain of polyglutamylated alpha 5-formyl-THF is branched.

[0043] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha 5,10-methenyl-THF (i.e., tetraglutamylated 5,10-methenyl-THF) comprising a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises tetraglutamylated alpha [6R]-5,10-methenyl-THF. In some embodiments, the liposome comprises tetraglutamylated alpha [6R,S]-5,10-methenyl-THF. In some embodiments, the liposome comprises tetraglutamylated alpha [6S]-5,10-methenyl-THF. In some embodiments, tetraglutamylated alpha 5,10-methenyl-THF comprises one, two, or three glutamyl groups having an alpha bond. In some embodiments, tetraglutamylated alpha 5,10-methenyl-THF comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamylated alpha 5,10-methenyl-THF comprises a D-type glutamyl group. In further embodiments, tetraglutamylated alpha 5,10-methenyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of 5,10-methenyl-THF tetrahydrofolic acid is linear. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5,10-methenyl-THF is branched.

[0044] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha 5,10-methenyl-THF (i.e., pentaglutamylated 5,10-methenyl-THF) comprising a chain of four glutamyl groups linked to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises pentaglutamylated alpha [6R]-5,10-methenyl-THF. In some embodiments, the liposome comprises pentaglutamylated alpha [6R,S]-5,10-methenyl-THF. In some embodiments, the liposome comprises pentaglutamylated alpha [6S]-5,10-methenyl-THF. In some embodiments, pentaglutamylated alpha 5,10-methenyl-THF comprises one, two, three, or four glutamyl groups having an alpha bond. In some embodiments, pentaglutamylated alpha 5,10-methenyl-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamylated alpha 5,10-methenyl-THF comprises a D-type glutamyl group. In further embodiments, pentaglutamylated alpha 5,10-methenyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5,10-methenyl-THF is linear. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5,10-methenyl-THF is branched.

[0045] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha 5,10-methenyl-THF (i.e., hexaglutamylated 5,10-methenyl-THF) comprising a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises hexaglutamylated alpha [6R]-5,10-methenyl-THF. In some embodiments, the liposome comprises hexaglutamylated alpha [6R,S]-5,10-methenyl-THF. In some embodiments, the liposome comprises hexaglutamylated alpha [6S]-5,10-methenyl-THF. In some embodiments, hexaglutamylated alpha 5,10-methenyl-THF comprises one, two, three, four, or five glutamyl groups having an alpha bond. In some embodiments, hexaglutamylated alpha 5,10-methenyl-THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamylated alpha 5,10-methenyl-THF comprises a D-type glutamyl group. In further embodiments, hexaglutamylated alpha 5,10-methenyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5,10-methenyl-THF is linear. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5,10-methenyl-THF is branched.

[0046] In some embodiments, the present disclosure provides a composition comprising liposomes encapsulated (filled) with polyglutamylated alpha 5-methyl-THF. In some embodiments, the liposomes comprise polyglutamylated alpha [6S]-5-methyl-THF. In some embodiments, the liposomes comprise polyglutamylated alpha [6R,S]-5-methyl-THF. In some embodiments, the liposomes comprise polyglutamylated alpha [6R]-5-methyl-THF. In some embodiments, the liposomes comprise polyglutamylated alpha 5-methyl-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5-methyl-THF). In some embodiments, the polyglutamylated alpha 5-methyl-THF comprises 1, 2, 3, or 4 or more glutamyl groups having an alpha bond. In some embodiments, the liposomes comprise polyglutamylated alpha 5-methyl-THF having 2 or more L-type glutamyl groups. In other embodiments, the liposomes comprise polyglutamylated alpha 5-methyl-THF having a D-type glutamyl group. In further embodiments, the liposomes comprise polyglutamylated alpha 5-methyl-THF having a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of polyglutamylated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamic acid chain of polyglutamylated alpha 5-methyl-THF is branched.

[0047] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha 5-methyl-THF (i.e., tetraglutamylated 5-methyl-THF) comprising a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises tetraglutamylated alpha [6S]-5-methyl-THF. In some embodiments, the liposome comprises tetraglutamylated alpha [6R,S]-5-methyl-THF. In some embodiments, the liposome comprises tetraglutamylated alpha [6R]-5-methyl-THF. In some embodiments, tetraglutamylated alpha 5-methyl-THF comprises one, two, or three glutamyl groups having an alpha bond. In some embodiments, tetraglutamylated alpha 5-methyl-THF comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamylated alpha 5-methyl-THF comprises a D-type glutamyl group. In further embodiments, tetraglutamylated alpha 5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5-methyl-THF is branched.

[0048] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha 5-methyl-THF (i.e., pentaglutamylated 5-methyl-THF) comprising a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises pentaglutamylated alpha [6S]-5-methyl-THF. In some embodiments, the liposome comprises pentaglutamylated alpha [6R,S]-5-methyl-THF. In some embodiments, the liposome comprises pentaglutamylated alpha [6R]-5-methyl-THF. In some embodiments, pentaglutamylated alpha 5-methyl-THF comprises one, two, three, or four glutamyl groups having an alpha bond. In some embodiments, pentaglutamylated alpha 5-methyl-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamylated alpha 5-methyl-THF comprises a D-type glutamyl group. In further embodiments, pentaglutamylated alpha 5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5-methyl-THF is branched.

[0049] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha 5-methyl-THF (i.e., hexaglutamylated 5-methyl-THF) comprising a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises hexaglutamylated alpha [6S]-5-methyl-THF. In some embodiments, the liposome comprises hexaglutamylated alpha [6R,S]-5-methyl-THF. In some embodiments, the liposome comprises hexaglutamylated alpha [6R]-5-methyl-THF. In some embodiments, hexaglutamylated alpha 5-methyl-THF comprises one, two, three, four, or five glutamyl groups having an alpha bond. In some embodiments, hexaglutamylated alpha 5-methyl-THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamylated alpha 5-methyl-THF comprises a D-type glutamyl group. In further embodiments, hexaglutamylated alpha 5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5-methyl-THF is branched.

[0050] In some embodiments, the present disclosure provides a composition comprising liposomes encapsulated (filled) with polyglutamylated alpha-tetrahydrofolic acid (THF). In some embodiments, the liposomes comprise polyglutamylated alpha-[6S]-tetrahydrofolic acid (THF). In some embodiments, the liposomes comprise polyglutamylated alpha-[6R,S]-tetrahydrofolic acid (THF). In some embodiments, the liposomes comprise polyglutamylated alpha-[6R]-tetrahydrofolic acid (THF). In some embodiments, the liposomes comprise polyglutamylated alpha-tetrahydrofolic acid (THF) having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in tetrahydrofolic acid (THF)). In some embodiments, the polyglutamylated alpha-tetrahydrofolic acid (THF) comprises 1, 2, 3, or 4 or more glutamyl groups having an alpha bond. In some embodiments, the liposomes comprise polyglutamylated alpha-tetrahydrofolic acid (THF) having 2 or more L-type glutamyl groups. In other embodiments, the liposomes comprise polyglutamylated alpha-tetrahydrofolic acid (THF) having a D-type glutamyl group. In further embodiments, the liposomes comprise polyglutamylated alpha-tetrahydrofolic acid (THF) having a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of the polyglutamylated alpha-tetrahydrofolic acid (THF) is linear. In some embodiments, the polyglutamic acid chain of the polyglutamylated alpha-tetrahydrofolic acid (THF) is branched.

[0051] In one embodiment, the Lp-αP tetrahydrofolic acid (THF) composition comprises polyglutamylated alpha tetrahydrofolic acid (i.e., tetraglutamylated tetrahydrofolic acid) containing a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid (THF). In some embodiments, the liposome comprises tetraglutamylated alpha [6S] tetrahydrofolic acid. In some embodiments, the liposome comprises tetraglutamylated alpha [6R,S]-tetrahydrofolic acid (THF). In some embodiments, the liposome comprises tetraglutamylated alpha [6R]-tetrahydrofolic acid (THF). In some embodiments, the tetraglutamylated alpha tetrahydrofolic acid (THF) contains one, two, or three glutamyl groups having an alpha bond. In some embodiments, the tetraglutamylated alpha tetrahydrofolic acid (THF) contains two or more L-type glutamyl groups. In other embodiments, the tetraglutamylated alpha tetrahydrofolic acid (THF) contains a D-type glutamyl group. In further embodiments, the tetraglutamylated alpha tetrahydrofolic acid (THF) contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of the tetraglutamylated alpha tetrahydrofolic acid is linear. In some embodiments, the polyglutamic acid chain of the tetraglutamylated alpha tetrahydrofolic acid is branched.

[0052] In one embodiment, the Lp-αP tetrahydrofolic acid (THF) composition comprises polyglutamylated alpha tetrahydrofolic acid (i.e., pentaglutamylated tetrahydrofolic acid) comprising a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid (THF). In some embodiments, the liposome comprises pentaglutamylated alpha [6S] tetrahydrofolic acid. In some embodiments, the liposome comprises pentaglutamylated alpha [6R,S]-tetrahydrofolic acid (THF). In some embodiments, the liposome comprises pentaglutamylated alpha [6R]-tetrahydrofolic acid (THF). In some embodiments, pentaglutamylated alpha tetrahydrofolic acid (THF) comprises one, two, three, or four glutamyl groups having an alpha bond. In some embodiments, pentaglutamylated alpha tetrahydrofolic acid (THF) comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamylated alpha tetrahydrofolic acid (THF) comprises a D-type glutamyl group. In further embodiments, pentaglutamylated alpha tetrahydrofolic acid (THF) comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha tetrahydrofolic acid (THF) is linear. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha tetrahydrofolic acid (THF) is branched.

[0053] In one embodiment, the Lp-αP tetrahydrofolic acid (THF) composition comprises polyglutamylated alpha tetrahydrofolic acid (i.e., hexaglutamylated tetrahydrofolic acid) comprising a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises hexaglutamylated alpha [6S] tetrahydrofolic acid. In some embodiments, the liposome comprises hexaglutamylated alpha [6R,S]-tetrahydrofolic acid (THF). In some embodiments, the liposome comprises hexaglutamylated alpha [6R]-tetrahydrofolic acid (THF). In some embodiments, the hexaglutamylated alpha tetrahydrofolic acid (THF) comprises one, two, three, four, or five glutamyl groups having an alpha bond. In some embodiments, the hexaglutamylated alpha tetrahydrofolic acid (THF) comprises two or more L-type glutamyl groups. In other embodiments, the hexaglutamylated tetrahydrofolic acid (THF) comprises a D-type glutamyl group. In further embodiments, the hexaglutamylated alpha tetrahydrofolic acid (THF) comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha tetrahydrofolic acid is linear. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha tetrahydrofolic acid is branched.

[0054] In some embodiments, the present disclosure provides a composition comprising liposomes encapsulated (filled) with polyglutamylated alpha 5,10-methylene-THF. In some embodiments, the liposomes comprise polyglutamylated alpha [6R]-5,10-methylene-THF. In some embodiments, the liposomes comprise polyglutamylated alpha [6R,S]-5,10-methylene-THF. In some embodiments, the liposomes comprise polyglutamylated alpha [6S]-5,10-methylene-THF. In some embodiments, the liposomes comprise polyglutamylated alpha 5,10-methylene-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5,10-methylene-THF). In some embodiments, the polyglutamylated alpha 5,10-methylene-THF comprises 1, 2, 3, or 4 or more glutamyl groups having an alpha bond. In some embodiments, the liposomes comprise polyglutamylated alpha 5,10-methylene-THF having 2 or more L-type glutamyl groups. In other embodiments, the liposomes comprise polyglutamylated alpha 5,10-methylene-THF having a D-type glutamyl group. In further embodiments, the liposomes comprise polyglutamylated alpha 5,10-methylene-THF having a D-type glutamyl group and 2 or more glutamyls. In some embodiments, the polyglutamic acid chain of polyglutamylated alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of polyglutamylated alpha 5,10-methylene-THF is branched.

[0055] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha 5,10-methylene-THF (i.e., tetraglutamylated 5,10-methylene-THF) comprising a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises tetraglutamylated alpha [6R]-5,10-methylene-THF. In some embodiments, the liposome comprises tetraglutamylated alpha [6R,S]-5,10-methylene-THF. In some embodiments, the liposome comprises tetraglutamylated alpha [6S]-5,10-methylene-THF. In some embodiments, tetraglutamylated alpha 5,10-methylene-THF comprises one, two, or three glutamyl groups having an alpha bond. In some embodiments, tetraglutamylated alpha 5,10-methylene-THF comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamylated alpha 5,10-methylene-THF comprises a D-type glutamyl group. In further embodiments, tetraglutamylated alpha 5,10-methylene-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5,10-methylene-THF is branched.

[0056] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha 5,10-methylene-THF (i.e., pentaglutamylated 10-methylene-THF) comprising a chain of four glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises pentaglutamylated alpha [6R]5,10-methylene-THF. In some embodiments, the liposome comprises pentaglutamylated alpha [6R,S]-5,10-methylene-THF. In some embodiments, the liposome comprises pentaglutamylated alpha [6S]-5,10-methylene-THF. In some embodiments, pentaglutamylated alpha 5,10-methylene-THF comprises one, two, three, or four glutamyl groups having an alpha bond. In some embodiments, pentaglutamylated alpha 5,10-methylene-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamylated alpha 5,10-methylene-THF comprises a D-type glutamyl group. In further embodiments, pentaglutamylated alpha 5,10-methylene-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5,10-methylene-THF is branched.

[0057] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha 5,10-methylene-THF (i.e., hexaglutamylated 5,10-methylene-THF) comprising a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises hexaglutamylated alpha [6R]5,10-methylene-THF. In some embodiments, the liposome comprises hexaglutamylated alpha [6R]5,10-methylene-THF. In some embodiments, the liposome comprises hexaglutamylated alpha [6R,S]-5,10-methylene-THF. In some embodiments, the liposome comprises hexaglutamylated alpha [6S]-5,10-methylene-THF. In some embodiments, hexaglutamylated alpha 5,10-methylene-THF comprises one, two, three, four, or five glutamyl groups having an alpha bond. In some embodiments, hexaglutamylated alpha 5,10-methylene-THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamylated alpha 5,10-methylene-THF comprises a D-type glutamyl group. In further embodiments, hexaglutamylated alpha 5,10-methylene-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5,10-methylene-THF is branched.

[0058] In some embodiments, the present disclosure provides a composition comprising liposomes encapsulated (filled) with polyglutaminylated alpha 5-formimino-THF. In some embodiments, the liposomes comprise polyglutaminylated alpha [6S]-5-formimino-THF. In some embodiments, the liposomes comprise polyglutaminylated alpha [6R,S]-5-formimino-THF. In some embodiments, the liposomes comprise polyglutaminylated alpha [6R]-5-formimino-THF. In some embodiments, the liposomes comprise polyglutaminylated alpha 5-formimino-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl group in 5-formimino-THF). In some embodiments, polyglutaminylated alpha 5-formimino-THF comprises 1, 2, 3, or 4 or more glutamyl groups having an alpha bond. In some embodiments, the liposomes comprise polyglutaminylated alpha 5-formimino-THF having 2 or more L-type glutamyl groups. In other embodiments, the liposomes comprise polyglutaminylated alpha 5-formimino-THF having a D-type glutamyl group. In further embodiments, the liposomes comprise polyglutaminylated alpha 5-formimino-THF having a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of polyglutaminylated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamic acid chain of polyglutaminylated alpha 5-formimino-THF is branched.

[0059] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha 5-formimino-THF (i.e., tetraglutamylated 5-formimino-THF) comprising a chain of three glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises tetraglutamylated alpha [6S]-5-formimino-THF. In some embodiments, the liposome comprises tetraglutamylated alpha [6R,S]-5-formimino-THF. In some embodiments, the liposome comprises tetraglutamylated alpha [6R]5-formimino-THF. In some embodiments, tetraglutamylated alpha 5-formimino-THF comprises one, two, or three glutamyl groups having an alpha bond. In some embodiments, tetraglutamylated alpha 5-formimino-THF comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamylated alpha 5-formimino-THF comprises a D-type glutamyl group. In further embodiments, tetraglutamylated alpha 5-formimino-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamic acid chain of tetraglutamylated alpha 5-formimino-THF is branched.

[0060] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha 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 pentaglutamylated alpha [6S]-5-formiminotetrahydrofolate. In some embodiments, the liposome comprises pentaglutamylated alpha [6R,S]-5-formiminotetrahydrofolate. In some embodiments, the liposome comprises pentaglutamylated alpha [6R]5-formiminotetrahydrofolate. In some embodiments, pentaglutamylated alpha 5-formiminotetrahydrofolate contains one, two, three, or four glutamyl groups having an alpha bond. In some embodiments, pentaglutamylated alpha 5-formiminotetrahydrofolate contains two or more L-type glutamyl groups. In other embodiments, pentaglutamylated alpha 5-formiminotetrahydrofolate contains a D-type glutamyl group. In further embodiments, pentaglutamylated alpha 5-formiminotetrahydrofolate contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5-formiminotetrahydrofolate is linear. In some embodiments, the polyglutamic acid chain of pentaglutamylated alpha 5-formiminotetrahydrofolate is branched.

[0061] In one embodiment, the Lp-αPTHF composition comprises polyglutamylated alpha-5-formimino-THF (i.e., hexaglutamylated 5-formimino-THF) comprising a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises hexaglutamylated alpha-[6S]-5-formimino-THF. In some embodiments, the liposome comprises hexaglutamylated alpha-[6R,S]-5-formimino-THF. In some embodiments, the liposome comprises hexaglutamylated alpha-[6R]5-formimino-THF. In some embodiments, hexaglutamylated alpha 5-formimino-THF comprises one, two, three, four, or five glutamyl groups having an alpha bond. In some embodiments, hexaglutamylated alpha 5-formimino-THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamylated alpha 5-formimino-THF comprises a D-type glutamyl group. In further embodiments, hexaglutamylated alpha 5-formimino-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamic acid chain of hexaglutamylated alpha 5-formimino-THF is branched.

[0062] In some embodiments, the Lp-αPTHF composition is cationic. In some embodiments, the Lp-αPTHF 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-αPTHF 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-αPTHF 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 polyglutamylated alpha THF. In some embodiments, in the process of preparing Lp-αPTHF, 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 polyglutamylated alpha THF is encapsulated (enclosed) in the cationic Lp-αPTHF. In further embodiments, the polyglutamylated alpha tetrahydrofolic acid encapsulated by the liposomes is present in the HEPES buffer within the liposomes.

[0063] In other embodiments, the Lp-αPTHF composition is anionic or neutral. In some embodiments, the Lp-αPTHF 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-αPTHF 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-αPTHF 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-αPTHF 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-αPTHF 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-αPTHF 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 polyglutamine oxidized alpha THF. In some embodiments, in the process of preparing Lp-αPTHF, 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 the starting material of polyglutamine oxidized alpha THF is encapsulated (enclosed) in the anionic or neutral Lp-αPTHF. In some embodiments, the anionic or neutral Lp-αPTHF 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 tetraglutamine oxidized alpha THF.In some embodiments, the anionic or neutral Lp-αPTHF 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 pentaglutamylated alpha THF. In some embodiments, the anionic or neutral Lp-αPTHF 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 hexaglutamylated alpha THF. In further embodiments, the polyglutamylated alpha tetrahydrofolic acid encapsulated by the liposome is present in the HEPES buffer solution within the liposome.

[0064] In further embodiments, the liposome polyglutamylated alpha tetrahydrofolic acid composition is pegylated (PLp-αPTHF).

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

[0066] In other embodiments, the liposomal polyglutamylated alpha tetrahydrofolate composition is targeted (TLp-αPTHF). That is, the TLp-αPTHF composition includes a targeting moiety having specific affinity for an epitope (surface antigen) on the target cell of interest. In some embodiments, the targeting moiety of TLp-αPTHF or TPLp-αPTHF is covalently bound to the liposome. In other embodiments, the targeting moiety of TLp-αPTHF or TPLp-αPTHF is bound to one or both of the liposome's PEG and the outside. The targeted liposomal polyglutamylated alpha tetrahydrofolate compositions (TLp-αPTHF and TPLp-αPTHF) provide further improvement in the efficacy and safety profile of tetrahydrofolate by specifically delivering polyglutamylated alpha (e.g., alpha-pentaglutamylated and / or alpha-hexaglutamylated) tetrahydrofolate to target cells such as cancer cells. In some embodiments, the targeted liposomal polyglutamylated alpha tetrahydrofolate composition is pegylated (TPLp-αPTHF). In some embodiments, the targeting moiety of TLp-αPTHF or TPLp-αPTHF is bound to one or both of the liposome's PEG and the outside. In some embodiments, the targeting moiety of TLp-αPTHF or TPLp-αPTHF is covalently bound to the liposome. The functions of the targeting moiety of the TLp-αPTHF and / or TPLp-αPTHF composition are not particularly limited, but include 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 specific affinity; and delivering the liposomal payload (αPTHF) to the cell. Suitable targeting moieties are known in the art and are not particularly limited, but include 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-αPTHF or TPLp-αPTHF 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-αPTHF or TPLp-αPTHF 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 on non-tumor cells or is difficult to access. In some embodiments, the targeting moiety binds to the target epitope with an equilibrium dissociation constant (Kd) in the range of 0.5×10 -10 to 10×10 -6 .

[0068] In certain embodiments, the TLp-αPTHF or TPLp-αPTHF targeting moiety comprises a polypeptide that specifically binds to the 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 specifically binds to 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 further embodiments, the Lp-αPTHF composition comprises one or more of an immunostimulant, a detectable marker, and a maleimide disposed on at least one of the PEG and the outer side of the liposome. In some embodiments, the liposomal αPTHF composition (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF) is cationic. In other embodiments, the liposomal αPTHF composition (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF) is anionic or neutral. In further embodiments, the liposomes of the liposomal αPTHF composition (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF) 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-αPTHF 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 αPTHF composition have a diameter in the range of 80 nm to 120 nm or any range therebetween. In some embodiments, the liposomal αPTHF composition is pegylated (e.g., PLp-αPTHF, NTPLp-αPTHF, or TPLp-αPTHF). In some embodiments, the liposomal αPTHF composition comprises a targeting moiety (e.g., TLp-αPTHF or TPLp-αPTHF). In further embodiments, the liposomal αPTHF composition is pegylated and targeted (e.g., TPLp-αPTHF). In some embodiments, the liposomal αPTHF composition comprises polyglutamylated alpha tetrahydrofolate containing 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups. In some embodiments, the liposomal αPTHF composition comprises tetraglutamylated alpha tetrahydrofolate.In some embodiments, the liposomal αPTHF composition comprises pentaglutamyl oxidized alpha-tetrahydrofolic acid. In other embodiments, the liposomal αPTHF composition comprises hexaglutamyl oxidized alpha-tetrahydrofolic acid.

[0070] In some embodiments, the liposomal composition comprises polyglutamyl oxidized alpha-tetrahydrofolic acid having 4, 5, 2 to 10, 4 to 6, or 6 or more 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 polyglutamyl oxidized alpha-THF. In some embodiments, the Lp-αPTHF composition comprises polyglutamyl oxidized alpha-tetrahydrofolic acid having 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups, and 1% to 98.5% w / w of polyglutamyl oxidized alpha-THF. In some embodiments, the liposome comprises polyglutamyl oxidized alpha-tetrahydrofolic acid having 4, 5, 2 to 10, 4 to 6, or 6 or more 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 the starting material of polyglutamyl oxidized alpha-THF is encapsulated (enclosed) in the Lp-αPTHF during the process of preparing the Lp-αPTHF.

[0071] In some embodiments, the liposome composition comprises tetraglutamyl oxidized alpha 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 tetraglutamyl oxidized alpha THF. In some embodiments, the Lp-αPTHF composition comprises tetraglutamyl oxidized alpha tetrahydrofolic acid and 1% - 98.5% w / w of tetraglutamyl oxidized alpha THF. In some embodiments, the liposome comprises tetraglutamyl oxidized alpha tetrahydrofolic acid, and in the process of preparing Lp-αPTHF, 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 the starting material of tetraglutamyl oxidized alpha THF is encapsulated (enclosed) in Lp-αPTHF.

[0072] In some embodiments, the liposome composition comprises pentaglutamyl oxidized alpha 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 pentaglutamyl oxidized alpha THF. In some embodiments, the Lp-αPTHF composition comprises pentaglutamyl oxidized alpha tetrahydrofolic acid and 1% - 98.5% w / w of pentaglutamyl oxidized alpha THF. In some embodiments, the liposome comprises pentaglutamyl oxidized alpha tetrahydrofolic acid, and in the process of preparing Lp-αPTHF, 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 the starting material of pentaglutamyl oxidized alpha THF is encapsulated (enclosed) in Lp-αPTHF.

[0073] In some embodiments, the liposome composition comprises hexaglutaminyl oxidized alpha 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 hexaglutaminyl oxidized alpha THF. In some embodiments, the Lp-αPTHF composition comprises hexaglutaminyl oxidized alpha tetrahydrofolate and 1% - 98.5% w / w of hexaglutaminyl oxidized alpha THF. In some embodiments, the liposome comprises hexaglutaminyl oxidized alpha tetrahydrofolate, and in the process of preparing Lp-αPTHF, 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 the starting material of pentaglutaminyl oxidized alpha THF is encapsulated (enclosed) in Lp-αPTHF.

[0074] Also provided is a liposome composition comprising liposomes encapsulating αPTHF. In some embodiments, the liposome composition comprises a pegylated αPTHF composition. In some embodiments, the liposome composition comprises an αPTHF composition that is bound to a targeting moiety or otherwise associated therewith. In further embodiments, the liposome composition comprises a pegylated αPTHF composition that is bound to a targeting moiety or otherwise associated therewith. In some embodiments, the liposome composition comprises an αPTHF comprising 4, 5, 2 - 10, 4 - 6, or 6 or more glutamyl groups. In some embodiments, the liposome composition comprises tetraglutaminyl oxidized alpha tetrahydrofolate. In some embodiments, the liposome composition comprises pentaglutaminyl oxidized alpha tetrahydrofolate. In other embodiments, the liposome composition comprises hexaglutaminyl oxidized alpha tetrahydrofolate.

[0075] In some embodiments, the liposome composition comprises liposomal αPTHF (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, and TPLp-αPTHF). In some embodiments, the liposomal αPTHF is pegylated (e.g., NTPLp-αPTHF, and TPLp-αPTHF). In some embodiments, the liposomal αPTHF 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-αPTHF or TPLp-αPTHF). In further embodiments, the liposome composition comprises pegylated liposomal αPTHF 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-αPTHF). In some embodiments, the liposome composition comprises liposomal αPTHF that is cationic. In other embodiments, the liposome composition comprises liposomal αPTHF that is anionic or neutral. In further embodiments, the liposome composition comprises liposomal αPTHF 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 αPTHF has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

[0076] Also provided are pharmaceutical compositions comprising polyglutamylated alpha-tetrahydrofolate (αPTHF) including a delivery medium such as liposomal αPTHF. In some embodiments, the pharmaceutical composition comprises a pegylated αPTHF composition. In some embodiments, the pharmaceutical composition comprises an αPTHF composition linked to or otherwise associated with a targeting moiety. In further embodiments, the pharmaceutical composition comprises a pegylated αPTHF composition linked to or otherwise associated with a targeting moiety. In some embodiments, the pharmaceutical composition comprises an αPTHF having 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups. In some embodiments, the pharmaceutical composition comprises tetraglutamylated alpha-tetrahydrofolate. In some embodiments, the pharmaceutical composition comprises pentaglutamylated alpha-tetrahydrofolate. In other embodiments, the pharmaceutical composition comprises hexaglutamylated alpha-tetrahydrofolate.

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

[0078] In further embodiments, the present disclosure provides a method of modulating cell activation, chemokine production, or metabolic activity, the method comprising contacting a cell with a composition comprising a polyglutamylated alpha-tetrahydrofolate (αPTHF) composition. In some embodiments, the cell to be contacted is a mammalian cell. In further embodiments, the cell to be contacted is a human cell. In some embodiments, the cell to be contacted is a hyperproliferative cell. In further embodiments, the cell is an immune cell. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, αPTHF contains 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the αPTHF composition comprises tetraglutamylated alpha-tetrahydrofolate. In some embodiments, the αPTHF composition comprises pentaglutamylated alpha-tetrahydrofolate. In other embodiments, the αPTHF composition comprises hexaglutamylated alpha-tetrahydrofolate.

[0079] In further embodiments, the present disclosure provides a method of modulating cell activation, chemokine production, or metabolic activity, the method comprising contacting a cell with a liposome comprising a polyglutamylated alpha-tetrahydrofolate (αPTHF) composition. In some embodiments, the cell to be contacted is a mammalian cell. In further embodiments, the cell to be contacted is a human cell. In some embodiments, the cell to be contacted is a hyperproliferative cell. In further embodiments, the cell is an immune cell. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, αPTHF contains 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the αPTHF composition comprises tetraglutamylated alpha-tetrahydrofolate. In some embodiments, the αPTHF composition comprises pentaglutamylated alpha-tetrahydrofolate. In other embodiments, the αPTHF composition comprises hexaglutamylated alpha-tetrahydrofolate.

[0080] In further embodiments, the present disclosure provides a method of killing cells, the method comprising contacting a cell with a composition comprising a polyglutamylated alpha-tetrahydrofolic acid (αPTHF) composition (e.g., the αPTHF herein). In some embodiments, the cells to be contacted are mammalian cells. In further embodiments, the cells to be contacted are human cells. In some embodiments, the cells to be contacted are hyperproliferative cells. In further embodiments, the hyperproliferative cells are cancer cells. In further embodiments, the cancer cells to be contacted are, 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 primary cells or cells derived from a cell line obtained / derived from a cancer selected from the group consisting of hematological tumors such as leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dysplasias or cachexia. In further embodiments, the cancer cells to be contacted are primary cells or cells derived from a cell line obtained / derived from a 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, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, central nervous system (CNS) lymphoma. In further embodiments, the cancer cells are primary cells or cells derived from a cell line obtained / derived from a cancer selected from colorectal cancer, breast cancer, gastric cancer (e.g., gastric carcinoma), 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 cells derived from a cell line obtained / derived from colorectal cancer. In some embodiments, the αPTHF contains 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the αPTHF contains 4 glutamyl groups. In some embodiments, the αPTHF contains 5 glutamyl groups. In some embodiments, the αPTHF contains 6 glutamyl groups. In some embodiments, this method is carried out in vivo.In other embodiments, this method is performed in vitro.

[0081] In a further embodiment, the present disclosure provides a method of killing cells, the method comprising contacting the cells with liposomes comprising polyglutamylated alpha-tetrahydrofolate (e.g., Lp-αPTHF such as PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF or TPLp-αPTHF). In a further embodiment, the proliferating cells to be contacted are cancer cells. In a further embodiment, the cancer cells are, 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, primary cells or cells derived from cell lines obtained / induced from cancers selected from the group consisting of hematological tumors such as leukemia, lymphoma and other B cell malignancies, multiple myeloma and other plasma cell dyscrasias or cachexia. In a further embodiment, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained / induced 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, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, central nervous system (CNS) lymphoma. In some embodiments, the cancer cells are primary cells or cells derived from cell lines obtained / induced from cancers selected from colorectal cancer, breast cancer, gastric cancer (e.g., gastric carcinoma), 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 a particular embodiment, the cancer cells are primary cells or cells derived from cell lines obtained / induced 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 comprise αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the liposomes comprise αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups.In some embodiments, the liposome comprises an αPTHF containing 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups. In some embodiments, the liposome comprises an αPTHF containing 4 glutamyl groups. In some embodiments, the liposome comprises an αPTHF containing 5 glutamyl groups. In some embodiments, the liposome comprises an αPTHF containing 6 glutamyl groups. In some embodiments, the αPTHF contains 1, 2, 3, or 4 or more glutamyl groups having a gamma bond.

[0082] In a further embodiment, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having cancer or at risk of having cancer an effective amount of a delivery medium (e.g., an antibody immune complex or liposome) comprising polyglutamylated alpha-tetrahydrofolate. In some embodiments, the delivery medium is an antibody-containing immune complex (e.g., comprising a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery medium is a liposome (e.g., an Lp-αPTHF such as PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF). In some embodiments, the delivery medium to be administered is pegylated. In some embodiments, the delivery medium to be administered is not pegylated. In a further embodiment, the delivery medium to be administered comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of cancer cells. In a further embodiment, the delivery medium to be administered comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of cancer cells.In a further embodiment, 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 external 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 and an 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, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery medium comprises a targeting moiety that specifically binds to a cell surface antigen that is derived from or is determined to be expressed on a particular subject's cancer (tumor), such as a neoantigen. In some embodiments, the targeting moiety specifically binds to a cell surface antigen that is derived from or is determined to be expressed on a particular tumor of the subject, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the delivery medium to be administered comprises an αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the delivery medium to be administered comprises an αPTHF containing 4 glutamyl groups. In some embodiments, the delivery medium to be administered comprises an αPTHF containing 5 glutamyl groups. In some embodiments, the delivery medium to be administered comprises an αPTHF containing 6 glutamyl groups. In some embodiments, the αPTHF is a member selected from: (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 αPTHF is polyglutamylated 5,10-methylene-THF. In a further embodiment, the αPTHF is polyglutamylated [6R]-5,10-methylene-THF. In other embodiments, the αPTHF is polyglutamylated [6R,S]-5,10-methylene-THF.In some embodiments, αPTHF is polyglutamylated 5-methyl-THF. In further embodiments, αPTHF is [6S]-5-methyl-THF. In other embodiments, αPTHF is [6R,S]-5-methyl-THF. In some embodiments, αPTHF is polyglutamylated 5-formyl-THF. In further embodiments, αPTHF is polyglutamylated [6S]-5-formyl-THF. In other embodiments, αPTHF is polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the cancer is selected from the group consisting of non-hematological tumors such as, 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, 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 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, aleukemic meningitis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, central nervous system (CNS) cancer. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, breast cancer, gastric cancer (e.g., gastric carcinoma), 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 a further embodiment, the present disclosure provides a method of treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of liposomes comprising polyglutamylated alpha-tetrahydrofolate (e.g., Lp-αPTHF, e.g., PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF). In some embodiments, the liposomes are pegylated. In some embodiments, the liposomes are not pegylated. In a further embodiment, the liposomes comprise a targeting moiety having a specific affinity for an epitope of an antigen on the cancer cell surface.In a further embodiment, 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 external 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 and an 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, PDGFR alpha, PDGFR beta, 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, as well as CD15. In some embodiments, the liposome comprises a targeting moiety having a specific affinity for an epitope of a cell surface antigen that is derived from or expressed in a particular subject's tumor, 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 an αPTHF containing 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups. In some embodiments, the liposome to be administered comprises an αPTHF selected from: (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 liposome to be administered comprises polyglutamylated 5,10-methylene-THF. In a further embodiment, the liposome to be administered comprises polyglutamylated [6R]-5,10-methylene-THF. In other embodiments, the liposome to be administered comprises polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the liposome to be administered comprises polyglutamylated 5-methyl-THF. In a further embodiment, the liposome to be administered comprises [6S]-5-methyl-THF. In other embodiments, the liposome to be administered 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 liposome composition to be administered contains tetraglutamylated αPTHF. In some embodiments, the liposome composition to be administered contains pentaglutamylated αPTHF. In some embodiments, the liposome composition to be administered contains hexaglutamylated αPTHF. In some embodiments, the liposomes of the liposome composition to be administered contain αPTHF having 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups. In some embodiments, the liposomes of the liposome composition to be administered contain αPTHF having 1, 2, 3, or 4 or more glutamyl groups having a gamma bond. In some embodiments, the cancer is selected from the group consisting of lung (e.g., non-small 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 tumor, central nervous system cancer, melanoma, and hematological malignancies (e.g., leukemia or lymphoma). In further embodiments, the cancer cells are primary cells or cells derived from a cell line obtained / derived from a cancer selected from colorectal cancer, breast cancer, gastric cancer (e.g., gastric cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or lung adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In certain embodiments, the cancer cells are primary cells or cells derived from a cell line obtained / derived from colorectal cancer.

[0084] In a further embodiment, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having cancer or at risk of having cancer an effective amount of a liposomal composition comprising a liposome containing polyglutamylated alpha-tetrahydrofolate and a targeting moiety having specific affinity for an epitope of an antigen on the cancer surface.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 and an 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, PDGFR alpha, PDGFR beta, 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 a specific affinity for an epitope of a cell surface antigen that is derived from or has been shown to be expressed on a particular subject's cancer (tumor), 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 an αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the liposome to be administered comprises an αPTHF containing 4 glutamyl groups. In some embodiments, the liposome to be administered comprises an αPTHF containing 5 glutamyl groups. In some embodiments, the liposome to be administered comprises an αPTHF containing 6 glutamyl groups. In some embodiments, the liposome to be administered comprises an αPTHF selected from (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 liposome to be administered comprises polyglutamylated 5,10-methylene-THF. In a further embodiment, the liposome to be administered comprises polyglutamylated [6R]-5,10-methylene-THF. In another embodiment, the liposome to be administered comprises polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the liposome to be administered comprises polyglutamylated 5-methyl-THF.In further embodiments, 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 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 contain αPTHF having one, two, three, or four or more glutamyl groups having a gamma linkage.

[0085] In some embodiments, the liposomal composition to be administered comprises pegylated liposomes (e.g., TPLp-αPTHF). 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 αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the liposomes to be administered comprise αPTHF containing 4 glutamyl groups. In some embodiments, the liposomes to be administered comprise αPTHF containing 5 glutamyl groups. In some embodiments, the liposomes to be administered comprise αPTHF containing 6 glutamyl groups. In some embodiments, the liposomes to be administered comprise an αPTHF selected from (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 a further embodiment, 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 a further embodiment, the liposomes to be administered comprise [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 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 αPTHF, which contains one, two, three, or four or more glutamyl groups having a gamma bond. 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 tumor, 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 a further embodiment, the present disclosure provides a method of treating cancer, comprising administering to a subject having or at risk of having cancer that expresses a folate receptor on the cell surface an effective amount of a liposomal composition, the liposomal composition comprising liposomes that include (a) polyglutamylated alpha-tetrahydrofolate (αPTHF) and (b) a targeting moiety having specific 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-αPTHF). 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 αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the liposomes to be administered comprise αPTHF containing 4 glutamyl groups. In some embodiments, the liposomes to be administered comprise αPTHF containing 5 glutamyl groups. In some embodiments, the liposomes to be administered comprise αPTHF containing 6 glutamyl groups.In some embodiments, the liposomes to be administered comprise an αPTHF selected from: (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 one, two, three, or four or more glutamyl groups including a gamma bond.In some embodiments, the liposomal composition is administered to treat cancer selected from the group consisting of, for example, non-blood malignancies 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 tumor, central nervous system cancer, and melanoma; and blood malignancies such as, for example, leukemia, lymphoma, and other B-cell malignancies, multiple myeloma and other plasma cell dysplasia 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, 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 a further embodiment, the present disclosure provides a method of maintenance therapy for cancer, the method comprising administering to a subject who is undergoing or has undergone cancer treatment, an effective amount of a liposomal composition comprising liposomes containing polyglutamylated alpha-tetrahydrofolate (Lp-αPTHF). In some embodiments, the liposomal composition to be administered is PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF. In some embodiments, the liposomal composition to be administered comprises pegylated liposomes (e.g., PLp-αPTHF, NTPLp-αPTHF, or TPLp-αPTHF). In some embodiments, the liposomal composition to be administered comprises targeted liposomes (e.g., TLp-αPTHF or TPLp-αPTHF). In some embodiments, the liposomal composition to be administered comprises liposomes that are pegylated and comprise a targeting moiety (e.g., TPLp-αPTHF). In some embodiments, the liposomes of the liposomal composition to be administered comprise polyglutamylated alpha-tetrahydrofolate containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the liposomes to be administered comprise αPTHF containing 4 glutamyl groups. In some embodiments, the liposomes to be administered comprise αPTHF containing 5 glutamyl groups. In some embodiments, the liposomes to be administered comprise αPTHF containing 6 glutamyl groups.In some embodiments, the liposomes to be administered comprise an αPTHF selected from: (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 1, 2, 3, or 4 or more glutamyl groups including a gamma bond.

[0088] In a further embodiment, the present disclosure provides a method of treating a disorder of the immune system, the method comprising administering to a subject having or at risk of having a disorder of the immune system an effective amount of a liposomal composition comprising polyglutamylated alpha-tetrahydrofolate (e.g., Lp-αPTHF such as PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF or TPLp-αPTHF) in liposomes. In some embodiments, the liposomal composition is administered to treat an autoimmune disease. In a further embodiment, 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 to be administered comprises pegylated liposomes (e.g., PLp-αPTHF, NTPLp-αPTHF, or TPLp-αPTHF). In some embodiments, the liposomal composition to be administered comprises targeted liposomes (e.g., TLp-αPTHF or TPLp-αPTHF) comprising a targeting moiety having specific affinity for a surface antigen on a target cell of interest (e.g., an immune cell). In a further embodiment, the liposomal composition to be administered comprises liposomes that are pegylated and comprise a targeting moiety (e.g., TPLp-αPTHF). In some embodiments, the liposomes of the liposomal composition to be administered comprise polyglutamylated alpha-tetrahydrofolate having 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the liposomal composition to be administered comprises αPTHF having 4 glutamyl groups. In some embodiments, the liposomes to be administered comprise αPTHF having 5 glutamyl groups. In some embodiments, the liposomes to be administered comprise αPTHF having 6 glutamyl groups.In some embodiments, the liposomes to be administered comprise an αPTHF selected from: (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 one, two, three, or four or more glutamyl groups containing a gamma bond.

[0089] The present disclosure also provides a method for delivering polyglutamylated alpha-tetrahydrofolate to tumors and / or cancer cells, the method comprising administering to a subject having a tumor a composition comprising polyglutamylated alpha-tetrahydrofolate (L-αPTHF) and a targeting moiety having specific binding affinity for an epitope of a surface antigen of a tumor cell or cancer cell. In some embodiments, the targeting moiety to be administered is bound 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 polyglutamylated alpha-tetrahydrofolate containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the composition to be administered comprises tetraglutamylated alpha-tetrahydrofolate. In some embodiments, the composition to be administered comprises pentaglutamylated alpha-tetrahydrofolate. In other embodiments, the composition to be administered comprises hexaglutamylated alpha-tetrahydrofolate. In some embodiments, the composition to be administered comprises an αPTHF selected from (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 composition to be administered comprises polyglutamylated [6R]-5,10-methylene-THF. In other embodiments, the composition to be administered comprises polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the composition to be administered comprises polyglutamylated 5-methyl-THF. In further embodiments, the composition to be administered comprises [6S]-5-methyl-THF. In other embodiments, the composition to be administered comprises [6R,S]-5-methyl-THF. In some embodiments, the composition to be administered comprises polyglutamylated 5-formyl-THF. In further embodiments, the composition to be administered comprises polyglutamylated [6S]-5-formyl-THF. In other embodiments, the composition to be administered comprises polyglutamylated [6R,S]-5-formyl-THF.

[0090] In a further embodiment, the present disclosure provides a method for preparing a liposomal composition comprising a liposomal polyglutamylated alpha-tetrahydrofolic acid (αPTHF) composition, the method comprising: forming a mixture comprising liposomal components and alpha-polyglutamylated tetrahydrofolic acid in a solution; homogenizing the mixture in the solution to form liposomes; and treating the mixture to form liposomes comprising polyglutamylated tetrahydrofolic acid. In some embodiments, the polyglutamylated alpha-tetrahydrofolic acid comprises 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the αPTHF composition comprises pentaglutamylated alpha-tetrahydrofolic acid. In some embodiments, the αPTHF composition comprises tetraglutamylated alpha-tetrahydrofolic acid. In other embodiments, the αPTHF composition comprises hexaglutamylated alpha-tetrahydrofolic acid. In some embodiments, the polyglutamylated alpha-tetrahydrofolic acid comprises 1, 2, 3, or 4 or more glutamyl groups comprising a gamma bond. In some embodiments, the αPTHF composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more D-type glutamyl groups. In some embodiments, the αPTHF composition comprises 2, 3, 4, 5, or 6 or more L-type glutamyl groups. In some embodiments, the αPTHF composition comprises 2, 3, 4, 5, or 6 or more L-type glutamyl groups and 1, 2, 3, 4, 5, or 6 or more D-type glutamyl groups.In some embodiments, the composition comprises an αPTHF selected from: (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 composition to be administered comprises polyglutamylated [6R,S]-5-formyl-THF.

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

[0092]

Fig. 1A - 1N

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Fig. 20A - 20F

BRIEF DESCRIPTION OF THE DRAWINGS

[0093] The present disclosure generally relates to polyglutamylated alpha-tetrahydrofolate compositions. These compositions represent an advancement over previous treatments for hyperproliferative diseases such as cancer. Methods of making, delivering, and using polyglutamylated alpha-tetrahydrofolate compositions are also provided. Polyglutamylated alpha compositions have uses including, but not limited to, the treatment (e.g., treatment and / or prevention) of hyperproliferative diseases such as cancer, immune system disorders such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria. Polyglutamylated alpha compositions have uses in combination therapies with one or more therapeutic agents (e.g., 5-fluorouracil) that enhance the effect of the therapeutic agent, or as "chemoprotective agents" that reduce toxic side effects associated with the therapeutic agent (e.g., in combination with folate antagonists such as methotrexate).

[0094] I. Definitions Unless otherwise defined, all technical and scientific terms used herein 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 herein with the word "comprising", it is understood that other similar embodiments are also provided, which are always described with the terms "containing", "consisting of", and / or "consisting essentially of". However, when used as a transitional phrase in a claim, each should be interpreted separately and in an appropriate legal and factual context (e.g., in a claim, the transitional phrase "comprising" is considered a more open-ended phrase, the transitional phrase "consisting of" is more exclusive, and "consisting essentially of" is intermediate between these).

[0096] As used herein, the singular forms "a", "an", and "the" include plural referents unless specifically stated otherwise or the context clearly indicates that only a single referent is intended.

[0097] The term "and / or" as used in phrases such as "A and / or B" is intended herein to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include 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 only for convenience and / or for compliance with official regulations, and do not limit the subject technology, nor are they referred to in connection with the interpretation of the description of the subject technology. Features described under one heading or subheading of the subject disclosure may be combined with features described under other headings or subheadings in various embodiments. Further, not all of the features under a single heading or subheading are necessarily used together in the embodiments.

[0099] The terms "tetrahydrofolic acid" and "THF" are used interchangeably to include tetrahydrofolic acid in salt, acid, and / or free base form (e.g., tetrahydrofolic acid disodium). Unless otherwise specified or clearly evident from the context, "THF" and "tetrahydrofolic acid" include natural and non-natural forms of THF, including one-carbon substituted THF derivatives. In particular, unless otherwise specified or clearly evident from the context, "THF" and "tetrahydrofolic acid" include diasteromeric compositions having a [6R] configuration at the C-6 atom of the tetrahydropterin moiety of THF, diasteromeric compositions having a [6S] configuration at the C-6 atom, and / or mixtures of [6,R,S] diastereomers (e.g., 1:1). Unless otherwise specified or clearly evident from the context, "THF" and "tetrahydrofolic acid" include (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 comprising a THF diastereomer selected from (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 THF; (f) [6R]-5,10-methylene-THF; and (g) [6S]-5-formimino-THF. In some embodiments, the present disclosure provides a composition comprising 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). Compositions containing THF salts can further include any of various cations such as Na. + , Mg 2+ , K + , NH4 + , and / or Ca 2+ and the like. In certain embodiments, the salt is a pharmaceutically acceptable salt. In further particular embodiments, the THF salt comprises Na + . Tetrahydrofolic acid contains one L-gamma-glutamyl group and is thus considered 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" may also be referred to herein as a type of tetrahydrofolic acid (THF).

[0101] The terms "polyglutamic acid", "polyglutamination", or variations thereof, mean a composition comprising at least one chain to which two or more glutamyl groups are attached. The polyglutamic acid chain can be linear or branched. A linear polyglutamic acid chain can include glutamyl groups, for example, including an alpha carboxyl group or a gamma carboxyl group bond. A branched polyglutamic acid chain includes one or more glutamyl groups that include both an alpha carboxyl group bond and a gamma carboxyl group bond with another glutamyl group, thereby creating a branch point of polyglutamic acid. Representative branched polyglutamic acids are shown in FIGS. 1R-1U. The polyglutamic acid chain includes an N-terminal glutamyl group and one or more C-terminal glutamyl groups. The N-terminal glutamyl group of the polyglutamic acid 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 tetrahydrofolate is the glutamyl group of tetrahydrofolate. The C-terminal glutamyl group of the polyglutamic acid chain binds to another glutamyl group via its amino group, but does not bind to another glutamyl group via its carboxylic acid group.

[0102] The terms "polyglutamylated-tetrahydrofolate", "polyglutamylated-THF", "THF-PG", "PTHF" and iterations thereof, as used herein, refer to a tetrahydrofolate composition (i.e., THF-PG) that includes at least one glutamyl group in addition to the glutamyl group of tetrahydrofolate n, where n ≧ 1) are used interchangeably to mean. When referring to the number of glutamyl groups in αPTHF (αTHF-PG) herein, the glutamyl groups of tetrahydrofolic acid are taken into account. For example, a THF-PG composition containing 5 glutamyl residues in addition to the glutamyl group of THF is referred to herein as hexaglutamylated tetrahydrofolic acid or tetrahydrofolic acid hexaglutamate. In some embodiments, polyglutamylated-tetrahydrofolic acid is (a) polyglutamylated 5-formyl-THF; (b) polyglutamylated 10-formyl-THF; (c) polyglutamylated 5,10-methenyl-THF; (d) polyglutamylated 5-methyl-THF; (e) polyglutamylated tetrahydrofolic acid ((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-tetrahydrofolic acid is a member selected from (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]-tetrahydrofolic acid THF; (f) polyglutamylated [6R]-5,10-methylene-THF; and (g) polyglutamic acid [6S]-5-formimino-THF. In some embodiments, polyglutamylated-tetrahydrofolic acid is [6R]-5,10-methylene-THF. In some embodiments, polyglutamylated-tetrahydrofolic acid is [6S]-5-methyl-THF. In some embodiments, polyglutamylated-tetrahydrofolic acid is [6S]-5-formyl-THF.In other embodiments, the 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., in a 1:1 weight ratio).

[0103] The terms "alpha-glutamyl group", "alpha-glutamic acid", and "alpha bond", when referring to the bond of a glutamyl group, mean a glutamyl group including an alpha-carboxyl group bond. In some embodiments, the alpha bond is an amide bond between the alpha-carboxyl group of one glutamyl group and a second glutamyl group. The alpha bond can be a bond between a glutamyl group and a glutamyl group in tetrahydrofolate, or a bond between a glutamyl group and a second glutamyl group not present in tetrahydrofolate, such as a glutamyl group within a polyglutamic acid chain bound to tetrahydrofolate. In some embodiments, the "alpha-glutamyl group" of the provided polyglutamylated alpha-tetrahydrofolate has both an alpha-carboxyl group bond and a gamma-carboxyl group bond. In some embodiments, the alpha-glutamyl group is of the L-type. In some embodiments, the alpha-glutamyl group is of the D-type. In some embodiments, the glutamyl group is of the L-type. In some embodiments, one or more glutamyl groups in the polyglutamylated alpha-tetrahydrofolate are of the L-type and one or more glutamyl groups in the polyglutamylated alpha-tetrahydrofolate are of the D-type.

[0104] The terms "polyglutamylated alpha-tetrahydrofolate", "α-polyglutamylated tetrahydrofolate", "αPTHF", "polyglutamylated alpha-tetrahydrofolate", "polyglutamylated alpha THF", "αTHF-PG" and iterations thereof, as used herein, refer to a tetrahydrofolate composition (e.g., THF-PG) that includes at least one glutamyl group having an alpha-carboxyl group bond. n, where n ≧ 1, are used interchangeably to mean an α-glutamyl group). When referring to the number of glutamyl groups in αPTHF (αTHF-PG) herein, the glutamyl groups of tetrahydrofolic acid are taken into account. For example, an αTHF-PG composition containing 5 glutamyl groups in addition to the glutamyl group of THF, with at least 1 of the glutamyl groups having an alpha carboxyl bond, may be referred to herein as alpha hexaglutamylated tetrahydrofolic acid, hexaglutamylated alpha tetrahydrofolic acid, or alpha tetrahydrofolic acid hexaglutamate.

[0105] The terms "gamma-glutamyl group", "gamma-glutamic acid", and "gamma bond", when they are related to the bond of a glutamyl group, mean a glutamyl group containing a gamma carboxyl group bond. In some embodiments, a gamma bond is an amide bond between the gamma carboxyl group of one glutamyl group and a second glutamyl group. A gamma bond can be between a glutamyl group and the glutamyl group of tetrahydrofolic acid, or between a glutamyl group present in tetrahydrofolic acid and a second glutamyl group, such as a glutamyl group within a polyglutamic acid chain bound to tetrahydrofolic acid. In some embodiments, one or more gamma-bonded glutamyl groups in polyglutamylated alpha tetrahydrofolic acid are of the L type. In some embodiments, one or more gamma-bonded glutamyl groups in polyglutamylated alpha tetrahydrofolic acid are of the D type. In some embodiments, one or more gamma-bonded glutamyl groups in polyglutamylated alpha tetrahydrofolic acid are of the L type and one or more gamma-bonded glutamyl groups in polyglutamylated alpha tetrahydrofolic acid are of the D type.

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

[0107] As used herein, the term "targeting moiety" means a molecule that enhances affinity for a selected target, such as a cell, cell type, tissue, organ, body region, or compartment, such as a compartment of a cell, tissue or organ. A targeting moiety can include a wide variety of entities. Targeting moieties include natural molecules, or recombinant or synthetic molecules. In some embodiments, the targeting moiety is an antibody, an antigen-binding antibody fragment, a bispecific antibody or other antibody-based molecule or compound. In some embodiments, the targeting moiety is an aptamer, an affimer, 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 of time, with higher 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 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 "specific binding" 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 require (although it can include) exclusive binding, e.g., binding to an epitope on a single target. Thus, in certain embodiments, a targeting moiety can specifically bind to an epitope present on two or more targets. In certain embodiments, the same targeting moiety that specifically binds to epitopes present on multiple targets can bind to 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 moiety), 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 even when the protein is denatured, whereas epitopes formed by tertiary folding are usually lost when the protein is denatured. An epitope typically contains at least 3 amino acids, more commonly 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", and similar expressions known in the art refer to properties of the 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 particular limitation, other methods such as competitive analysis, equilibrium analysis, and microcalorimetry, as well as real-time interaction analysis based on surface plasmon resonance interaction (e.g., those using a Biacore® device) can be used to characterize intermolecular interactions. 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 aids, promotes, or facilitates the entry of polyglutamate oxidized alpha-tetrahydrofolate into cells. Such delivery vehicles are known in the art and include, without particular limitation, liposomes, lipospheres, polymers (e.g., polymer conjugates), peptides, proteins such as antibodies (e.g., immunoconjugates such as antibody-drug conjugates (ADCs), as well as antigen-binding antibody fragments and their derivatives), cell components, cyclic oligosaccharides (e.g., cyclodextrin), micelles, microparticles (e.g., microspheres), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), hydrogels, lipoprotein particles, viral sequences, viral substances, or lipid or liposome formulations, and combinations thereof. The delivery vehicle can be directly or indirectly bound 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 human or, without particular limitation, a vertebrate mammal including a dog, a cat, a horse, a goat, and a primate such as a monkey. Thus, the present invention can also be used to treat diseases or conditions in non-human subjects. For example, cancer is one of the major causes of death in companion animals (e.g., cats and dogs). In some embodiments of the present invention, the subject is a human. In the present disclosure, the terms "subject" and "patient" are used interchangeably and have the same meaning. Generally, it is preferred to use the maximum dose, i.e., the maximum safe dose according to sound medical judgment.

[0113] As used herein, "effective amount" means a dosage of a drug sufficient to obtain a medically desirable result. The effective amount can vary depending on the desired outcome, the specific condition to be treated or prevented, the age and health status of the subject to be treated, the severity of the condition, the treatment period, the nature of any concurrent or combined therapies (if any), the specific route of administration, and similar factors within the knowledge and expertise of the medical practitioner. The "effective amount" can be determined experimentally in a routine manner in relation to the indicated purpose. In the case of cancer, the effective amount of a drug is one that can reduce the number of cancer cells; reduce the size of the tumor; inhibit the invasion of cancer cells into surrounding organs (i.e., slow it down to some extent and preferably stop it); inhibit the metastasis of the tumor (i.e., slow it down to some extent and preferably stop it); inhibit the growth of the tumor to some extent; and / or alleviate one or more of the symptoms associated with the disorder to some extent. As long as the drug can prevent the proliferation of existing cancer cells and / or kill cancer cells, it can be cytostatic and / or cytotoxic. In cancer treatment, 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, regardless of in vitro or in vivo, in connection with unwanted or abnormal cell growth, such as neoplastic or hyperplastic growth, of unwanted or uncontrolled cells. In some embodiments, the proliferative disease is a cancer, tumor, and / or any tumor metastasis, regardless of the location of the cancer, tumor, and / or tumor metastasis, including benign or cancerous cancer or tumor diseases. In some embodiments, the proliferative disease is a benign or malignant tumor. In some embodiments, the proliferative disease is a non-cancerous disease. In some embodiments, the proliferative disease is a hyperproliferative condition, such as hyperplasia, fibrosis (especially other types of fibrosis such as renal fibrosis in addition to pulmonary fibrosis), angiogenesis, psoriasis, atherosclerosis, and smooth muscle proliferation of 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 and abnormal growth of cells, the ability of the affected cells to spread (metastasize) locally or to other parts of the body via the bloodstream and lymphatic system, and / or any characteristic structures and / or molecular features known to be associated with the cell type or disease. As used herein, "tumor" means 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 with specific structural characteristics, lacking differentiation, and capable of invasion and metastasis. Cancers that can be treated with the αPTHF compositions provided herein include, but are not limited to, non-blood system tumors such as, 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; 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., gastric carcinoma), 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 αPTHF compositions are those described herein or known in the art. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder", and "tumor" are not mutually exclusive as used herein.

[0117] The terms "treating," "treatment," or "treat" mean both (a) therapeutic means for curing, suppressing, alleviating symptoms of, and / or halting the progression of a diagnosed medical condition or disorder, and (b) prophylactic or preventive means for preventing and / or delaying the onset of a targeted disease or medical condition. Accordingly, subjects in need of treatment include subjects already having cancer, a disorder or disease, subjects at risk of having cancer or a medical condition, and subjects in whom an infection or medical condition should be prevented. A subject is confirmed to be "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 "well treated" by the methods provided herein if, for example, total, partial, or temporary remission or disappearance of symptoms associated with a disease or medical condition (e.g., cancer, inflammation, and rheumatoid arthritis) is observed. 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 necessarily be identifiable by the patient. In other embodiments, the term "treating" or "treatment" or "treat" means suppressing the progression of a proliferative disorder, for example, physically by stabilization of an identifiable symptom, physiologically by stabilization of a physical parameter, or both. In other embodiments, the term "treating" or "treatment" or "treat" means a decrease or stabilization in tumor size, tumor cell proliferation or survival, or cancer cell number. Treatment can be with the αPTHF composition alone or in combination with additional therapeutic agents.

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

[0119] "Treatment of a proliferative disorder" is used herein to include maintenance or reduction of tumor size, induction of (partial or complete) tumor regression, inhibition of tumor growth, and / or extension of lifespan in a subject with a proliferative disorder. 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 caused by 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, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, inflammation, and rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis.

[0121] The term "therapeutic agent" is used to mean a drug or its derivative that can interact with hyperproliferative cells such as cancer cells or immune cells, thereby suppressing 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 drugs further include, but are not limited to, the anticancer drugs trimethoprim, temozolomide, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), bis-chloronitrosourea (BCNU) and camptothecin (trademark), or any of their therapeutic derivatives. Further examples of therapeutic agents suitable for use by the methods of the present disclosure include, but are not limited to, anti-restenosis drugs, promoting or anti-proliferative agents, anti-inflammatory drugs, anti-neoplastic drugs, anti-mitotic drugs, antiplatelet agents, anticoagulants, antifibrin drugs, antithrombin drugs, cell growth inhibitory drugs, antibiotics and other anti-infective drugs, anti-enzyme drugs, antimetabolites, angiogenesis drugs, cytoprotective drugs, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists and / or cardioprotective drugs. "Therapeutic agent" also means salts, acids, and free base forms of the above drugs.

[0122] As used herein, the term "chemotherapeutic agent," when used in the context of cancer treatment, 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 polyglutamylated alpha-tetrahydrofolate compositions of the present disclosure are used in combination with a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent 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 agent is a pyrimidine analog (e.g., a fluoropyrimidine such as 5-fluorouracil (5-FU)).

[0123] The term "antimetabolite" as used herein means a therapeutic agent that inhibits the utilization of a metabolite or its prodrug. Examples of antimetabolites include 5-FU, as well as 5-FU metabolites and / or prodrugs, such as 5-FUMP, 5-FUDP, 5-FdUMP, capecitabine, tegafur 5-fluorodeoxyuridine monophosphate, etc.; and cytarabine, and cytarabine prodrugs such as nelarabine, 5-azacitidine, gemcitabine, mercaptopurine, thioguanine, azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, and cladribine. Antimetabolites useful for practicing the methods of the present disclosure include nucleoside analogs, including purine or pyrimidine analogs. In some embodiments, the polyglutamylated alpha-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 examples, the nucleoside analogs are selected from 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 (JP-A-55059173), N'-(2-furanyl)-5-fluorouracil (JP-A-53149985), and 1-(2-tetrahydrofuryl)-5-fluorouracil, or a derivative thereof. In certain embodiments, the antimetabolite is a pyrimidine analog or a pyrimidine analog prodrug (e.g., a fluoropyrimidine). In certain embodiments, the antimetabolite is 5-fluorouracil.

[0124] As used herein, a "taxane" is an anti-cancer agent that interferes with or disrupts the stability, formation, and / or function of microtubules. Taxane agents include paclitaxel and docetaxel, and derivatives thereof, which function in the same mode of action as the taxane from which they are derived with respect to microtubules. 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 preparation 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 solid or liquid fillers, diluents, or encapsulating substances that are suitable for and compatible with administration to humans or other subjects.

[0126] The present disclosure generally relates to polyglutamylated alpha-tetrahydrofolate (αPTHF) compositions, and methods of making and using 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. Gamma polyglutamylated compositions also have uses in combination therapies with one or more therapeutic agents (such as chemotherapeutic agents, e.g., 5-fluorouracil) that enhance the effect of the therapeutic agent, or as "chemoprotective agents" (such as in combination with folate antagonists, e.g., methotrexate) that reduce toxic side effects associated with the therapeutic agent.

[0127] In some embodiments, the present disclosure provides the following: [1] A composition comprising polyglutamylated alpha-tetrahydrofolate. [2] The polyglutamylated alpha-tetrahydrofolate is (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 (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) The composition according to item [1], selected from the group consisting of [3] The composition according to item [1] or [2], wherein the polyglutamylated alpha-tetrahydrofolic acid contains 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups having an alpha-carboxyl group bond. [4] The composition according to any one of items [1] to [3], wherein the polyglutamylated alpha-tetrahydrofolic acid is tetraglutamylated alpha-tetrahydrofolic acid (e.g., [6R]-5,10-methenyl-THF, [6S]-5-formyl-THF, and / or [6R]-10-formyl-THF). [5] The composition according to any one of items [1] to [3], wherein the polyglutamylated alpha-tetrahydrofolic acid is pentaglutamylated alpha-tetrahydrofolic acid (e.g., [6R]-5,10-methenyl-THF, [6S]-5-formyl-THF, and / or [6R]-10-formyl-THF). [6] The composition according to any one of items [1] to [3], wherein the polyglutamylated alpha-tetrahydrofolic acid is hexaglutamylated alpha-tetrahydrofolic acid (e.g., [6R]-5,10-methenyl-THF, [6S]-5-formyl-THF, and / or [6R]-10-formyl-THF). [7](a) Two or more glutamyl groups have an alpha-carboxyl group bond, (b) Glutamyl groups other than the glutamyl group of tetrahydrofolic acid each have an alpha-carboxyl group bond; or (c) Two or more glutamyl groups have a gamma-carboxyl group bond, The composition according to any one of items [1] to [6]. [8](a) The glutamyl groups other than the C-terminal glutamyl group and the glutamyl group of tetrahydrofolic acid each have an alpha-carboxyl group bond; or (b) The glutamyl groups other than the C-terminal glutamyl group each have an alpha-carboxyl group bond, The composition according to any one of items [1] to [6]. [9] The composition according to any one of items [1] to [8], wherein at least one glutamyl group has both an alpha-carboxyl group bond and a gamma-carboxyl group bond;

[10] (a) At least two of the glutamyl groups of alpha-polyglutamylated tetrahydrofolic acid are of the L-form, (b) The glutamyl groups of alpha-polyglutamylated tetrahydrofolic acid are each of the L-form, (c) At least one of the glutamyl groups of alpha-polyglutamylated tetrahydrofolic acid is of the D-form, (d) The glutamyl groups of alpha-polyglutamylated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid are each of the D-form, or (e) At least two of the glutamyl groups of alpha-polyglutamylated tetrahydrofolic acid are of the L-form and at least one of the glutamyl groups is of the D-form, The composition according to any one of items [1] to [9];

[11] The composition according to any one of items [1] to

[10] , wherein polyglutamic acid is linear;

[12] The composition according to any one of items [1] to

[10] , wherein polyglutamic acid is branched;

[13] A liposome composition (Lp-αPTHF) containing the alpha-polyglutamylated tetrahydrofolic acid according to any one of items [1] to

[12] ;

[14] The LαPP composition according to item

[13] , wherein alpha-polyglutamylated tetrahydrofolic acid contains an L-form glutamyl group having an alpha-carboxyl group bond;

[15] The Lp-αPTHF composition according to item

[13] or

[14] , wherein the glutamyl groups of alpha-polyglutamylated tetrahydrofolic acid are each in the L-form;

[16] The Lp-αPTHF composition according to item

[13] or

[14] , wherein at least one of the glutamyl groups of alpha-polyglutamylated tetrahydrofolic acid is in the D-form;

[17] The Lp-αPTHF composition according to any one of items

[13] to

[16] , wherein the liposome contains alpha-polyglutamylated tetrahydrofolic acid containing 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups;

[18] The Lp-αPTHF composition according to any one of items

[13] to

[17] , wherein at least one of the glutamyl groups of alpha-polyglutamylated tetrahydrofolic acid has a gamma-carboxyl group bond;

[19] The composition according to any one of items

[13] to

[18] , wherein at least one glutamyl group has both an alpha-carboxyl group bond and a gamma-carboxyl group bond;

[20] The composition according to any one of items

[13] to

[19] , containing 2, 3, 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups having both an alpha-carboxyl group bond and a gamma-carboxyl group bond;

[21] The Lp-αPTHF composition according to any one of items

[13] to

[20] , wherein the liposome contains alpha-polyglutamylated tetrahydrofolic acid containing alpha-tetraglutamylated tetrahydrofolic acid, alpha-pentaglutamylated tetrahydrofolic acid, or alpha-hexaglutamylated tetrahydrofolic acid;

[22] The Lp-αPTHF composition according to any one of items

[13] to

[21] , wherein the polyglutamic acid is linear or branched;

[23] The Lp-αPTHF composition according to any one of items

[13] to

[22] , wherein the liposome is pegylated (PαLp-αPTHF);

[24] The Lp-αPTHF composition according to any one of items

[13] to

[23] , wherein the liposome contains at least 1% by weight of alpha-polyglutamyl oxidized tetrahydrofolic acid, or at least 1% of the starting material of alpha-polyglutamyl oxidized THF is encapsulated (enclosed) in the Lp-αPTHF in the process of preparing Lp-αPTHF;

[25] The Lp-αPTHF composition according to any one of items

[13] to

[24] , wherein the liposome has a diameter in the range of 20 nm to 500 nm or 20 nm to 200 nm;

[26] The Lp-αPTHF composition according to any one of items

[13] to

[25] , wherein the liposome has a diameter in the range of 80 nm to 120 nm;

[27] The Lp-αPTHF composition according to any one of items

[13] to

[26] , wherein the liposome is formed from liposome components;

[28] The Lp-αPTHF composition according to item

[27] , wherein the liposome components include at least one anionic lipid and neutral lipid;

[29] The Lp-αPTHF composition according to item

[27] or

[28] , wherein the liposome components include at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide;

[30] The Lp-αPTHF composition according to any one of items

[27] to

[29] , wherein the liposome components include at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC;

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

[27] to

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

[32] The Lp-αPTHF composition according to item

[31] , wherein the steric stabilizer is at least one 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 polymer; oligoglycerin, a copolymer containing polyethylene glycol and polypropylene oxide, poloxamer 188, and polyvinyl alcohol;

[33] The Lp-αPTHF composition according to item

[32] , wherein the steric stabilizer is PEG and PEG has a number average molecular weight (Mn) of 200 to 5000 daltons;

[34] The Lp-αPTHF composition according to any one of items

[13] to

[33] , wherein the liposome is anionic or neutral;

[35] The Lp-αPTHF composition according to any one of items

[13] to

[33] , wherein the liposome has a zeta potential of zero or less;

[36] The Lp-αPTHF composition according to any one of items

[13] to

[33] , wherein the liposome has a zeta potential of 0 to -150 mV;

[37] The Lp-αPTHF composition according to any one of items

[13] to

[33] , wherein the liposome has a zeta potential of -30 to -50 mV;

[38] The Lp-αPTHF composition according to any one of items

[13] to

[33] , wherein the liposome is cationic;

[39] The Lp-αPTHF composition according to any one of items

[13] to

[38] , wherein the liposome has an internal space containing polyglutamic acid oxidized alpha-tetrahydrofolic acid and an aqueous pharmaceutically acceptable carrier;

[40] The Lp-αPTHF composition according to item

[39] , wherein the pharmaceutically acceptable carrier contains an isotonic agent such as dextrose, mannitol, glycerol, potassium chloride, sodium chloride, etc. at a concentration of more than 1%;

[41] The Lp-αPTHF composition according to item

[39] , wherein the aqueous pharmaceutically acceptable carrier is trehalose;

[42] The Lp-αPTHF composition according to item

[41] , wherein the pharmaceutically acceptable carrier contains 5% to 20% by weight of trehalose;

[43] The Lp-αPTHF composition according to any one of items

[39] to

[42] , wherein the pharmaceutically acceptable carrier contains 1% to 15% by weight of dextrose;

[44] The Lp-αPTHF composition according to any one of items

[39] to

[43] , wherein the internal space of the liposome contains 5% dextrose suspended in HEPES buffer;

[45] The Lp-αPTHF composition according to any one of items

[39] to

[44] , wherein the pharmaceutically acceptable carrier contains a buffer solution 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 Lp-αPTHF composition according to any one of items

[39] to

[45] , wherein the pharmaceutically acceptable carrier contains sodium acetate and calcium acetate at a total concentration of 50 mM to 500 mM;

[47] The Lp-αPTHF composition according to any one of items

[13] to

[46] , wherein the internal space of the liposome has a pH of 5 to 8 or 6 to 7, or any range therebetween;

[48] The Lp-αPTHF composition according to any one of items

[13] to

[47] , wherein the liposome contains less than 500,000 molecules or less than 200,000 molecules of alphapolyg lutamyl oxidized tetrahydrofolic acid;

[49] The Lp-αPTHF composition according to any one of items

[13] to

[48] , wherein the liposome contains 10 to 100,000 molecules or any range therebetween of alphapolyg lutamyl oxidized tetrahydrofolic acid;

[50] The Lp-αPTHF composition according to any one of items

[13] to

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

[51] The targeting moiety is bound to one or both of the PEG and the outer side of the liposome, and optionally, the targeting moiety is covalently bound to one or both of the PEG and the outer side of the liposome. The Lp-αPTHF composition according to item

[50] ;

[52] The Lp-αPTHF composition according to item

[50] or

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

[53] The Lp-αPTHF composition according to any one of items

[50] to

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

[54] The targeting moiety binds to the surface antigen with an equilibrium dissociation constant (Kd) in the range of 0.5×10 -10 ~10×10 -6 as measured using BIACORE (registered trademark) analysis. The Lp-αPTHF composition according to any one of items

[50] to

[53] ;

[55] The Lp-αPTHF composition according to any one of items

[50] to

[55] , wherein the targeting moiety specifically binds to 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);

[56] The Lp-αPTHF composition according to any one of items

[50] to

[56] , wherein the targeting moiety comprises one or more selected from the group consisting 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;

[57] Each pegylated liposome contains 1 to 1000 or 30 to 200 targeting moieties. The Lp-αPTHF composition according to any one of items

[50] to

[56] ;

[58] Further comprising one or more of an immunostimulant, a detectable marker, and maleimide, wherein the immunostimulant, the detectable marker, or maleimide is bound to the PEG or the outer side of the liposome. The Lp-αPTHF composition according to any one of items

[39] to

[57] ;

[59] The Lp-αPTHF composition according to item

[58] , wherein the immunostimulant is at least one selected from the group consisting of a protein immunostimulant, a nucleic acid immunostimulant, a chemical immunostimulant, a hapten, and an adjuvant;

[60] The immunostimulant is at least one selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan; resorbin (e.g., D n-6DPA or D n-3DPA , such as resorbin D, resorbin E, or T-series resorbin), and toll-like receptor (TLR) regulators such as oxidized low-density lipoprotein (e.g., OXPAC, PGPC) and erythran lipids (e.g., E5564), etc., the Lp-αPTHF composition according to item

[58] or

[59] ;

[61] The Lp-αPTHF composition according to any one of items

[58] to

[60] , wherein the immunostimulant and the detectable marker are the same;

[62] The Lp-αPTHF composition according to any one of items

[58] to

[61] , further comprising a hapten;

[63] The Lp-αPTHF composition according to item

[62] , wherein the hapten comprises one or more of fluorescein or beta-1,6-glucan;

[64] The Lp-αPTHF composition according to any one of

[13] to

[63] , further comprising at least one cryoprotective substance selected from the group consisting of mannitol; trehalose; sorbitol; and sucrose in the internal space, the outer space, or both internal spaces; 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 one of items [1] to

[64] ;

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

[49] ;

[67] The Lp-αPTHF composition according to any one of items

[13] to

[66] , further comprising carboplatin and / or pembrolizumab;

[68] A pharmaceutical composition comprising the liposomal alpha-polyglutamylated tetrahydrofolic acid composition according to any one of items

[13] to

[67] ;

[69] A pharmaceutical composition comprising alpha-polyglutamylated tetrahydrofolic acid according to any one of items [1] to [8];

[70] The composition according to any one of items [1] to

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

[71] Use of the composition according to any one of items [1] to

[70] in the manufacture of a drug for the treatment of a disease;

[72] For the treatment of a disease and / or for enhancing the effect of one or more therapeutic agents, for use in combination therapy with one or more therapeutic agents such as chemotherapeutic drugs (e.g., 5-fluorouracil), or for use in the manufacture of a drug for use as a "chemoprotective agent" to reduce the toxic side effects associated with the therapeutic agent(s) (e.g., in combination with folic acid antagonists such as methotrexate) of the composition according to any one of [1] to

[70] ;

[73] A method for treating or preventing a disease in a subject in need thereof, comprising administering to the subject a composition according to any one of [1] to

[69] ;

[74] A method for treating or preventing a disease in a subject in need thereof, comprising administering to the subject a liposomal polyglutamylated alpha-tetrahydrofolic acid composition according to any one of

[13] to

[69] ;

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

[69] ;

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

[13] to

[69] ;

[77] The method according to item

[75] or

[76] , wherein the proliferating cells are cancer cells, mammalian cells, and / or human cells;

[78] A method for treating cancer, comprising administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having cancer or at risk of having cancer;

[79] A method for treating cancer, comprising administering an effective amount of the liposomal polyglutamylated alpha-tetrahydrofolic acid composition according to any one of items

[13] to

[68] to a subject having cancer or at risk of having cancer;

[80] A method for treating or preventing cancer, wherein the cancer is a non-hematological tumor including, 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; and, for example, a hematological tumor such as leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias, the method according to item

[78] or

[79] , which is selected from the group consisting of;

[81] A method for treating or preventing cancer, wherein the cancer is a member selected from the group consisting of lung cancer, breast cancer, colon cancer, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, ovarian cancer, and cervical cancer, the method according to item

[78] or

[79] ;

[82] A method for treating or preventing cancer, wherein the cancer is a member selected from the group consisting of colorectal cancer, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer, the method according to item

[78] or

[79] ;

[83] A method for treating or preventing cancer, wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma, the method according to item

[78] or

[79] ;

[84] A method for treating cancer, comprising administering an effective amount of the Lp-αPTHF composition according to any one of items

[50] to

[66] to a subject having or at risk of having cancer cells expressing folate receptor to which the targeting moiety binds;

[85] A maintenance therapy for a subject who is receiving or has received cancer treatment, comprising administering an effective amount of the composition according to any one of items [1] to

[69] to a subject who is receiving or has received cancer treatment;

[86] Maintenance therapy for a subject who is receiving or has received cancer treatment, the maintenance therapy comprising administering an effective amount of the liposomal polyglutamate oxidized alpha-tetrahydrofolic acid composition according to any one of items

[13] to

[69] to a subject who is receiving or has received cancer treatment;

[87] A method for treating an immune system disorder, the method comprising administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having an immune system disorder, and optionally, the immune system disorder is selected from inflammation (e.g., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, systemic lupus erythematosus, and Takayasu's disease, and psoriasis;

[88] A method for treating an immune system disorder, the method comprising administering an effective amount of the liposomal polyglutamate oxidized alpha-tetrahydrofolic acid composition according to any one of items

[13] to

[69] to a subject having or at risk of having an immune system disorder, and optionally, the immune system disorder is selected from inflammation (e.g., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, systemic lupus erythematosus, and Takayasu's disease, and psoriasis;

[89] The following treatment methods: (a) A method for treating neutropenia, the method comprising administering an effective amount of the composition according to any one of items [1] to

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

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

[69] to a subject having or at risk of having an infectious disease, a cardiovascular disease, or another disease, and the disease is a member selected from 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 an effective amount of the composition according to any one of items [1] to

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

[69] to a subject having or at risk of having rheumatoid arthritis; (f) A method for treating an inflammatory condition, comprising administering an effective amount of the composition according to any one of items [1] to

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

[69] to a subject having or at risk of having a skin disease;

[90] A method for treating an infectious disease, comprising administering an effective amount of the liposomal alpha-polyglutamylated tetrahydrofolic acid composition according to any one of items

[13] to

[69] to a subject having or at risk of having an infectious disease;

[91] A method for delivering polyglutamylated alpha-tetrahydrofolic acid to a tumor expressing a folate receptor on its surface, comprising administering to a subject having a tumor the Lp-αPTHF composition according to any one of items [1] to

[69] in an amount such that a therapeutically effective amount of alpha-polyglutamylated tetrahydrofolic acid is delivered to the tumor;

[92] A method for preparing an alpha-polyglutamylated tetrahydrofolic acid composition comprising the liposomal alpha-polyglutamylated tetrahydrofolic acid composition according to any one of items

[13] to

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

[93] A method for preparing alpha-polyglutamyl oxidized tetrahydrofolic acid comprising the liposomal alpha-polyglutamyl oxidized tetrahydrofolic acid composition according to any one of items

[13] to

[69] , comprising: forming a mixture comprising a liposomal component and alpha-polyglutamyl oxidized tetrahydrofolic acid in a solution; and treating the mixture to form liposomes containing alpha-polyglutamyl oxidized tetrahydrofolic acid.

[94] The method according to item

[93] , wherein treating the mixture comprises homogenizing the mixture in a solution to form liposomes.

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

[51] to

[70] , comprising: forming a mixture comprising a liposomal component and alpha-polyglutamyl oxidized tetrahydrofolic acid in a solution; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes in which alpha-polyglutamyl oxidized tetrahydrofolic acid is encapsulated and / or enclosed; and imparting a targeting moiety to the surface of the liposomes, wherein the targeting moiety has specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ).

[96] A method for preparing the composition according to any one of items

[51] to

[70] , comprising: forming a mixture comprising a liposomal component and alpha-polyglutamyl oxidized tetrahydrofolic acid in a solution; treating the mixture to form liposomes in which alpha-polyglutamyl oxidized tetrahydrofolic acid is encapsulated and / or enclosed; and imparting a targeting moiety to the surface of the liposomes, wherein the targeting moiety has specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ).

[97] The method according to item

[96] , wherein the treating step comprises homogenizing the mixture in a solution to form liposomes.

[98] The method according to item

[93] , wherein the processing step includes one or more steps of thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw method, reverse phase evaporation method, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, freeze-dried double emulsion, 3D printing, membrane contactor method, and stirring; and / or

[99] The method according to any one of items

[96] to

[98] , wherein the processing step includes one or more steps of changing the size of liposomes by one or more steps of extrusion, high pressure microfluidization, and / or ultrasonic treatment; and / or

[0100] The method according to any one of items

[92] to

[99] , wherein at least 1% of the starting material of alpha polyglutamine oxidized tetrahydrofolic acid is encapsulated or enclosed in liposomes.

[0128] II. Polyglutamine oxidized alpha tetrahydrofolic acid (αPTHF) The present disclosure generally relates to polyglutamylated alpha tetrahydrofolate (αPTHF) compositions. The αPTHF compositions have at least one glutamyl group having an alpha carboxyl group bond. These compositions are structurally different from L-gamma polyglutamylated tetrahydrofolate (produced by the enzyme holylpoly-gamma-). In some embodiments, the αPTHF composition contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, 2 to 6, or 6 or more glutamyl groups (including the glutamyl groups in tetrahydrofolate). In some embodiments, each of the glutamyl groups in αPTHF other than the glutamyl groups of tetrahydrofolate has an alpha bond. In some embodiments, each of the glutamyl groups in αPTHF other than the C-terminal glutamyl group and the glutamyl groups in tetrahydrofolate has an alpha bond. In some embodiments, each of the glutamyl groups in αPTHF other than the C-terminal glutamyl group has an alpha bond. In some embodiments, two or more of the glutamyl groups in αPTHF have a gamma bond. In some embodiments, at least one glutamyl group of alpha polyglutamylated tetrahydrofolate has both an alpha carboxyl group bond and a gamma carboxyl group bond. In some embodiments, the glutamyl groups in αPTHF are each of the L-type. In some embodiments, the glutamyl groups in αPTHF other than the glutamyl groups of tetrahydrofolate are of the D-type. In some embodiments, αPTHF contains two or more L-type glutamyl groups and one or more D-type glutamyl groups. In some embodiments, the polyglutamic acid chain of αPTHF is linear (not branched). In some embodiments, the polyglutamic acid chain of αPTHF is branched.

[0129] In some embodiments, alpha-polyglutamylated tetrahydrofolate is diglutamylated. That is, alpha-polyglutamylated tetrahydrofolate contains one additional glutamyl group in addition to the glutamyl group of tetrahydrofolate (αTHF-PG1), and the additional glutamyl group is linked to the glutamyl group in tetrahydrofolate via an alpha bond. In some embodiments, the glutamyl groups of alpha-diglutamylated tetrahydrofolate are each of the L-type. In other embodiments, alpha-diglutamylated THF contains a D-type glutamyl group.

[0130] In some embodiments, alpha-polyglutamylated tetrahydrofolate is triglutamylated. That is, alpha-polyglutamylated tetrahydrofolate contains two additional glutamyl groups in addition to the glutamyl group of tetrahydrofolate (αTHF-PG2). In some embodiments, each of the two glutamyl groups has an alpha bond. In other embodiments, one of the two additional glutamyl groups has an alpha bond and the other glutamyl group has a gamma bond. In some embodiments, one of the two additional glutamyl groups has an alpha bond. In some embodiments, one of the two additional glutamyl groups has a gamma bond. In some embodiments, two of the three glutamyl groups have an alpha bond. In other embodiments, one of the three glutamyl groups has an alpha bond and another glutamyl group has a gamma bond. In some embodiments, one glutamyl group has both an alpha bond and a gamma bond. In some embodiments, the alpha-triglutamylated tetrahydrofolate glutamyl groups are each of the L-type. In other embodiments, alpha-triglutamylated THF contains a D-type glutamyl group. In further embodiments, the glutamyl groups of alpha-triglutamylated tetrahydrofolate other than the glutamyl group of tetrahydrofolate are each of the D-type. In further embodiments, triglutamylated THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0131] In some embodiments, alpha-polyglutamyl oxidized tetrahydrofolate is tetraglutamylated and thus contains three additional glutamyl groups in addition to the glutamyl group in tetrahydrofolate (αTHF-PG3). In some embodiments, each of the three glutamyl groups has an alpha bond. In other embodiments, one or two of the three additional glutamyl groups have an alpha bond and the remaining two or one glutamyl groups each have a gamma bond. In some embodiments, two of the three additional glutamyl groups have an alpha bond. In other embodiments, one of the three glutamyl groups has an alpha bond and another additional glutamyl group has a gamma bond. In other embodiments, one of the three additional glutamyl groups has both an alpha bond and a gamma bond. In other embodiments, three of the four glutamyl groups have an alpha bond. In some embodiments, at least one glutamyl group has both an alpha bond and a gamma bond. In some embodiments, alpha-tetraglutamyl oxidized THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha-tetraglutamyl oxidized tetrahydrofolate are each of the L-type. In other embodiments, alpha-tetraglutamyl oxidized THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha-tetraglutamyl oxidized tetrahydrofolate other than the glutamyl group of tetrahydrofolate are each of the D-type. In a further embodiment, tetraglutamyl oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0132] In some embodiments, polyglutamyl oxidized alpha tetrahydrofolate is pentaglutamylated (αTHF-PG4) and contains a chain of 4 additional glutamyl groups attached to the glutamyl groups in tetrahydrofolate. In some embodiments, each of the 4 additional glutamyl groups in the chain has an alpha bond. In some embodiments, each of the 4 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In other embodiments, 1, 2, or 3 of the 4 additional glutamyl groups have an alpha bond and the remaining 3, 2, or 1 glutamyl groups are each attached to the glutamyl group of the molecule via a gamma bond. In other embodiments, 1 or 2 of the 4 additional glutamyl groups have an alpha bond and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least 1 additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least 1 of the 5 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 5 glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, alpha pentaglutamylated THF contains 2 or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha pentaglutamylated tetrahydrofolate are each L-type. In other embodiments, alpha pentaglutamylated THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha pentaglutamylated tetrahydrofolate other than the glutamyl groups of tetrahydrofolate are each D-type. In a further embodiment, pentaglutamylated THF contains a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0133] In some embodiments, alpha-polyglutamylated tetrahydrofolate is hexaglutamylated (αTHF-PG5) and contains a chain of 5 additional glutamyl groups attached to the glutamyl group in tetrahydrofolate. In some embodiments, each of the 5 additional glutamyl groups in the chain has an alpha bond. In some embodiments, each of the 5 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 4 of the 5 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, or 4 of the 5 additional glutamyl groups are attached to the glutamyl group of the molecule via an alpha bond, and the remaining 4, 3, 2, or 1 glutamyl groups are each attached to the glutamyl group of the molecule via a gamma bond. In other embodiments, 1, 2, 3, or 4 of the 5 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 6 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 6 glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 5 of the 6 glutamyl groups have an alpha bond. In some embodiments, alpha-hexaglutamylated THF contains 2 or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha-hexaglutamylated tetrahydrofolate are each of the L-type. In some embodiments, alpha-hexaglutamylated THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha-hexaglutamylated tetrahydrofolate other than the glutamyl groups of tetrahydrofolate are each of the D-type. In a further embodiment, hexaglutamylated THF contains a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0134] In some embodiments, alpha-polyglutamyl oxidized tetrahydrofolate is heptaglutamylated (αTHF-PG6) and thus contains a chain of six additional glutamyl groups attached to the glutamyl group of tetrahydrofolate. In some embodiments, each of the six additional glutamyl groups has an alpha bond. In some embodiments, each of the six additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, five of the six additional glutamyl groups in the chain have an alpha bond. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have an alpha bond and the remaining five, four, three, two, or one glutamyl group has a gamma bond, respectively. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have an alpha bond and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the seven glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the seven glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, six of the seven glutamyl groups have an alpha bond. In some embodiments, alpha-heptaglutamyl oxidized THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha-heptaglutamyl oxidized tetrahydrofolate are each of the L-type. In other embodiments, alpha-heptaglutamyl oxidized THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha-heptaglutamyl oxidized tetrahydrofolate other than the glutamyl groups of tetrahydrofolate are each of the D-type. In a further embodiment, heptaglutamyl oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0135] In some embodiments, alpha-polyglutamylated tetrahydrofolate is octaglutamylated (αTHF-PG7) and thus contains a chain of seven additional glutamyl groups attached to the glutamyl group of tetrahydrofolate. In some embodiments, each of the seven additional glutamyl groups in the chain, other than the C-terminal glutamyl group, has an alpha bond. In some embodiments, six of the seven additional glutamyl groups in the chain have an alpha bond. In some embodiments, each of the seven additional glutamyl groups has an alpha bond. In other embodiments, one, two, three, four, five, or six of the seven additional glutamyl groups have an alpha bond and the remaining six, five, four, three, two, or one glutamyl group has a gamma bond, respectively. In other embodiments, one, two, three, four, five, or six of the seven additional glutamyl groups have an alpha bond and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the eight glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the eight glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, seven of the eight glutamyl groups have an alpha bond. In some embodiments, alpha-octaglutamylated THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha-octaglutamylated tetrahydrofolate are each L-type. In other embodiments, alpha-octaglutamylated THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha-octaglutamylated folic acid tetrahydrofolate other than the glutamyl groups of tetrahydrofolate are each D-type. In a further embodiment, octaglutamylated THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0136] In some embodiments, alpha-polyglutamylated tetrahydrofolate is nonaglutamylated (αTHF-PG8) and contains a chain of eight additional glutamyl groups attached to the glutamyl group of tetrahydrofolate. In some embodiments, each of the eight additional glutamyl groups in the chain, other than the C-terminal glutamyl group, has an alpha bond. In some embodiments, seven of the eight additional glutamyl groups in the chain have an alpha bond. In some embodiments, each of the eight additional glutamyl groups has an alpha bond. In other embodiments, one, two, three, four, five, six, or seven of the eight additional glutamyl groups have an alpha bond, and the remaining seven, six, five, four, three, two, or one glutamyl groups each have a gamma bond. In other embodiments, one, two, three, four, five, six, or seven of the eight additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the nine glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the nine glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, eight of the nine glutamyl groups have an alpha bond. In some embodiments, alpha nonaglutamylated THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha nonaglutamylated tetrahydrofolate are each L-type. In other embodiments, alpha nonaglutamylated THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha nonaglutamylated tetrahydrofolate other than the glutamyl groups of tetrahydrofolate are each D-type. In a further embodiment, nonaglutamylated THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0137] In some embodiments, alpha-polyglutamyl oxidized tetrahydrofolate is decaglutamylated (αTHF-PG9) (i.e., it contains a chain of 9 additional glutamyl groups attached to the glutamyl group of tetrahydrofolate). In some embodiments, each of the 9 additional glutamyl groups has an alpha bond. In some embodiments, each of the 9 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 8 of the 9 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 of the 9 additional glutamyl groups have an alpha bond, and the remaining 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 of the 9 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 10 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 10 glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 9 of the 10 glutamyl groups have an alpha bond. In some embodiments, alpha-decaglutamylated THF contains 2 or more L-type glutamyl groups. In further embodiments, the glutamyl groups of alpha-decaglutamylated tetrahydrofolate are each of the L-type. In other embodiments, alpha-decaglutamylated THF contains a D-type glutamyl group. In further embodiments, the glutamyl groups of alpha-decaglutamylated tetrahydrofolate other than the glutamyl groups of tetrahydrofolate are each of the D-type. In further embodiments, decaglutamylated THF contains a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0138] In some embodiments, alpha-polyglytamylated tetrahydrofolate is undecaglutamylated (αTHF-PG 10)。In some embodiments, each of the 10 additional glutamyl groups has an alpha bond. In some embodiments, each of the 10 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 9 of the 10 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an alpha bond, and the remaining 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bound to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 11 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 11 glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 10 of the 11 glutamyl groups have an alpha bond. In some embodiments, alpha-undecaglutamyl oxidized THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha-undecaglutamyl oxidized tetrahydrofolate are each of the L-type. In other embodiments, alpha-undecaglutamyl oxidized THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha-undecaglutamyl oxidized tetrahydrofolate other than the glutamyl groups of tetrahydrofolate are each of the D-type. In a further embodiment, undecaglutamyl oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0139] In some embodiments, alpha-polyglutamyl oxidized tetrahydrofolate is dodecaglutamylated (αTHF-PG 11 ). In some embodiments, each of the 11 additional glutamyl groups has an alpha bond. In some embodiments, each of the 11 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 10 of the 11 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alpha bond, and the remaining 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bonded to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 12 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 12 glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 11 of the 12 glutamyl groups have an alpha bond. In some embodiments, alpha dodecaglutamylated THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha dodecaglutamylated tetrahydrofolate are each of the L-type. In other embodiments, alpha dodecaglutamylated THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha dodecaglutamylated tetrahydrofolate other than the glutamyl groups of tetrahydrofolate are each of the D-type. In a further embodiment, dodecaglutamylated THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0140] In some embodiments, alpha-polyglutamyl oxidized tetrahydrofolate is tris-decaglutamylated (αTHF-PG 12)。In some embodiments, each of the 12 additional glutamyl groups has an alpha bond. In some embodiments, each of the 12 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 11 of the 12 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alpha bond, and the remaining 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group has a gamma bond, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bound to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 13 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 13 glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 12 of the 13 glutamyl groups have an alpha bond. In some embodiments, alpha-triskadecakaglutamyl oxidized THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha-triskadecakaglutamyl oxidized tetrahydrofolic acid are each L-type. In other embodiments, alpha-triskadecakaglutamyl oxidized THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha-triskadecakaglutamyl oxidized tetrahydrofolic acid other than the glutamyl groups of tetrahydrofolic acid are each D-type. In a further embodiment, triskadecakaglutamyl oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0141] In some embodiments, alpha-polyglutamyl oxidized tetrahydrofolate is tetradeca-glutamylated (αTHF-PG 13)。In some embodiments, each of the 13 additional glutamyl groups has an alpha bond. In some embodiments, each of the 13 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 12 of the 13 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have an alpha bond, and the remaining 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bound to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 14 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 14 glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 13 of the 14 glutamyl groups have an alpha bond. In some embodiments, alpha-tetradecaglutamyl oxidized THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha-tetradecaglutamyl oxidized tetrahydrofolic acid are each of the L-type. In other embodiments, alpha-tetradecaglutamyl oxidized THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha-tetradecaglutamyl oxidized tetrahydrofolic acid other than the glutamyl groups of tetrahydrofolic acid are each of the D-type. In a further embodiment, tetradecaglutamyl oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0142] In some embodiments, alpha-polyglutamyl oxidized tetrahydrofolate is pentadeca-glutamylated (αTHF-PG 14)。In some embodiments, each of the 14 additional glutamyl groups has an alpha bond. In some embodiments, each of the 14 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 13 of the 14 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14 additional glutamyl groups have an alpha bond, and the remaining 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each has a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bonded to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 15 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 15 glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 14 of the 15 glutamyl groups have an alpha bond. In some embodiments, alpha-pentadeca glutamyl oxidized THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha-pentadeca glutamyl oxidized tetrahydrofolic acid are each of the L-type. In other embodiments, alpha-pentadeca glutamyl oxidized THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha-pentadeca glutamyl oxidized tetrahydrofolic acid other than the glutamyl groups of tetrahydrofolic acid are each of the D-type. In a further embodiment, pentadeca glutamyl oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0143] In some embodiments, the alpha-polyglutamylated tetrahydrofolate is hexadeca-glutamylated (αTHF-PG 15)。In some embodiments, each of the 15 additional glutamyl groups has an alpha bond. In some embodiments, each of the 15 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 14 of the 15 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15 additional glutamyl groups have an alpha bond, and the remaining 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bound to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 16 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 16 glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 15 of the 16 glutamyl groups have an alpha bond. In some embodiments, alpha-hexadeca glutamyl oxidized THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha-hexadeca glutamyl oxidized tetrahydrofolic acid are each of the L-type. In other embodiments, alpha-hexadeca glutamyl oxidized THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha-hexadeca glutamyl oxidized tetrahydrofolic acid other than the glutamyl groups of tetrahydrofolic acid are each of the D-type. In a further embodiment, hexadeca glutamyl oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0144] In some embodiments, alpha-polyglutamylated tetrahydrofolate is heptadeca-glutamylated (αTHF-PG 16)。In some embodiments, each of the 16 additional glutamyl groups has an alpha bond. In some embodiments, each of the 16 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 15 of the 16 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the 16 additional glutamyl groups have an alpha bond, and the remaining 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the 16 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bound to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 17 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 17 glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 16 of the 17 glutamyl groups have an alpha bond. In some embodiments, alpha-heptadeca glutamyl oxidized THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha-heptadeca glutamyl oxidized tetrahydrofolic acid are each of the L-type. In other embodiments, alpha-heptadeca glutamyl oxidized THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha-heptadeca glutamyl oxidized tetrahydrofolic acid other than the glutamyl groups of tetrahydrofolic acid are each of the D-type. In a further embodiment, heptadeca glutamyl oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0145] In some embodiments, alpha-polyglutamyl oxidized tetrahydrofolate is octadeca-glutamylated (αTHF-PG 17)。In some embodiments, each of the 17 additional glutamyl groups has an alpha bond. In some embodiments, each of the 17 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 16 of the 17 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the 17 additional glutamyl groups have an alpha bond, and the remaining 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the 17 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bound to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 18 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 18 glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 17 of the 18 glutamyl groups have an alpha bond. In some embodiments, alpha-octadeca-glutamyl oxidized THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha-octadeca-glutamyl oxidized tetrahydrofolic acid are each of the L-type. In other embodiments, alpha-octadeca-glutamyl oxidized THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha-octadeca-glutamyl oxidized tetrahydrofolic acid other than the glutamyl groups of tetrahydrofolic acid are each of the D-type. In a further embodiment, octadeca-glutamyl oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0146] In some embodiments, alpha-polyglutamyl oxidized tetrahydrofolate is enniadecakaglutamylated (αTHF-PG 18)。In some embodiments, each of the 18 additional glutamyl groups has an alpha bond. In some embodiments, each of the 18 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 17 of the 18 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have an alpha bond, and the remaining 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bound to the glutamyl groups of the molecule via gamma bonds. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 19 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 19 glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 18 of the 19 glutamyl groups have an alpha bond. In some embodiments, alpha-enniadecakisglutamyl oxidized THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alpha-enniadecakisglutamyl oxidized tetrahydrofolic acid are each of the L-type. In other embodiments, alpha-enniadecakisglutamyl oxidized THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alpha-enniadecakisglutamyl oxidized tetrahydrofolic acid other than the glutamyl groups of tetrahydrofolic acid are each of the D-type. In a further embodiment, enniadecakisglutamyl oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear.In other embodiments, the polyglutamic acid chain is a branched chain.

[0147] In some embodiments, alpha-polyglutamylated tetrahydrofolate is eicosaglutamylated (αTHF-PG). 19)。In some embodiments, each of the 19 additional glutamyl groups has an alpha bond. In some embodiments, each of the 19 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 18 of the 19 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups have an alpha bond, and the remaining 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bound to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 20 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 20 glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 19 of the 20 glutamyl groups have an alpha bond. In some embodiments, alphai cosiglutamine oxidized THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alphai cosiglutamine oxidized tetrahydrofolic acid are each L-type. In other embodiments, alphai cosiglutamine oxidized THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alphai cosiglutamine oxidized tetrahydrofolic acid other than the glutamyl groups of tetrahydrofolic acid are each D-type. In a further embodiment, icosiglutamine oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain is linear.In other embodiments, the polyglutamic acid chain is a branched chain.

[0148] In some embodiments, alpha-polyglutamylated tetrahydrofolate is eicosakifolylglutamylated (αTHF-PG). 20)。In some embodiments, each of the 20 additional glutamyl groups has an alpha bond. In some embodiments, each of the 20 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 19 of the 20 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have an alpha bond, and the remaining 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bound to the glutamyl groups of the molecule via gamma bonds. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 21 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 21 glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 20 of the 21 glutamyl groups have an alpha bond. In some embodiments, alphaiocosikahenaglutamine oxidized THF contains two or more L-type glutamyl groups. In a further embodiment, the glutamyl groups of alphaiocosikahenaglutamine oxidized tetrahydrofolic acid are each of the L-type. In other embodiments, alphaiocosikahenaglutamine oxidized THF contains a D-type glutamyl group. In a further embodiment, the glutamyl groups of alphaiocosikahenaglutamine oxidized tetrahydrofolic acid other than the glutamyl groups of tetrahydrofolic acid are each of the D-type. In a further embodiment, icosikahenaglutamine oxidized THF contains a D-type glutamyl group and two or more L-type glutamyl groups.In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0149] In some embodiments, alpha-polyglutamylated tetrahydrofolate contains 4 to 7 glutamyl groups attached to tetrahydrofolate (i.e., αTHF-PGn, n = 4 to 7), and each of the 4 to 7 attached glutamyl groups has an alpha bond. In some embodiments, alpha-polyglutamylated tetrahydrofolate contains 4 to 7 glutamyl groups attached to tetrahydrofolate (i.e., αTHF-PGn, n = 4 to 7), and each of the 4 to 7 attached glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, each of the 4 to 7 attached glutamyl groups is of the L-form. In other embodiments, each of the 4 to 7 attached glutamyl groups is of the D-form. In other embodiments, the 4 to 7 attached glutamyl groups are of the L-form and D-form. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0150] In one embodiment, alpha-polyglutamylated tetrahydrofolate is tetraglutamylated, and each of the 3 glutamyl groups in the polyglutamic acid chain attached to tetrahydrofolate contains an alpha bond. In one embodiment, alpha-polyglutamylated tetrahydrofolate is tetraglutamylated, and each of the 3 glutamyl groups in the polyglutamic acid chain attached to tetrahydrofolate other than the C-terminal glutamyl group contains an alpha bond. In some embodiments, each of the 4 glutamyl groups is of the L-form. In some embodiments, each of the glutamyl groups in alpha-tetraglutamylated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is of the D-form. In other embodiments, at least 2 of the glutamyl groups in alpha-tetraglutamylated tetrahydrofolate are of the L-form and at least 1 of the glutamyl groups is of the D-form. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0151] In one embodiment, alpha-pentaglutamyl oxidized tetrahydrofolic acid is pentaglutamylated, and each of the four glutamyl groups in the polyglutamic acid chain bound to tetrahydrofolic acid contains an alpha bond. In one embodiment, alpha-pentaglutamyl oxidized tetrahydrofolic acid is pentaglutamylated, and each of the four glutamyl groups in the polyglutamic acid chain bound to tetrahydrofolic acid other than the C-terminal glutamyl group contains an alpha bond. In some embodiments, each of the four glutamyl groups is of the L-type. In some embodiments, the glutamyl groups in alpha-pentaglutamyl oxidized tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid are each of the D-type. In other embodiments, at least two of the glutamyl groups in alpha-pentaglutamyl oxidized tetrahydrofolic acid are of the L-type and at least one of the glutamyl groups is of the D-type. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0152] In one embodiment, alpha-hexaglutamyl oxidized tetrahydrofolic acid is hexaglutamylated, and each of the five glutamyl groups in the polyglutamic acid chain bound to tetrahydrofolic acid contains an alpha bond. In one embodiment, alpha-hexaglutamyl oxidized tetrahydrofolic acid is hexaglutamylated, and each of the five glutamyl groups in the polyglutamic acid chain bound to tetrahydrofolic acid other than the C-terminal glutamyl group contains an alpha bond. In some embodiments, each of the five glutamyl groups is of the L-type. In some embodiments, the glutamyl groups in alpha-hexaglutamyl oxidized tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid are each of the D-type. In other embodiments, at least two of the glutamyl groups in alpha-hexaglutamyl oxidized tetrahydrofolic acid are of the L-type and at least one of the glutamyl groups is of the D-type. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0153] In another embodiment, alpha-polyglutamyl oxidized tetrahydrofolate is heptaglutamylated, and each of the six glutamyl groups in the polyglutamic acid chain bound to tetrahydrofolate contains an alpha bond. In another embodiment, alpha-polyglutamyl oxidized tetrahydrofolate is heptaglutamylated, and each of the six glutamyl groups in the polyglutamic acid chain bound to tetrahydrofolate other than the C-terminal glutamyl group contains an alpha bond. In some embodiments, each of the six glutamyl groups is of the L-type. In some embodiments, the glutamyl groups in alpha-heptaglutamylated tetrahydrofolate other than the glutamyl group of tetrahydrofolate are each of the D-type. In other embodiments, at least two of the glutamyl groups in alpha-heptaglutamylated tetrahydrofolate are of the L-type and at least one of the glutamyl groups is of the D-type. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0154] In some embodiments, alpha-polyglutamyl oxidized tetrahydrofolate (αPTHF) contains a total of 1 to 15, 1 to 10, 2 to 15, 2 to 10, 3 to 15, 3 to 10, 3 to 6, 3 to 5, 4 to 10, 4 to 7, or 4 to 6, or any range therebetween of glutamyl groups, including the glutamyl group of tetrahydrofolate. In some embodiments, each glutamyl group in αPTHF other than the glutamyl group of tetrahydrofolate has an alpha bond. In some embodiments, each glutamyl group in αPTHF other than the C-terminal glutamyl group and the glutamyl group of tetrahydrofolate has an alpha bond. In some embodiments, each glutamyl group in αPTHF other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPTHF have an alpha bond. In some embodiments, αPTHF contains L-type and D-type glutamyl groups. In a further embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPTHF have an alpha bond and 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 of the glutamyl groups each have a gamma bond. In some embodiments, the glutamyl groups in the polyglutamic acid structure of polyglutamyl oxidized tetrahydrofolate are each of the L-type. In some embodiments, the glutamyl groups in αPTHF other than the glutamyl group of tetrahydrofolate are each of the D-type. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the glutamyl groups in αPTHF are of the L-type. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the glutamyl groups in αPTHF are of the D-type. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0155] In some embodiments, alpha-polyglutamyl oxidized tetrahydrofolate (αPTHF) contains a total of 2 to 20, 2 to 15, 2 to 10, 2 to 5, or any range therebetween of glutamyl groups, including the glutamyl group of tetrahydrofolate. In some embodiments, each glutamyl group in αPTHF other than the glutamyl group of tetrahydrofolate has an alpha bond. In some embodiments, each glutamyl group in αPTHF other than the C-terminal glutamyl group and the glutamyl group of tetrahydrofolate has an alpha bond. In some embodiments, each glutamyl group in αPTHF other than the C-terminal glutamyl group has an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups have an alpha bond. In some embodiments, αPTHF contains two or more glutamyl groups having a gamma bond. In a further embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups in αPTHF other than the glutamyl group of tetrahydrofolate have an alpha bond, and 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 glutamyl groups each have a gamma bond. In some embodiments, the glutamyl groups in αPTHF are each of the L-type. In some embodiments, the glutamyl groups in αPTHF other than the glutamyl group of tetrahydrofolate are each of the D-type. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the glutamyl groups in αPTHF are of the L-type. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the glutamyl groups in αPTHF are of the D-type.

[0156] In some embodiments, alpha-polyglutamylated tetrahydrofolate contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in addition to the glutamyl group of tetrahydrofolate. In further embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additional glutamyl groups have an alpha bond. In further embodiments, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the glutamyl groups in alpha-polyglutamylated tetrahydrofolate have a gamma bond. In some embodiments, at least one glutamyl group has both an alpha bond and a gamma bond. In some embodiments, the glutamyl group in tetrahydrofolate has an alpha bond. In some embodiments, the glutamyl group in tetrahydrofolate has both an alpha bond and a gamma bond.

[0157] In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen of the total glutamyl groups in alpha-polyglutamyl oxidized tetrahydrofolate are of the L-form, D-form, or a combination of L-form and D-form. In some embodiments, the glutamyl groups of polyglutamyl oxidized alpha-tetrahydrofolate are each of the L-form. In other embodiments, the glutamyl groups of polyglutamyl oxidized alpha-tetrahydrofolate other than the glutamyl groups of tetrahydrofolate are each of the D-form. In alternative embodiments, at least two of the glutamyl groups of polyglutamyl oxidized alpha-tetrahydrofolate are of the L-form, and at least one of the glutamyl groups in polyglutamyl oxidized alpha-tetrahydrofolate is of the D-form. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen of the glutamyl groups in polyglutamyl oxidized alpha-tetrahydrofolate are of the L-form. In other embodiments, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen of the glutamyl groups in polyglutamyl oxidized alpha-tetrahydrofolate are of the D-form. In some embodiments, at least one glutamyl group has both an alpha bond and a gamma bond.

[0158] In further embodiments, polyglutamyl oxidized alpha-tetrahydrofolate contains 20 to 100, 20 to 75, 20 to 50, 20 to 40, 20 to 30, 20 to 25, or 101 or more glutamyl groups, or any range therebetween. In some embodiments, the glutamyl groups of αPTHF are each of the L-form. In other embodiments, the glutamyl groups of αPTHF other than the glutamyl groups of tetrahydrofolate are each of the D-form. In alternative embodiments, at least two of the glutamyl groups in αPTHF are of the L-form, and at least one of the glutamyl groups in αPTHF is of the D-form. In some embodiments, at least one glutamyl group has both an alpha bond and a gamma bond.

[0159] In a further embodiment, the provided composition comprises αPTHF having 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 glutamyl groups having an alpha bond. In some embodiments, αPTHF comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 L-type glutamyl groups. In some embodiments, αPTHF comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 D-type glutamyl groups. In some embodiments, αPTHF comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 L-type glutamyl groups and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 D-type glutamyl groups. In other embodiments, polyglutamylated alpha tetrahydrofolate comprises at least 1 glutamyl group having both an alpha bond and a gamma bond. In some embodiments, polyglutamylated alpha tetrahydrofolate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 11 or more glutamyl groups having both an alpha bond and a gamma bond.

[0160] In some embodiments, alpha-polyglutamylated tetrahydrofolate contains at least 1 glutamyl group having an alpha bond and 2, 3, 4, 5, 6, 7, 8, 9, 1-10, 1-20, or more glutamyl groups having a gamma bond. For example, in some embodiments, polyglutamylated alpha-tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10 L-alpha-glutamyl group bonds and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 L-gamma-glutamyl group bonds. In some further embodiments, polyglutamylated alpha-tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 L-alpha-glutamyl group bonds and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma-glutamyl group bonds. In additional further embodiments, polyglutamylated alpha-tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-alpha-glutamyl group bonds and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma-glutamyl group bonds. In other further embodiments, polyglutamylated alpha-tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma-glutamyl group bonds and further contains 1, 2, 3, 4, 5, 6, or 1-10 L-gamma-glutamyl group bonds. In other embodiments, polyglutamylated alpha-tetrahydrofolate contains at least 1 glutamyl group having both an alpha bond and a gamma bond. In some embodiments, polyglutamylated alpha-tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 11 or more glutamyl groups having both an alpha bond and a gamma bond.

[0161] In some embodiments, the αPTHF provided herein can have one or more additional glutamyl groups added thereto, i.e., the composition can serve as a substrate for FPGS (folylpolyglutamate synthetase). Reagents and assays for measuring the ability of αPTHF to act as a substrate for FPGS (e.g., human FPGS, or rat liver FPGS) are readily available and can be routinely performed.

[0162] In some embodiments, the naked alpha PTHF composition disclosed herein (e.g., alpha PTHF not bound to a delivery vehicle) is taken up by liver cells at a significantly slower rate compared to the uptake rate of tetrahydrofolic acid under the same physiological conditions. In some embodiments, the liver cell uptake rate of the naked alpha PTHF 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 alpha PTHF composition disclosed herein from liver cells occurs at a rate significantly slower (less than 30%, 20%, 15%, or 10%) than that of tetrahydrofolic acid compared to the rate of tetrahydrofolic acid.

[0163] In some embodiments, the alpha polyglutamylated tetrahydrofolic acid composition provided herein has higher cytotoxicity against proliferating cells than tetrahydrofolic acid. In some embodiments, the proliferating cells are cancer cells. In some embodiments, the proliferating cells are colorectal cancer cells, colon cancer cells, breast cancer cells, or ovarian cancer cells. In some embodiments, the cancer cells are mesothelioma cells or non-small cell lung cancer cells. In some embodiments, the cytotoxicity is measured in an in vitro assay. In some embodiments, the alpha polyglutamylated tetrahydrofolic acid is hexaglutamylated tetrahydrofolic acid.

[0164] In some embodiments, the alpha-polyglutamylated tetrahydrofolate compositions provided herein have lower toxic side effects than tetrahydrofolate. In some embodiments, the alpha-polyglutamylated tetrahydrofolate compositions provided herein are less toxic to non-proliferating cells than tetrahydrofolate. In some embodiments, the alpha-polyglutamylated tetrahydrofolate compositions provided herein are less toxic to neutrophils, liver cells, or colon epithelial cells than tetrahydrofolate. In some embodiments, the neutrophils are human neutrophils, differentiated human neutrophils, or neutrophils differentiated from CD34+ cells. In some embodiments, the liver cells are AML12 liver cells. In some embodiments, the colon epithelial cells are CCD841 colon epithelial cells. In some embodiments, toxicity is measured in an in vitro assay. In some embodiments, the alpha-polyglutamylated tetrahydrofolate is hexaglutamylated tetrahydrofolate.

[0165] In some embodiments, the alpha-polyglutamylated tetrahydrofolate compositions provided herein have lower toxic side effects than tetrahydrofolate. In some embodiments, the alpha-polyglutamylated tetrahydrofolate compositions provided herein have fewer or less severe toxic side effects than tetrahydrofolate in an in vivo assay. In some embodiments, the in vivo assay is performed in an in vivo mouse model. In some embodiments, the alpha-polyglutamylated tetrahydrofolate compositions provided herein have fewer or less severe hematological or liver toxic side effects than tetrahydrofolate. In some embodiments, hematological side effects are evaluated by mean neutrophils, mean white blood cells, or mean platelet count. In some embodiments, liver toxic side effects are evaluated by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the in vivo assay comprises administering the alpha-polyglutamylated tetrahydrofolate composition at 40 mg / kg or 80 mg / kg once a week for 4 weeks. In some embodiments, the alpha-polyglutamylated tetrahydrofolate is hexaglutamylated tetrahydrofolate.

[0166] In some embodiments, treatment with the alpha-polyglutamylated tetrahydrofolic acid compositions provided herein does not induce significant hematological or liver toxicity side effects in an in vivo mouse model. In some embodiments, hematological side effects are evaluated by mean neutrophils, mean white blood cells, or mean platelet counts. In some embodiments, liver toxicity side effects are evaluated by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the alpha-polyglutamylated tetrahydrofolic acid compositions provided herein do not significantly decrease mean neutrophils, mean white blood cells, or mean platelet counts. In some embodiments, the alpha-polyglutamylated tetrahydrofolic acid compositions provided herein do not significantly increase serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. In some embodiments, the alpha-polyglutamylated tetrahydrofolic acid compositions provided herein do not significantly decrease serum albumin levels. In some embodiments, the in vivo assay comprises administering the polyglutamylated alpha-tetrahydrofolic acid composition at 40 mg / kg or 80 mg / kg once a week for 4 weeks. In some embodiments, the alpha-polyglutamylated tetrahydrofolic acid is hexaglutamylated tetrahydrofolic acid.

[0167] In some embodiments, the αPTHF composition does not contain a fluorine atom. In some embodiments, the αPTHF composition does not contain a 4-fluoroglutamyl group.

[0168] Regarding the polyglutamylated alpha-tetrahydrofolic acid (αPTHF) composition and its use, it is further disclosed in U.S. Patent Application Nos. 62 / 374,458, 62 / 583,432, 62 / 627,741, 62 / 630,820, 62 / 630,821, 62 / 630,824, 62 / 630,825, 15 / 675,695, and 15 / 675,701; International Application Nos. PCT / US2017 / 046666 and PCT / US2017 / 046667; the content of each of the above applications is hereby incorporated by reference in its entirety.

[0169] A. Polyglutamylated Tetrahydrofolic Acid Analogs and Derivatives The present disclosure also encompasses αPTHF derivatives and analogs. The compositions and methods disclosed herein are believed to be applicable to any and all known derivatives or analogs of oxidized tetrahydrofolic acid. In some embodiments, the polyglutamylated tetrahydrofolic acid analog or derivative compositions prepared and used in accordance with the compositions and methods of the present disclosure are those illustrated in FIGS. 1I - 1J. In some embodiments, the analog corresponds to a modified form of tetrahydrofolic acid in which the glutamyl group of tetrahydrofolic acid is not bound to the remainder of the tetrahydrofolic acid molecule via a gamma peptide bond. In some embodiments, the analog is a variant of tetrahydrofolic acid in which the glutamyl group of tetrahydrofolic acid is of the D type. In some embodiments, the polyglutamylated form of tetrahydrofolic acid or the polyglutamylated tetrahydrofolic acid analog or derivative is not fluorinated.

[0170] In further embodiments, the polyglutamylated alpha-tetrahydrofolic acid derivative or analog has a variant polyglutamate chain. In some embodiments, the polyglutamate chain contains one or more natural or synthetic residues other than glutamate. In some embodiments, the polyglutamate chain contains one or more glutamyl groups that do not contain an amide bond. In other embodiments, one or more glutamyl groups of the polyglutamate chain are derivatized.

[0171] Synthesis of THF-PG The tetrahydrofolic acid polyglutamate compositions provided herein are obtained by the following synthetic methods using available reagents and synthetic intermediates. The addition of glutamyl residues to the glutamyl residues of tetrahydrofolic acid can be carried out using synthetic methods known in the art. In some embodiments, glutamyl residues are continuously added to the glutamyl residues of tetrahydrofolic acid. In further embodiments, "click chemistry" methods or other bioconjugate chemistries known to those skilled in the art are used to add polyglutamic acid to the glutamyl residues of tetrahydrofolic acid. Alternatively, a peptide of glutamyl residues of a desired length can be prepared and added to a precursor without a glutamyl residue of tetrahydrofolic acid. The peptide can be prepared using methods known in the art. In some embodiments, the first glutamyl residue is attached to the wang resin, and additional glutamyl residues are continuously added by solid-phase peptide synthesis using F-moc chemistry. After adding the last glutamyl residue, the tetrahydrofolic acid precursor is coupled with the peptide, and the molecule is cleaved from the resin.

[0172] The addition of glutamyl residues to the glutamyl residues of tetrahydrofolic acid can be achieved using synthetic methods known in the art. In some embodiments, glutamyl residues are continuously added to the glutamyl residues of tetrahydrofolic acid. In further embodiments, "click chemistry" methods and other bioconjugate chemistries known to those skilled in the art are used to add polyglutamic acid to the glutamyl residues of tetrahydrofolic acid. Alternatively, a peptide of glutamyl residues of a desired length can be prepared and added to a precursor of tetrahydrofolic acid without a glutamyl residue. The peptide can be prepared using synthetic methods known in the art. In some embodiments, the first glutamyl residue is attached to the wang resin, and additional glutamyl residues are continuously added by solid-phase peptide synthesis using F-moc chemistry. After adding the last glutamyl residue, the tetrahydrofolic acid precursor is coupled with the peptide, and the molecule is cleaved from the resin.

[0173] C. Tetrahydrofolic acid-PG complex The inventors have surprisingly discovered that polyglutamylated folic acid antimetabolites that share substantially the same structure and chemical characteristics as tetrahydrofolic acid (αPTHF) can form complexes with other compositions including therapeutic agents such as cytotoxic compounds including platinum-based compounds. Thus, in some embodiments, the present disclosure provides a complex of αPTHF (e.g., αPTHF disclosed herein) with a therapeutic agent or a salt or acid thereof.

[0174] In some embodiments, the αPTHF / complex comprises αPTHF and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound such as a chemotherapeutic agent. In further embodiments, the αPTHF / complex comprises a platinum-based drug such as a platinum-based chemotherapeutic agent (e.g., cisplatin, carboplatin, and oxaliplatin). In other embodiments, the αPTHF / complex comprises a taxane-based chemotherapeutic agent (e.g., paclitaxel and docetaxel). In other embodiments, the αPTHF / complex comprises cyclodextrin. In further embodiments, the αPTHF / complex is encapsulated in liposomes.

[0175] In some embodiments, the present disclosure provides a composition comprising a complex of αPTH and a therapeutic agent or a salt or acid thereof. In further embodiments, the αPTHF / therapeutic agent complex comprises one or more αPTHFs having 2 to 150, 2 to 100, 2 to 75, 2 to 50, 2 to 24, 2 to 30, 2 to 20, 2 to 19, 2 to 15, 2 to 10, or 2 to 5 glutamyl groups. In some embodiments, the αPTHF / therapeutic agent complex comprises one or more αPTHFs having 3 to 10, 3 to 9, 3 to 8, or 3 to 7, or any number in between, glutamyl groups. In other embodiments, the αPTHF / therapeutic agent complex comprises one or more αPTHFs having 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5, or any number in between, glutamyl groups. In certain embodiments, the complex comprises one or more αPTHFs having 3 to 10 glutamyl groups. In further embodiments, the αPTHF / therapeutic agent complex comprises one or more αPTHFs having 3 to 7 glutamyl groups. In another embodiment, the αPTHF / therapeutic agent complex comprises one or more αPTHFs having 5 glutamyl groups. In another embodiment, the αPTHF / therapeutic agent complex comprises one or more αPTHFs having 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 further embodiments, the molar ratio of αPTHF / therapeutic agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of αPTHF / 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 to 50):1, or >50:1. In other embodiments, the molar ratio of αPTHF / therapeutic agent in the complex is 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range in between.In some embodiments, the molar ratio of αPPTHF / therapeutic agent in the complex 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 αPTHF / therapeutic agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0176] In alternative embodiments, the αPTHF complex comprises αPTHF and cyclodextrin. In some embodiments, the molar ratio of αPTHF (e.g., αPTHF salt) / cyclodextrin in the complex ranges from 1 - 20:1, or any range therebetween. In some embodiments, the molar ratio of αPTHF / cyclodextrin in the complex ranges from 1 - 10:1, or any range therebetween. In further embodiments, the molar ratio of αPTHF / cyclodextrin in the complex ranges from 2 - 8:1, or any range therebetween. In some embodiments, the molar ratio of αPTHF / 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 αPTHF / cyclodextrin 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 αPTHF / cyclodextrin in the complex 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 αPTHF / cyclodextrin complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0177] In some embodiments, the present disclosure provides a composition comprising an αPTHF / 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 αPTHF / platinum-based chemotherapeutic agent complex comprises a cisplatin, carboplatin, oxaliplatin analog, or salts or acids thereof. In some embodiments, the molar ratio of αPTHF / platinum-based agent in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPTHF / platinum-based agent in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of αPTHF / platinum-based agent in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPTHF / 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 to 50):1, or >50:1. In other embodiments, the molar ratio of αPTHF / platinum-based agent in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of αPTHF / platinum-based agent in the complex 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 to 50), or 1:>50. In some embodiments, the αPTHF / platinum-based agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0178] In a further embodiment, the αPTHF / platinum-based chemotherapeutic agent complex comprises cisplatin, carboplatin, an oxaliplatin analog, or a salt or acid thereof. In some embodiments, the molar ratio of αPTHF / platinum-based analog in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPTHF / platinum-based analog in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of αPTHF / platinum-based agent in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPTHF / 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 to 50):1, or >50:1. In other embodiments, the molar ratio of αPTHF / platinum-based analog in the complex is from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of αPTHF / platinum-based analog in the complex 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 to 50), or 1:>50. In some embodiments, the αPTHF / platinum-based analog complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0179] In a further embodiment, the present disclosure provides a complex comprising αPTHF and cisplatin or a salt or acid thereof. In some embodiments, the molar ratio of αP...

Claims

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

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