Alpha polyglutamylated 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 technology]

[0001] The present disclosure generally relates to polyglutamated alpha-tetrahydrofolate compositions, including delivery vehicles such as liposomes containing the polyglutamated alpha-tetrahydrofolate compositions, and methods of making and using the compositions to treat diseases, including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria. The polyglutamated alpha-tetrahydrofolate compositions also find use in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effectiveness of the therapeutic agent(s), or as "chemopreventive agents" (e.g., in combination with antifolates such as methotrexate) to reduce toxic side effects associated with the therapeutic agent(s).

[0002] Folate is an essential cofactor mediating the transfer of one-carbon units involved in nucleotide biosynthesis and DNA repair, homocysteine ​​remethylation (Hcy), and methylation of DNA, proteins, and lipids. The only circulating form of folate in the blood is monoglutamate, which is the only form of folate transported across cell membranes, as is the monoglutamate form of tetrahydrofolate. Upon uptake into cells, intracellular tetrahydrofolate is polyglutamated by the enzyme folylpolygammaglutamate synthase (FPGS). Tetrahydrofolate polyglutamation by FPGS serves at least two primary therapeutic purposes: (1) it significantly increases the affinity of tetrahydrofolate for DHFR; and (2) it facilitates the accumulation of polyglutamated tetrahydrofolate, which, unlike tetrahydrofolate (monoglutamate), is not readily transported out of cells by cellular efflux pumps.

[0003] The polyglutamated alpha-tetrahydrofolate compositions provided offer a strategy to improve the therapeutic efficacy of tetrahydrofolate. Summary of the Invention

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

[0005] In some embodiments, the present disclosure provides: [1] A composition containing polyglutamylated alpha-tetrahydrofolic acid. [2] Polyglutamylated alpha-tetrahydrofolate (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-tetrahydrofolate contains 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups with alpha carboxyl group bonds. [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 polyglutamated alpha-tetrahydrofolic acid is pentaglutamated 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 polyglutamated alpha-tetrahydrofolic acid is hexaglutamated 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 having an alpha carboxyl group bond; (b) each glutamyl group other than the glutamyl group of tetrahydrofolic acid has an alpha carboxyl linkage; or (c) two or more glutamyl groups have gamma carboxyl linkages; 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-tetrahydrofolate are in the L-configuration; (b) each glutamyl group of the polyglutamylated alpha-tetrahydrofolate is in the L-configuration; (c) at least one of the glutamyl groups of the polyglutamylated alpha-tetrahydrofolate is in the D-form; (d) each of the glutamyl groups of the polyglutamylated alpha-tetrahydrofolate other than the glutamyl group of the tetrahydrofolate is in the D-form; or (e) at least two of the glutamyl groups of the polyglutamylated alpha-tetrahydrofolate are in the L-configuration and at least one of the glutamyl groups is in the D-configuration; 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) containing the polyglutamic acid-containing alpha-tetrahydrofolate according to any one of items [1] to

[11] ;

[13] The LαPP composition according to item

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

[14] The Lp-αPTHF composition according to item

[12] or

[13] , wherein each glutamyl group of the polyglutamylated alpha-tetrahydrofolate is in the L-form;

[15] The Lp-αPTHF composition according to item

[12] or

[13] , wherein at least one of the glutamyl groups of the polyglutamylated alpha-tetrahydrofolate is in the D-form;

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

[12] to

[15] , wherein the liposome contains polyglutamylated alpha-tetrahydrofolate 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-tetrahydrofolate 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 alpha carboxyl group bonds and gamma carboxyl group bonds;

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

[12] to

[19] , wherein the liposome contains polyglutamated alpha-tetrahydrofolic acid, including tetraglutamated alpha-tetrahydrofolic acid, pentaglutamated alpha-tetrahydrofolic acid, or hexaglutamated alpha-tetrahydrofolic acid;

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

[12] to

[19] , wherein the liposome contains polyglutamated alpha-tetrahydrofolic acid, including tetraglutamated alpha-tetrahydrofolic acid, pentaglutamated alpha-tetrahydrofolic acid, or hexaglutamated 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% weight / weight (w / w) polyglutamylated alpha-tetrahydrofolic acid, or wherein in the process of preparing Lp-αPTHF, at least 1% of the starting material of polyglutamylated alpha-tetrahydrofolic acid is encapsulated in 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] The Lp-αPTHF composition according to any one of items

[12] to

[26] , wherein the liposome is formed from a liposome component;

[28] The Lp-αPTHF composition according to item

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

[29] The Lp-αPTHF composition according to item

[27] or

[28] , wherein the liposome component comprises 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 component comprises 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 comprise 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); phosphatidylpolyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymers; oligoglycerin, polyethylene glycol and polypropylene oxide-containing copolymers, poloxamer 188, and polyvinyl alcohol;

[33] The Lp-αPTHF composition according to item

[32] , wherein the steric stabilizer is PEG, and the 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 comprises an isotonicity agent such as dextrose, mannitol, glycerol, potassium chloride, or sodium chloride 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 comprises 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 inner space of the liposome contains 5% dextrose suspended in a HEPES buffer solution;

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

[39] to

[44] , wherein the pharmaceutically acceptable carrier comprises 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 comprises 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

[12] to

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

[48] ​​The 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 polyglutamated alpha-tetrahydrofolic acid;

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

[12] to

[48] , wherein the liposome contains 10 to 100,000 polyglutamylated alpha-tetrahydrofolate molecules or any range therebetween;

[50] The 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] The Lp-αPTHF composition according to item

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

[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 has a nucleotide sequence of 0.5 x 10 as measured using BIACORE® analysis. -10 ~10×10 -6 the Lp-αPTHF composition according to any one of items

[50] to

[53] , which binds to a surface antigen with an equilibrium dissociation constant (Kd) in the range of

[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 contains 1 to 1,000 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 immunostimulatory agent, a detectable marker, and a maleimide, wherein the immunostimulatory agent, the detectable marker, or the 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] Immunostimulants include fluorescein, fluorescein isothiocyanate (FITC), DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan; resolvins (e.g., D n-6DPA Or D n-3DPA the Lp-αPTHF composition according to item

[58] or

[59] , wherein the Lp-αPTHF composition is at least one selected from the group consisting of resolvin D, resolvin E, or T-series resolvins, and toll-like receptor (TLR) modulators, such as oxidized low-density lipoproteins (e.g., OXPAC, PGPC) and eritran lipids (e.g., E5564);

[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 items

[12] to

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

[65] A targeting 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

[12] to

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

[68] A pharmaceutical composition comprising the liposomal polyglutamated alpha-tetrahydrofolate composition according to any one of items

[12] to

[67] ;

[69] A pharmaceutical composition comprising the polyglutamic acid alpha-tetrahydrofolate according to any one of items [1] to [7];

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

[69] , for use in treating a disease;

[71] Use of a composition according to any of items [1] to

[70] in the manufacture of a drug for the treatment of a disease and / or for use in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effect of the therapeutic agent(s), or as a "chemopreventive agent" (e.g., in combination with an antifolate such as methotrexate) to reduce toxic side effects associated with the therapeutic agent(s);

[72] A method for 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, comprising administering to the subject the liposomal polyglutamated alpha-tetrahydrofolate composition according to any one of items

[12] to

[69] ;

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

[69] ;

[75] A method for killing hyperproliferative cells, comprising contacting the hyperproliferative cells with the liposomal polyglutamated 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, 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, comprising administering an effective amount of the liposomal polyglutamated alpha-tetrahydrofolate composition according to any one of items

[12] to

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

[79] The method according to item

[77] or

[78] , for treating or preventing cancer, wherein the cancer is selected from the group consisting of non-hematological tumors, 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, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors, such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias;

[80] The method according to item

[77] or

[78] , 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;

[81] The 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] The 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 described in any one of items

[50] to

[66] to a subject having or at risk of having cancer cells that express a folate receptor on their surface to which a targeting moiety binds;

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

[69] to the subject undergoing or having undergone cancer treatment;

[85] A maintenance therapy for a subject undergoing or having undergone cancer treatment, comprising administering to the subject undergoing or having undergone cancer treatment an effective amount of the liposomal polyglutamated alpha-tetrahydrofolate composition according to any one of items

[12] to

[69] ;

[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, wherein 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, Takayasu's disease, and psoriasis;

[87] A method for treating an immune system disorder, comprising administering an effective amount of the liposomal polyglutamated tetrahydrofolate composition according to any one of items [8] to

[69] to a subject having or at risk of having an immune system disorder, wherein 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, Takayasu's disease, and psoriasis;

[88] Treatment of: (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 or 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, wherein the inflammation is, optionally, 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 polyglutamated alpha-tetrahydrofolate 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 polyglutamated alpha-tetrahydrofolate to a tumor expressing a folate receptor on its surface, comprising administering to a subject having a tumor the Lp-αPTHF composition described in any of items [1] to

[69] in an amount sufficient to deliver a therapeutically effective amount of polyglutamated alpha-tetrahydrofolate to the tumor;

[91] A method for preparing a polyglutamated alpha-tetrahydrofolate composition comprising the liposomal polyglutamated alpha-tetrahydrofolate composition according to any one of items

[12] to

[69] , the method comprising: forming a mixture in a solution containing liposome components and a polyglutamated alpha-folate anti-metabolite; homogenizing the mixture in the solution to form liposomes; and treating the mixture to form liposomes containing polyglutamated alpha-tetrahydrofolate;

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

[12] to

[69] , comprising: forming a mixture in a solution containing liposome components and polyglutamated alpha-tetrahydrofolate; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes in which the polyglutamated alpha-tetrahydrofolate is entrapped and / or encapsulated; and providing a targeting moiety on the surface of the liposome, 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 processing step comprises 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

[94] The method of item

[92] , wherein the processing step comprises one or more steps of modifying the size of the liposomes by one or more of extrusion, high-pressure microfluidization, and / or sonication.

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

[0007] In one embodiment, the αPTHF composition comprises a chain of three glutamyl groups linked to the glutamyl group of tetrahydrofolate (i.e., tetraglutamated tetrahydrofolate). In some embodiments, the tetraglutamated THF comprises two or more glutamyl groups in the L-form. In other embodiments, the tetraglutamated THF comprises a glutamyl group in the D-form. In further embodiments, the tetraglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In still further embodiments, the tetraglutamated THF comprises two or more glutamyl groups with gamma linkages.

[0008] In one embodiment, the αPTHF composition comprises a chain of four glutamyl groups linked to a gamma-glutamyl group of tetrahydrofolate (e.g., α-pentaglutamated tetrahydrofolate). In some embodiments, the pentaglutamated alpha-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the pentaglutamated alpha-THF comprises a glutamyl group in the D-form. In further embodiments, the pentaglutamated alpha-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In still further embodiments, the pentaglutamated THF comprises two or more glutamyl groups with gamma bonds.

[0009] In one embodiment, the αPTHF composition comprises a chain of five glutamyl groups linked to a gamma-glutamyl group of tetrahydrofolate (e.g., α-hexaglutamated tetrahydrofolate). In some embodiments, the hexaglutamated alpha THF comprises two or more glutamyl groups in the L-form. In other embodiments, the hexaglutamated alpha THF comprises a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In still further embodiments, the hexaglutamated THF comprises two or more glutamyl groups with gamma linkages.

[0010] In further embodiments, the present disclosure provides compositions comprising delivery vehicles, such as liposomes, in which polyglutamated alpha-tetrahydrofolate is loaded (e.g., encapsulated) and / or otherwise associated, as well as methods of making and using αPTHF-loaded / associated delivery vehicle compositions (DV-αPTHF) for delivering polyglutamated alpha-tetrahydrofolate to diseased (e.g., cancerous) and / or target cells. These compositions have uses, including, but not limited to, treating (e.g., treating or preventing) diseases, including hyperproliferative diseases such as cancer, immune system disorders such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria. In some embodiments, the polyglutamated alpha-tetrahydrofolate in 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-loaded / 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 its monoglutamate state.

[0011] In some embodiments, the present disclosure provides for the use of a composition comprising a delivery vehicle, such as a liposome, in which polyglutamated alphatetrahydrofolate has been loaded (e.g., encapsulated) and / or otherwise associated, in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effect of the therapeutic agent(s), or as a "chemopreventive agent" (e.g., in combination with an antifolate, such as methotrexate) to reduce toxic side effects associated with the therapeutic agent(s). In some embodiments, the polyglutamated alphatetrahydrofolate in 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 those in tetrahydrofolate). The DV-αPTHF-loaded / 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 its monoglutamate state.

[0012] In a further embodiment, 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 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 with alpha linkages. In some embodiments, the composition comprises polyglutamylated alpha 5-formyl-THF having two or more glutamyl groups in the L-form. In other embodiments, the composition comprises polyglutamylated alpha 5-formyl-THF having glutamyl groups in the D-form. In further embodiments, the composition comprises polyglutamylated alpha 5-formyl-THF having glutamyl groups in the D-form and two or more glutamyl groups in the L-form. In further embodiments, the polyglutamylated alpha tetrahydrofolate in Lp-αPTHF comprises two or more glutamyl groups with alpha linkages. In further embodiments, the polyglutamylated alpha tetrahydrofolate in Lp-αPTHF comprises one or more glutamyl groups with both alpha and gamma linkages. In some embodiments, the polyglutamylated alpha tetrahydrofolate in Lp-αPTHF comprises 2 to 10 glutamyl groups with both alpha and gamma linkages, or any range therebetween. In some embodiments, the polyglutamic acid chain of the polyglutamic acid alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of the polyglutamic acid alpha 5-formyl-THF is branched.

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

[0015] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha 5-formyl-THF (i.e., pentaglutamylated 5-formyl-THF), which comprises a chain of four glutamyl groups linked to the glutamyl groups 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, the pentaglutamylated alpha 5-formyl-THF comprises one, two, three, or four glutamyl groups with alpha linkages. In some embodiments, the pentaglutamylated 5-formyl-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the pentaglutamylated 5-formyl-THF comprises a glutamyl group in the D-configuration. In further embodiments, the pentaglutamated 5-formyl-THF comprises a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of the pentaglutamated 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), which comprises a chain of five glutamyl groups linked to the glutamyl groups 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, the hexaglutamylated alpha 5-formyl-THF comprises one, two, three, four, or five glutamyl groups with alpha linkages. In some embodiments, the hexaglutamylated alpha 5-formyl-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the hexaglutamylated THF comprises glutamyl groups in the D-configuration. In further embodiments, the hexaglutamated alpha 5-formyl-THF comprises a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5-formyl-THF is branched.

[0017] In some embodiments, the present disclosure provides a composition comprising polyglutamated alpha 5,10-methenyl-THF. In some embodiments, the composition comprises polyglutamated alpha[6R]-5,10-methenyl-THF. In some embodiments, the composition comprises polyglutamated alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprises polyglutamated alpha[6S]-5,10-methenyl-THF. In some embodiments, the composition comprises polyglutamated 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 polyglutamated alpha 5,10-methenyl-THF comprises 1, 2, 3, or 4 or more alpha-linked glutamyl groups. In some embodiments, the composition comprises polyglutamylated alpha 5-formyl-THF having two or more glutamyl groups in the L-configuration. In other embodiments, the composition comprises polyglutamylated alpha 5,10-methenyl-THF having glutamyl groups in the D-configuration. In further embodiments, the composition comprises polyglutamylated alpha 5,10-methenyl-THF having glutamyl groups in the D-configuration and two or more glutamyl groups in the L-configuration. 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 compositions comprising polyglutamated alpha 5,10-methenyl-THF (i.e., tetraglutamated 5,10-methenyl-THF) comprising a chain of three glutamyl groups linked to the glutamyl groups of tetrahydrofolic acid. In some embodiments, the compositions comprise tetraglutamated alpha[6R]-5,10-methenyl-THF. In some embodiments, the compositions comprise tetraglutamated alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the compositions comprise tetraglutamated alpha[6S]-5,10-methenyl-THF. In some embodiments, the tetraglutamated alpha 5,10-methenyl-THF comprises one, two, or three glutamyl groups with alpha linkages. In some embodiments, the tetraglutamated alpha 5,10-methenyl-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the tetraglutamated alpha 5,10-methenyl-THF contains a glutamyl group in the D-form. In further embodiments, the tetraglutamated alpha 5,10-methenyl-THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the 5,10-methenyl-THF tetrahydrofolate is linear. In some embodiments, the polyglutamic acid chain of the tetraglutamated alpha 5,10-methenyl-THF is branched.

[0019] In some embodiments, the present disclosure provides compositions comprising polyglutamated alpha 5,10-methenyl-THF (i.e., pentaglutamated 5,10-methenyl-THF), which comprises a chain of four glutamyl groups linked to glutamyl groups of tetrahydrofolic acid. In some embodiments, the compositions comprise pentaglutamated alpha[6R]-5,10-methenyl-THF. In some embodiments, the compositions comprise pentaglutamated alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the compositions comprise pentaglutamated alpha[6S]-5,10-methenyl-THF. In some embodiments, the pentaglutamated alpha 5,10-methenyl-THF comprises one, two, three, or four glutamyl groups with alpha linkages. In some embodiments, the pentaglutamated alpha 5,10-methenyl-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the pentaglutamated alpha 5,10-methenyl-THF comprises a glutamyl group in the D-form. In further embodiments, the pentaglutamated alpha 5,10-methenyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha 5,10-methenyl-THF is linear. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha 5,10-methenyl-THF is branched.

[0020] In some embodiments, the present disclosure provides a composition comprising polyglutamated alpha 5,10-methenyl-THF (i.e., hexaglutamated 5,10-methenyl-THF), which comprises a chain of five glutamyl groups linked to the glutamyl groups of tetrahydrofolic acid. In some embodiments, the composition comprises hexaglutamated alpha[6R]-5,10-methenyl-THF. In some embodiments, the composition comprises hexaglutamated alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprises hexaglutamated alpha[6S]-5,10-methenyl-THF. In some embodiments, the hexaglutamated alpha 5,10-methenyl-THF comprises 1, 2, 3, 4, or 5 alpha-linked glutamyl groups. In some embodiments, the hexaglutamated alpha 5,10-methenyl-THF contains two or more glutamyl groups in the L-form. In other embodiments, the hexaglutamated alpha 5,10-methenyl-THF contains a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha 5,10-methenyl-THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5,10-methenyl-THF is linear. In some embodiments, the polyglutamic acid chain of the hexaglutamated 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, the polyglutamylated alpha 5-methyl-THF comprises 1, 2, 3, or 4 or more glutamyl groups with alpha linkages. In some embodiments, the composition comprises polyglutamylated alpha 5-methyl-THF having 2 or more glutamyl groups in the L-form. In other embodiments, the composition comprises polyglutamylated alpha 5-methyl-THF having glutamyl groups in the D-form. In further embodiments, the composition comprises polyglutamylated alpha 5-methyl-THF having glutamyl groups in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the polyglutamylated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamic acid chain of the polyglutamylated alpha 5-methyl-THF is branched.

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

[0023] In some embodiments, the present disclosure provides compositions comprising polyglutamylated alpha 5-methyl-THF (i.e., pentaglutamylated 5-methyl-THF), which comprises a chain of four glutamyl groups linked to the glutamyl groups of tetrahydrofolic acid. In some embodiments, the compositions comprise pentaglutamylated alpha[6S]-5-methyl-THF. In some embodiments, the compositions comprise pentaglutamylated alpha[6R,S]-5-methyl-THF. In some embodiments, the compositions comprise pentaglutamylated alpha[6R]-5-methyl-THF. In some embodiments, the pentaglutamylated alpha 5-methyl-THF comprises one, two, three, or four glutamyl groups with gamma linkages. In some embodiments, the pentaglutamylated alpha 5-methyl-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the pentaglutamylated alpha 5-methyl-THF comprises a glutamyl group in the D-configuration. In further embodiments, the pentaglutamated alpha 5-methyl-THF comprises a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha 5-methyl-THF is branched.

[0024] In some embodiments, the present disclosure provides compositions comprising polyglutamylated alpha 5-methyl-THF (i.e., hexaglutamylated 5-methyl-THF), which comprises a chain of five glutamyl groups linked to glutamyl groups of tetrahydrofolic acid. In some embodiments, the compositions comprise hexaglutamylated alpha[6S]-5-methyl-THF. In some embodiments, the compositions comprise hexaglutamylated alpha[6R,S]-5-methyl-THF. In some embodiments, the compositions comprise hexaglutamylated alpha[6R]-5-methyl-THF. In some embodiments, the hexaglutamylated alpha 5-methyl-THF comprises one, two, three, four, or five glutamyl groups with alpha linkages. In some embodiments, the hexaglutamylated alpha 5-methyl-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the hexaglutamylated alpha 5-methyl-THF comprises a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha 5-methyl-THF comprises a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5-methyl-THF is branched.

[0025] In some embodiments, the present disclosure provides a composition comprising polyglutamated alpha tetrahydrofolic acid THF. In some embodiments, the composition comprises polyglutamated alpha [6S]-tetrahydrofolic acid THF. In some embodiments, the composition comprises polyglutamated alpha [6R,S]-tetrahydrofolic acid THF. In some embodiments, the composition comprises polyglutamated alpha [6R]-tetrahydrofolic acid THF. In some embodiments, the composition comprises polyglutamated 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 alpha polyglutamated tetrahydrofolic acid-THF comprises 1, 2, 3, or 4 or more glutamyl groups in an alpha linkage. In some embodiments, the composition comprises polyglutamated alpha tetrahydrofolic acid THF having two or more glutamyl groups in the L-form. In other embodiments, the composition comprises a polyglutamic acid THF having a glutamic acid group in the D-form. In further embodiments, the composition comprises a polyglutamic acid THF having a glutamic acid group in the D-form and two or more glutamic acid groups in the L-form. In some embodiments, the polyglutamic acid chain of the polyglutamic acid THF is linear. In some embodiments, the polyglutamic acid chain of the polyglutamic acid THF is branched.

[0026] In some embodiments, the present disclosure provides compositions comprising polyglutamated alpha tetrahydrofolic acid THF (i.e., tetraglutamated tetrahydrofolic acid THF) comprising a chain of three glutamyl groups linked to the glutamyl groups of tetrahydrofolic acid THF. In some embodiments, the composition comprises tetraglutamated alpha[6S]tetrahydrofolic acid THF. In some embodiments, the composition comprises tetraglutamated alpha[6R,S]-tetrahydrofolic acid THF. In some embodiments, the composition comprises tetraglutamated alpha[6R]-tetrahydrofolic acid THF. In some embodiments, the tetraglutamated alpha tetrahydrofolic acid-THF comprises one, two, or three glutamyl groups with alpha linkages. In some embodiments, the tetraglutamated alpha tetrahydrofolic acid THF comprises two or more glutamyl groups in the L-form. In other embodiments, the tetraglutamated alpha tetrahydrofolic acid THF comprises a glutamyl group in the D-form. In further embodiments, the tetraglutamated alphatetrahydrofolic acid THF comprises a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamic acid chain of the tetraglutamated alphatetrahydrofolic acid THF is linear. In some embodiments, the polyglutamic acid chain of the tetraglutamated alphatetrahydrofolic acid THF is branched.

[0027] In some embodiments, the present disclosure provides compositions comprising polyglutamated alpha tetrahydrofolic acid THF (i.e., pentaglutamated tetrahydrofolic acid THF) comprising a chain of four glutamyl groups linked to the glutamyl groups of THF. In some embodiments, the compositions comprise pentaglutamated alpha[6S]tetrahydrofolic acid THF. In some embodiments, the compositions comprise pentaglutamated alpha[6R,S]-tetrahydrofolic acid THF. In some embodiments, the compositions comprise pentaglutamated alpha[6R]-tetrahydrofolic acid THF. In some embodiments, the pentaglutamated alpha tetrahydrofolic acid-THF comprises one, two, three, or four glutamyl groups with alpha linkages. In some embodiments, the pentaglutamated alpha THF comprises two or more glutamyl groups in the L-form. In other embodiments, the pentaglutamated alpha tetrahydrofolic acid THF comprises a glutamyl group in the D-form. In further embodiments, the pentaglutamated alpha tetrahydrofolic acid THF comprises a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha tetrahydrofolic acid THF is linear. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha THF is branched.

[0028] In some embodiments, the present disclosure provides compositions comprising polyglutamated alpha tetrahydrofolate THF (i.e., hexaglutamated tetrahydrofolate THF), which comprises a chain of five glutamyl groups linked to the glutamyl groups of tetrahydrofolate THF. In some embodiments, the compositions comprise hexaglutamated alpha[6S]tetrahydrofolate THF. In some embodiments, the compositions comprise hexaglutamated alpha[6R,S]-tetrahydrofolate THF. In some embodiments, the compositions comprise hexaglutamated alpha[6R]-tetrahydrofolate THF. In some embodiments, the hexaglutamated alpha tetrahydrofolate-THF comprises one, two, three, four, or five glutamyl groups with alpha linkages. In some embodiments, the hexaglutamated alpha tetrahydrofolate tetrahydrofolate THF comprises two or more glutamyl groups in the L-form. In other embodiments, the hexaglutamated tetrahydrofolic acid THF contains a D-glutamyl group. In further embodiments, the hexaglutamated alpha-tetrahydrofolic acid THF contains a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha-tetrahydrofolic acid THF is linear. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha-tetrahydrofolic acid 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, the polyglutamylated alpha 5,10-methylene-THF comprises 1, 2, 3, or 4 or more glutamyl groups with alpha linkages. In some embodiments, the composition comprises polyglutamylated alpha 5,10-methylene-THF having two or more glutamyl groups in the L-form. In other embodiments, the composition comprises polyglutamylated alpha 5,10-methylene-THF having glutamyl groups in the D-form. In further embodiments, the composition comprises polyglutamylated alpha 5,10-methylene-THF having glutamyl groups in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the polyglutamylated alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of the polyglutamylated alpha 5,10-methylene-THF is branched.

[0030] In some embodiments, the present disclosure provides compositions comprising polyglutamylated alpha 5,10-methylene-THF (i.e., tetraglutamylated 5,10-methylene-THF), which comprises a chain of three glutamyl groups linked to the glutamyl groups of tetrahydrofolic acid. In some embodiments, the compositions comprise tetraglutamylated alpha[6R]-5,10-methylene-THF. In some embodiments, the compositions comprise tetraglutamylated alpha[6R,S]-5,10-methylene-THF. In some embodiments, the compositions comprise tetraglutamylated alpha[6S]-5,10-methylene-THF. In some embodiments, the tetraglutamylated alpha 5,10-methylene-THF comprises one, two, or three glutamyl groups with alpha linkages. In some embodiments, the tetraglutamylated alpha 5,10-methylene-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the tetraglutamylated alpha 5,10-methylene-THF contains a glutamyl group in the D-form. In further embodiments, the tetraglutamylated alpha 5,10-methylene-THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the tetraglutamylated alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of the tetraglutamylated alpha 5,10-methylene-THF is branched.

[0031] In some embodiments, the present disclosure provides compositions comprising polyglutamated alpha 5,10-methylene-THF (i.e., pentaglutamated 10-methylene-THF), which comprises a chain of four glutamyl groups linked to glutamyl groups of tetrahydrofolic acid. In some embodiments, the compositions comprise pentaglutamated alpha[6R]5,10-methylene-THF. In some embodiments, the compositions comprise pentaglutamated alpha[6R,S]-5,10-methylene-THF. In some embodiments, the compositions comprise pentaglutamated alpha[6S]-5,10-methylene-THF. In some embodiments, the pentaglutamated alpha 5,10-methylene-THF comprises one, two, three, or four glutamyl groups with alpha linkages. In some embodiments, the pentaglutamated alpha 5,10-methylene-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the pentaglutamated alpha 5,10-methylene-THF comprises a D-glutamyl group. In further embodiments, the pentaglutamated alpha 5,10-methylene-THF comprises a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha 5,10-methylene-THF is branched.

[0032] In some embodiments, the present disclosure provides compositions comprising polyglutamated alpha 5,10-methylene-THF (i.e., hexaglutamated 5,10-methylene-THF), which comprises a chain of five glutamyl groups linked to glutamyl groups of tetrahydrofolic acid. In some embodiments, the compositions comprise hexaglutamated alpha[6R]5,10-methylene-THF. In some embodiments, the compositions comprise hexaglutamated alpha[6R]5,10-methylene-THF. In some embodiments, the compositions comprise hexaglutamated alpha[6R,S]-5,10-methylene-THF. In some embodiments, the compositions comprise hexaglutamated alpha[6S]-5,10-methylene-THF. In some embodiments, the alpha hexaglutamated 5,10-methylene-THF comprises 1, 2, 3, 4, or 5 alpha-linked glutamyl groups. In some embodiments, the hexaglutamated alpha 5,10-methylene-THF contains two or more glutamyl groups in the L-form. In other embodiments, the hexaglutamated alpha 5,10-methylene-THF contains a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha 5,10-methylene-THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5,10-methylene-THF is branched.

[0033] In some embodiments, the present disclosure provides a composition comprising polyglutamylated alpha 5-formimino-THF. In some embodiments, the composition comprises polyglutamylated alpha[6S]-5-formimino-THF. In some embodiments, the composition comprises polyglutamylated alpha[6R,S]-5-formimino-THF. In some embodiments, the composition comprises polyglutamylated alpha[6R]-5-formimino-THF. In some embodiments, the composition comprises polyglutamylated 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 groups in 5-formimino-THF). In some embodiments, the polyglutamylated alpha 5-formimino-THF comprises 1, 2, 3, or 4 or more glutamyl groups with alpha linkages. In some embodiments, the composition comprises polyglutamylated alpha 5-formimino-THF having two or more glutamyl groups in the L-configuration. In other embodiments, the composition comprises polyglutamylated alpha 5-formimino-THF having glutamyl groups in the D-configuration. In further embodiments, the composition comprises polyglutamylated alpha 5-formimino-THF having glutamyl groups in the D-configuration and two or more glutamyl groups in the L-configuration. In some embodiments, the polyglutamic acid chain of the polyglutamylated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamic acid chain of the polyglutamylated alpha 5-formimino-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 a 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, the tetraglutamylated alpha 5-formimino-THF comprises one, two, or three glutamyl groups with alpha linkages. In some embodiments, the tetraglutamylated alpha 5-formimino-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the tetraglutamylated alpha 5-formimino-THF comprises a glutamyl group in the D-form. In further embodiments, the tetraglutamylated alpha 5-formimino-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the tetraglutamylated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamic acid chain of the 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), which comprises a chain of four glutamyl groups linked to glutamyl groups 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, the pentaglutamylated alpha 5-formimino-THF comprises one, two, three, or four glutamyl groups with alpha linkages. In some embodiments, the pentaglutamylated alpha 5-formimino-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the pentaglutamylated alpha 5-formimino-THF comprises a glutamyl group in the D-form. In further embodiments, the pentaglutamylated alpha 5-formimino-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the pentaglutamylated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamic acid chain of the pentaglutamylated alpha 5-formimino-THF is branched.

[0036] In one embodiment, the composition comprises polyglutamated alpha 5-formimino-THF (i.e., hexaglutamated 5-formimino-THF), which comprises a chain of five glutamyl groups linked to glutamyl groups of tetrahydrofolic acid. In some embodiments, the composition comprises hexaglutamated alpha[6S]-5-formimino-THF. In some embodiments, the composition comprises hexaglutamated alpha[6R,S]-5-formimino-THF. In some embodiments, the composition comprises hexaglutamated alpha[6R]5-formimino-THF. In some embodiments, the hexaglutamated alpha 5-formimino-THF comprises one, two, three, four, or five glutamyl groups with alpha linkages. In some embodiments, the hexaglutamated alpha 5-formimino-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the hexaglutamated alpha 5-formimino-THF comprises a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha 5-formimino-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5-formimino-THF is branched.

[0037] In a further embodiment, the present disclosure provides a composition comprising polyglutamylated alphatetrahydrofolate encapsulated (loaded) liposomes (Lp-αPTHF).

[0038] In some embodiments, the present disclosure provides compositions comprising liposomes encapsulated (loaded) 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 2-20, 2-15, 2-10, 2-5, or 21 or more glutamyl groups (including the glutamyl group in 5-formyl-THF). In some embodiments, the polyglutamylated alpha 5-formyl-THF comprises 1, 2, 3, or 4 or more glutamyl groups with alpha linkages. In some embodiments, the liposome comprises polyglutamylated alpha 5-formyl-THF having two or more glutamyl groups in the L-form. In other embodiments, the liposome comprises polyglutamylated alpha 5-formyl-THF having glutamyl groups in the D-form. In further embodiments, the liposome comprises polyglutamylated alpha 5-formyl-THF having glutamyl groups in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the polyglutamylated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of the 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), which comprises a chain of three glutamyl groups linked to the glutamyl groups 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, the tetraglutamylated alpha 5-formyl-THF comprises one, two, or three glutamyl groups with gamma linkages. In some embodiments, the tetraglutamylated alpha 5-formyl-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the tetraglutamylated alpha 5-formyl-THF comprises a glutamyl group in the D-configuration. In further embodiments, the tetraglutamated alpha 5-formyl-THF comprises a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamic acid chain of the tetraglutamated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of the tetraglutamated 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), which comprises a chain of four glutamyl groups linked to the glutamyl groups 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, the pentaglutamylated alpha 5-formyl-THF comprises one, two, three, or four glutamyl groups with alpha linkages. In some embodiments, the pentaglutamylated 5-formyl-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the pentaglutamated 5-formyl-THF comprises a glutamyl group in the D-form. In further embodiments, the pentaglutamated 5-formyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha 5-formyl-THF is branched.

[0041] In one embodiment, the Lp-αPTHF composition comprises polyglutamated alpha 5-formyl-THF (i.e., hexaglutamated 5-formyl-THF), which comprises a chain of five glutamyl groups linked to the glutamyl groups of tetrahydrofolic acid. In some embodiments, the liposome comprises hexaglutamated alpha[6S]-5-formyl-THF. In some embodiments, the liposome comprises hexaglutamated alpha[6R,S]-5-formyl-THF. In some embodiments, the liposome comprises hexaglutamated alpha[6R]-5-formyl-THF. In some embodiments, the hexaglutamated alpha 5-formyl-THF comprises one, two, three, four, or five glutamyl groups with alpha linkages. In some embodiments, the hexaglutamated alpha 5-formyl-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the hexaglutamated THF comprises a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha 5-formyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5-formyl-THF is branched.

[0042] In some embodiments, the present disclosure provides compositions comprising liposomes encapsulated (loaded) 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 contains one, two, three, or more than three glutamyl groups with alpha linkages. In some embodiments, the liposome contains polyglutamylated alpha 5-formyl-THF containing two or more glutamyl groups in the L-configuration. In other embodiments, the liposome contains polyglutamylated alpha 5,10-methenyl-THF containing glutamyl groups in the D-configuration. In further embodiments, the liposome contains polyglutamylated alpha 5,10-methenyl-THF containing glutamyl groups in the D-configuration and two or more glutamyl groups in the L-configuration. 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-formyl-THF is branched.

[0043] In one embodiment, the Lp-αPTHF composition comprises polyglutamated alpha 5,10-methenyl-THF (i.e., tetraglutamated 5,10-methenyl-THF), which comprises a chain of three glutamyl groups linked to the glutamyl groups of tetrahydrofolic acid. In some embodiments, the liposome comprises tetraglutamated alpha[6R]-5,10-methenyl-THF. In some embodiments, the liposome comprises tetraglutamated alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the liposome comprises tetraglutamated alpha[6S]-5,10-methenyl-THF. In some embodiments, the tetraglutamated alpha 5,10-methenyl-THF comprises one, two, or three glutamyl groups with alpha linkages. In some embodiments, the tetraglutamated alpha 5,10-methenyl-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the tetraglutamated alpha 5,10-methenyl-THF contains a glutamyl group in the D-form. In further embodiments, the tetraglutamated alpha 5,10-methenyl-THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the 5,10-methenyl-THF tetrahydrofolate is linear. In some embodiments, the polyglutamic acid chain of the tetraglutamated alpha 5,10-methenyl-THF is branched.

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

[0045] In one embodiment, the Lp-αPTHF composition comprises polyglutamated alpha 5,10-methenyl-THF (i.e., hexaglutamated 5,10-methenyl-THF), which comprises a chain of five glutamyl groups linked to the glutamyl groups of tetrahydrofolic acid. In some embodiments, the liposome comprises hexaglutamated alpha[6R]-5,10-methenyl-THF. In some embodiments, the liposome comprises hexaglutamated alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the liposome comprises hexaglutamated alpha[6S]-5,10-methenyl-THF. In some embodiments, the hexaglutamated alpha 5,10-methenyl-THF comprises one, two, three, four, or five alpha-linked glutamyl groups. In some embodiments, the hexaglutamated alpha 5,10-methenyl-THF contains two or more glutamyl groups in the L-form. In other embodiments, the hexaglutamated alpha 5,10-methenyl-THF contains a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha 5,10-methenyl-THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5,10-methenyl-THF is linear. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5,10-methenyl-THF is branched.

[0046] In some embodiments, the present disclosure provides compositions comprising liposomes encapsulated (loaded) 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 2-20, 2-15, 2-10, 2-5, or 21 or more glutamyl groups (including the glutamyl group in 5-methyl-THF). In some embodiments, the polyglutamylated alpha 5-methyl-THF comprises 1, 2, 3, or 4 or more glutamyl groups with alpha linkages. In some embodiments, the liposome comprises polyglutamylated alpha 5-methyl-THF having two or more glutamyl groups in the L-form. In other embodiments, the liposome comprises polyglutamylated alpha 5-methyl-THF having glutamyl groups in the D-form. In further embodiments, the liposome comprises polyglutamylated alpha 5-methyl-THF having glutamyl groups in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the polyglutamylated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamic acid chain of the 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 linked to the glutamyl groups 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, the tetraglutamylated alpha 5-methyl-THF comprises one, two, or three glutamyl groups with alpha linkages. In some embodiments, the tetraglutamylated alpha 5-methyl-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the tetraglutamylated alpha 5-methyl-THF comprises a glutamyl group in the D-configuration. In further embodiments, the tetraglutamylated alpha 5-methyl-THF comprises a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamic acid chain of the tetraglutamylated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamic acid chain of the 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), which comprises a chain of four glutamyl groups linked to the glutamyl groups 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, the pentaglutamylated alpha 5-methyl-THF comprises one, two, three, or four glutamyl groups with alpha linkages. In some embodiments, the pentaglutamylated alpha 5-methyl-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the pentaglutamylated alpha 5-methyl-THF comprises a glutamyl group in the D-configuration. In further embodiments, the pentaglutamated alpha 5-methyl-THF comprises a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha 5-methyl-THF is branched.

[0049] In one embodiment, the Lp-αPTHF composition comprises polyglutamated alpha 5-methyl-THF (i.e., hexaglutamated 5-methyl-THF), which comprises a chain of five glutamyl groups linked to the glutamyl groups of tetrahydrofolic acid. In some embodiments, the liposome comprises hexaglutamated alpha[6S]-5-methyl-THF. In some embodiments, the liposome comprises hexaglutamated alpha[6R,S]-5-methyl-THF. In some embodiments, the liposome comprises hexaglutamated alpha[6R]-5-methyl-THF. In some embodiments, the hexaglutamated alpha 5-methyl-THF comprises one, two, three, four, or five glutamyl groups with alpha linkages. In some embodiments, the hexaglutamated alpha 5-methyl-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the hexaglutamated alpha 5-methyl-THF comprises a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha 5-methyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5-methyl-THF is branched.

[0050] In some embodiments, the present disclosure provides compositions comprising liposomes encapsulated (loaded) with 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[6R]-tetrahydrofolic acid THF. In some embodiments, the liposomes comprise polyglutamylated alpha[6R]-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 group in tetrahydrofolic acid THF). In some embodiments, the polyglutamylated alpha[6R]-tetrahydrofolic acid THF comprises 1, 2, 3, or 4 or more glutamyl groups with alpha linkages. In some embodiments, the liposome comprises polyglutamylated alpha-tetrahydrofolic acid THF having two or more glutamyl groups in the L-form. In other embodiments, the liposome comprises polyglutamylated alpha-tetrahydrofolic acid THF having glutamyl groups in the D-form. In further embodiments, the liposome comprises polyglutamylated alpha-tetrahydrofolic acid THF having glutamyl groups in the D-form and two or more glutamyl groups in the L-form. 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 tetrahydrofolate THF composition comprises a polyglutamated alpha-tetrahydrofolate (i.e., tetraglutamated tetrahydrofolate) comprising a chain of three glutamyl groups linked to a glutamyl group of tetrahydrofolate THF. In some embodiments, the liposome comprises tetraglutamated alpha[6S]tetrahydrofolate. In some embodiments, the liposome comprises tetraglutamated alpha[6R,S]-tetrahydrofolate THF. In some embodiments, the liposome comprises tetraglutamated alpha[6R]-tetrahydrofolate THF. In some embodiments, the tetraglutamated alpha-tetrahydrofolate THF comprises one, two, or three glutamyl groups with alpha linkages. In some embodiments, the tetraglutamated alpha-tetrahydrofolate THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the tetraglutamated alpha-tetrahydrofolate THF comprises a glutamyl group in the D-configuration. In further embodiments, the tetraglutamated alphatetrahydrofolate THF comprises a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamate chain of the tetraglutamated alphatetrahydrofolate is linear. In some embodiments, the polyglutamate chain of the tetraglutamated alphatetrahydrofolate is branched.

[0052] In one embodiment, the Lp-αP tetrahydrofolate THF composition comprises a polyglutamated alpha tetrahydrofolate (i.e., pentaglutamated tetrahydrofolate) comprising a chain of four glutamyl groups linked to a glutamyl group of tetrahydrofolate THF. In some embodiments, the liposome comprises pentaglutamated alpha[6S] tetrahydrofolate. In some embodiments, the liposome comprises pentaglutamated alpha[6R,S]-tetrahydrofolate THF. In some embodiments, the liposome comprises pentaglutamated alpha[6R]-tetrahydrofolate THF. In some embodiments, the pentaglutamated alpha tetrahydrofolate THF comprises one, two, three, or four glutamyl groups with alpha linkages. In some embodiments, the pentaglutamated alpha tetrahydrofolate THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the pentaglutamated alpha tetrahydrofolate THF comprises a glutamyl group in the D-configuration. In further embodiments, the pentaglutamated alphatetrahydrofolic acid THF comprises a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamic acid chain of the pentaglutamated alphatetrahydrofolic acid THF is linear. In some embodiments, the polyglutamic acid chain of the pentaglutamated alphatetrahydrofolic acid THF is branched.

[0053] In one embodiment, the Lp-αP tetrahydrofolate THF composition comprises a polyglutamated alpha tetrahydrofolate (i.e., hexaglutamated tetrahydrofolate) containing a chain of five glutamyl groups linked to a glutamyl group of tetrahydrofolate. In some embodiments, the liposome comprises hexaglutamated alpha[6S]tetrahydrofolate. In some embodiments, the liposome comprises hexaglutamated alpha[6R,S]-tetrahydrofolate THF. In some embodiments, the liposome comprises hexaglutamated alpha[6R]-tetrahydrofolate THF. In some embodiments, the hexaglutamated alpha tetrahydrofolate THF comprises one, two, three, four, or five glutamyl groups with alpha linkages. In some embodiments, the hexaglutamated alpha tetrahydrofolate THF comprises two or more glutamyl groups in the L-form. In other embodiments, the hexaglutamated tetrahydrofolic acid THF contains a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha-tetrahydrofolic acid THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha-tetrahydrofolic acid is linear. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha-tetrahydrofolic acid is branched.

[0054] In some embodiments, the present disclosure provides compositions comprising liposomes encapsulated (loaded) 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 polyglutamic acid alpha 5,10-methylene-THF contains one, two, three, or more than three glutamic acid groups with alpha linkages. In some embodiments, the liposome contains polyglutamic acid alpha 5,10-methylene-THF containing two or more glutamic acid groups in the L-form. In other embodiments, the liposome contains polyglutamic acid alpha 5,10-methylene-THF containing glutamic acid groups in the D-form. In further embodiments, the liposome contains polyglutamic acid alpha 5,10-methylene-THF containing glutamic acid groups in the D-form and two or more glutamic acid groups. In some embodiments, the polyglutamic acid chain of the polyglutamic acid alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of the polyglutamic acid 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), which comprises a chain of three glutamyl groups linked to the glutamyl groups 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, the tetraglutamylated alpha 5,10-methylene-THF comprises one, two, or three glutamyl groups with alpha linkages. In some embodiments, the tetraglutamylated alpha 5,10-methylene-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the tetraglutamylated alpha 5,10-methylene-THF contains a glutamyl group in the D-form. In further embodiments, the tetraglutamylated alpha 5,10-methylene-THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the tetraglutamylated alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of the tetraglutamylated alpha 5,10-methylene-THF is branched.

[0056] In one embodiment, the Lp-αPTHF composition comprises polyglutamated alpha 5,10-methylene-THF (i.e., pentaglutamated 10-methylene-THF), which comprises a chain of four glutamyl groups linked to the glutamyl groups of tetrahydrofolic acid. In some embodiments, the liposome comprises pentaglutamated alpha[6R]5,10-methylene-THF. In some embodiments, the liposome comprises pentaglutamated alpha[6R,S]-5,10-methylene-THF. In some embodiments, the liposome comprises pentaglutamated alpha[6S]-5,10-methylene-THF. In some embodiments, the pentaglutamated alpha 5,10-methylene-THF comprises one, two, three, or four alpha-linked glutamyl groups. In some embodiments, the pentaglutamated alpha 5,10-methylene-THF contains two or more glutamyl groups in the L-form. In other embodiments, the pentaglutamated alpha 5,10-methylene-THF contains a glutamyl group in the D-form. In further embodiments, the pentaglutamated alpha 5,10-methylene-THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of the pentaglutamated alpha 5,10-methylene-THF is branched.

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

[0058] In some embodiments, the present disclosure provides compositions comprising liposomes encapsulated (loaded) with polyglutamylated alpha 5-formimino-THF. In some embodiments, the liposomes comprise polyglutamylated alpha[6S]-5-formimino-THF. In some embodiments, the liposomes comprise polyglutamylated alpha[6R,S]-5-formimino-THF. In some embodiments, the liposomes comprise polyglutamylated alpha[6R]-5-formimino-THF. In some embodiments, the liposomes comprise polyglutamylated 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 groups in 5-formimino-THF). In some embodiments, the polyglutamylated alpha 5-formimino-THF contains one, two, three, or more than three glutamyl groups with alpha linkages. In some embodiments, the liposome contains polyglutamylated alpha 5-formimino-THF containing two or more glutamyl groups in the L-form. In other embodiments, the liposome contains polyglutamylated alpha 5-formimino-THF containing glutamyl groups in the D-form. In further embodiments, the liposome contains polyglutamylated alpha 5-formimino-THF containing glutamyl groups in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the polyglutamylated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamic acid chain of the polyglutamylated alpha 5-formimino-THF is branched.

[0059] In one embodiment, the Lp-αPTHF composition comprises polyglutamated alpha 5-formimino-THF (i.e., tetraglutamated 5-formimino-THF) comprising a chain of three glutamyl groups linked to the glutamyl groups of tetrahydrofolic acid. In some embodiments, the liposome comprises tetraglutamated alpha[6S]-5-formimino-THF. In some embodiments, the liposome comprises tetraglutamated alpha[6R,S]-5-formimino-THF. In some embodiments, the liposome comprises tetraglutamated alpha[6R]5-formimino-THF. In some embodiments, the tetraglutamated alpha 5-formimino-THF comprises one, two, or three glutamyl groups with alpha linkages. In some embodiments, the tetraglutamated alpha 5-formimino-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the tetraglutamylated alpha 5-formimino-THF comprises a glutamyl group in the D-form. In further embodiments, the tetraglutamylated alpha 5-formimino-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the tetraglutamylated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamic acid chain of the tetraglutamylated alpha 5-formimino-THF is branched.

[0060] In one embodiment, the Lp-αPTHF composition comprises polyglutamated alpha 5-formimino-THF (i.e., pentaglutamated 5-formimino-THF), which comprises a chain of four glutamyl groups linked to the glutamyl groups of tetrahydrofolic acid. In some embodiments, the liposome comprises pentaglutamated alpha[6S]-5-formimino-THF. In some embodiments, the liposome comprises pentaglutamated alpha[6R,S]-5-formimino-THF. In some embodiments, the liposome comprises pentaglutamated alpha[6R]5-formimino-THF. In some embodiments, the pentaglutamated alpha 5-formimino-THF comprises one, two, three, or four glutamyl groups with alpha linkages. In some embodiments, the pentaglutamated alpha 5-formimino-THF comprises two or more glutamyl groups in the L-configuration. In other embodiments, the pentaglutamylated alpha 5-formimino-THF comprises a glutamyl group in the D-form. In further embodiments, the pentaglutamylated alpha 5-formimino-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain of the pentaglutamylated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamic acid chain of the pentaglutamylated alpha 5-formimino-THF is branched.

[0061] In one embodiment, the Lp-αPTHF composition comprises polyglutamated alpha-5-formimino-THF (i.e., hexaglutamated 5-formimino-THF), which comprises a chain of five glutamyl groups linked to the glutamyl groups of tetrahydrofolic acid. In some embodiments, the liposome comprises hexaglutamated alpha[6S]-5-formimino-THF. In some embodiments, the liposome comprises hexaglutamated alpha[6R,S]-5-formimino-THF. In some embodiments, the liposome comprises hexaglutamated alpha[6R]5-formimino-THF. In some embodiments, the hexaglutamated alpha 5-formimino-THF comprises 1, 2, 3, 4, or 5 alpha-linked glutamyl groups. In some embodiments, the hexaglutamated alpha 5-formimino-THF contains two or more glutamyl groups in the L-configuration. In other embodiments, the hexaglutamated alpha 5-formimino-THF contains a glutamyl group in the D-configuration. In further embodiments, the hexaglutamated alpha 5-formimino-THF contains a glutamyl group in the D-configuration and two or more glutamyl groups in the L-configuration. In some embodiments, the polyglutamic acid chain of the hexaglutamated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamic acid chain of the hexaglutamated 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 diameters ranging from 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 ranging from 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 polyglutamated 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 polyglutamylated alpha-THF starting material is encapsulated in cationic Lp-αPTHF. In further embodiments, the polyglutamylated alpha-tetrahydrofolate encapsulated by the liposomes is present in a HEPES buffer solution 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 diameters ranging from 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 diameters ranging from 80 nm to 120 nm, or any range therebetween. In some embodiments, the Lp-αPTHF liposomes are anionic and have diameters ranging from 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 diameters ranging from 80 nm to 120 nm, or any range therebetween. In some embodiments, Lp-αPTHF liposomes are neutral and have diameters ranging from 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, anionic or neutral Lp-αPTHF compositions comprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w 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% w / w of the polyglutamylated alpha THF starting material is encapsulated 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 tetraglutamylated alpha THF.In some embodiments, anionic or neutral Lp-αPTHF compositions contain 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 pentaglutamated alpha-THF. In some embodiments, anionic or neutral Lp-αPTHF compositions contain 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 hexaglutamated alpha-THF. In further embodiments, the polyglutamated alpha-tetrahydrofolate encapsulated by the liposomes is present in a HEPES-buffered solution within the liposomes.

[0064] In a further embodiment, the liposomal polyglutamated alphatetrahydrofolate composition is PEGylated (PLp-αPTHF).

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

[0066] In other embodiments, the liposomal polyglutamated alpha-tetrahydrofolate composition (TLp-αPTHF) is targeted. That is, the TLp-αPTHF composition includes a targeting moiety that has specific affinity for an epitope (surface antigen) on a target cell of interest. In some embodiments, the targeting moiety of TLp-αPTHF or TPLp-αPTHF is covalently attached to the liposome. In other embodiments, the targeting moiety of TLp-αPTHF or TPLp-αPTHF is attached to one or both of the PEG and the exterior of the liposome. The targeted liposomal polyglutamated alpha-tetrahydrofolate compositions (TLp-αPTHF and TPLp-αPTHF) further improve the efficacy and safety profile of tetrahydrofolate by specifically delivering polyglutamated alpha (e.g., α-pentaglutamated and / or α-hexaglutamated) tetrahydrofolate to target cells, such as cancer cells. In some embodiments, the targeted liposomal polyglutamated alpha-tetrahydrofolate composition is PEGylated (TPLp-αPTHF). In some embodiments, the targeting moiety of TLp-αPTHF or TPLp-αPTHF is attached to one or both of the PEG and the exterior of the liposome. In some embodiments, the targeting moiety of TLp-αPTHF or TPLp-αPTHF is attached to the liposome via a covalent bond. The function of the targeting moiety of TLp-αPTHF and / or TPLp-αPTHF composition includes, but is not limited to, targeting the liposome to a desired target cell in vivo or in vitro; interacting with a 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 include, but are not limited to, antibodies, antigen-binding antibody fragments, scaffold proteins, polypeptides, and peptides. In some embodiments, the targeting moiety is a polypeptide.In further embodiments, 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 further embodiments, 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, compared to normal cells or non-tumor cells. In some embodiments, the targeting moiety has specific affinity for an epitope on a tumor cell surface antigen that is present on tumor cells but not present or inaccessible on non-tumor cells. In some embodiments, the targeting moiety has a specific affinity of 0.5×10 as measured using BIACORE® analysis. -10 ~10×10 -6 It binds to the epitope of interest with an equilibrium dissociation constant (Kd) in the range of

[0068] In certain embodiments, the TLp-αPTHF or TPLp-αPTHF targeting moiety comprises a polypeptide that specifically binds to a folate receptor. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the folate receptor to which the targeting moiety binds 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 immunostimulatory agent, a detectable marker, and a maleimide disposed on at least one of the PEG and the exterior 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 diameters ranging from 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 liposomal αPTHF composition have diameters ranging from 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 diameters ranging from 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 a polyglutamylated alpha-tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the liposomal αPTHF composition comprises a tetraglutamylated alpha-tetrahydrofolate.In some embodiments, the liposomal αPTHF composition comprises pentaglutamated alpha-tetrahydrofolate, hi other embodiments, the liposomal αPTHF composition comprises hexaglutamated alpha-tetrahydrofolate.

[0070] In some embodiments, the liposome composition comprises polyglutamylated alpha-tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 6 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 polyglutamylated alpha-THF. In some embodiments, the Lp-αPTHF composition comprises polyglutamylated alpha-tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 6 glutamyl groups and 1% to 98.5% w / w polyglutamylated alpha-THF. In some embodiments, the liposomes comprise polyglutamylated alpha-tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 6 glutamyl groups, 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 polyglutamylated alpha-tetrahydrofolate starting material is encapsulated (encapsulated) in the Lp-αPTHF.

[0071] In some embodiments, the liposome composition comprises tetraglutamated 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 tetraglutamated alpha-THF. In some embodiments, the Lp-αPTHF composition comprises tetraglutamated alpha-tetrahydrofolic acid and 1% to 98.5% w / w tetraglutamated alpha-THF. In some embodiments, the liposomes comprise tetraglutamylated 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 tetraglutamylated alpha-THF starting material is encapsulated (enclosed) in the Lp-αPTHF.

[0072] In some embodiments, the liposome composition comprises pentaglutamated 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 pentaglutamated alpha-THF. In some embodiments, the Lp-αPTHF composition comprises pentaglutamated alpha-tetrahydrofolate and 1% to 98.5% w / w pentaglutamated alpha-THF. In some embodiments, the liposome comprises pentaglutamylated 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 pentaglutamylated alpha THF starting material is encapsulated (encapsulated) in the Lp-αPTHF.

[0073] In some embodiments, the liposome composition comprises hexaglutamated 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 hexaglutamated alpha-THF. In some embodiments, the Lp-αPTHF composition comprises hexaglutamated alpha-tetrahydrofolic acid and 1% to 98.5% w / w hexaglutamated alpha-THF. In some embodiments, the liposomes contain hexaglutamated alphatetrahydrofolic 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 pentaglutamated alphaTHF starting material is encapsulated (enclosed) in the Lp-αPTHF.

[0074] Also provided are liposome compositions 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 conjugated to or otherwise associated with a targeting moiety. In further embodiments, the liposome composition comprises an αPTHF composition that is PEGylated and conjugated to or otherwise associated with a targeting moiety. In some embodiments, the liposome composition comprises αPTHF comprising 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the liposome composition comprises tetraglutamated alpha-tetrahydrofolate. In some embodiments, the liposome composition comprises pentaglutamated alpha-tetrahydrofolate. In other embodiments, the liposome composition comprises hexaglutamated alpha-tetrahydrofolate.

[0075] In some embodiments, the liposome composition comprises liposomal αPTHF (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-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 of interest, 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 of interest, such as a cancer cell (e.g., TPLp-αPTHF). In some embodiments, the liposome composition comprises cationic liposomal αPTHF. In other embodiments, the liposome composition comprises liposomal αPTHF that is anionic or neutral. In further embodiments, the liposome composition comprises liposomal αPTHF with a diameter of 20 nm to 500 nm, 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposomal αPTHF has a diameter of 80 nm to 120 nm, or any range therebetween.

[0076] Pharmaceutical compositions are also provided that include polyglutamated alpha-tetrahydrofolate (αPTHF) in a delivery vehicle, such as liposomal αPTHF. In some embodiments, the pharmaceutical composition includes a pegylated αPTHF composition. In some embodiments, the pharmaceutical composition includes an αPTHF composition linked to or otherwise associated with a targeting moiety. In further embodiments, the pharmaceutical composition includes an αPTHF composition that is pegylated and linked to or otherwise associated with a targeting moiety. In some embodiments, the pharmaceutical composition includes αPTHF containing 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the pharmaceutical composition includes tetraglutamated alpha-tetrahydrofolate. In some embodiments, the pharmaceutical composition includes pentaglutamated alpha-tetrahydrofolate. In other embodiments, the pharmaceutical composition includes hexaglutamated alpha-tetrahydrofolate.

[0077] In some embodiments, the pharmaceutical composition comprises liposomal αPTHF (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-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 cationic liposomal αPTHF. In other embodiments, the pharmaceutical composition comprises anionic or neutral liposomal αPTHF. In further embodiments, the pharmaceutical composition comprises liposomal αPTHF having a diameter 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 methods for modulating cellular activation, chemokine production, or metabolic activity, the method comprising contacting a cell with a composition comprising a polyglutamated alpha-tetrahydrofolate (αPTHF) composition. In some embodiments, the contacted cell is a mammalian cell. In further embodiments, the contacted cell is a human cell. In some embodiments, the contacted cell 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, the αPTHF composition comprises 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the αPTHF composition comprises tetraglutamated alpha-tetrahydrofolate. In some embodiments, the αPTHF composition comprises pentaglutamated alpha-tetrahydrofolate. In other embodiments, the αPTHF composition comprises hexaglutamated alpha-tetrahydrofolate.

[0079] In further embodiments, the present disclosure provides methods for modulating cellular activation, chemokine production, or metabolic activity, comprising contacting a cell with a liposome comprising a polyglutamated alpha-tetrahydrofolate (αPTHF) composition. In some embodiments, the contacted cell is a mammalian cell. In further embodiments, the contacted cell is a human cell. In some embodiments, the contacted cell 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, the αPTHF comprises 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the αPTHF composition comprises tetraglutamated alpha-tetrahydrofolate. In some embodiments, the αPTHF composition comprises pentaglutamated alpha-tetrahydrofolate. In other embodiments, the αPTHF composition comprises hexaglutamated alpha-tetrahydrofolate.

[0080] In further embodiments, the present disclosure provides a method of killing a cell, the method comprising contacting the cell with a composition comprising a polyglutamylated alphatetrahydrofolate (αPTHF) composition (e.g., αPTHF herein). In some embodiments, the contacted cell is a mammalian cell. In further embodiments, the contacted cell is a human cell. In some embodiments, the contacted cell is a hyperproliferative cell. In further embodiments, the hyperproliferative cell is a cancer cell. In further embodiments, the cancer cells contacted are primary cells or cells from a cell line obtained / derived from a cancer selected from the group consisting of non-hematologic tumors 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, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematologic tumors such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. In further embodiments, the cancer cells contacted are primary cells or cells 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's lymphoma (NHL), acute lymphocytic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, and villous adenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (demenoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In further embodiments, the cancer cells are primary cells or cells 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 adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In certain embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from colorectal cancer. In some embodiments, αPTHF contains 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, αPTHF contains 4 glutamyl groups. In some embodiments, αPTHF contains 5 glutamyl groups. In some embodiments, αPTHF contains 6 glutamyl groups. In some embodiments, the method is performed in vivo.In another embodiment, the method is performed in vitro.

[0081] In a further embodiment, the present disclosure provides a method of killing a cell, the method comprising contacting the cell with a liposome comprising polyglutamylated alphatetrahydrofolate (e.g., Lp-αPTHF, such as PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF). In a further embodiment, the contacted hyperproliferative cell is a cancer cell. In further embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from the group consisting of non-hematologic tumors 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, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematologic tumors such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. In further embodiments, the cancer cells contacted are primary cells or cells 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's lymphoma (NHL), acute lymphocytic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, and villous adenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (demenoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the cancer cells are primary cells or cells 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 adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In certain embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from colorectal cancer. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the liposomes comprise αPTHF containing 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the liposomes comprise αPTHF containing 4, 5, 2-10, 4-6, or more than 6 glutamyl groups.In some embodiments, the liposome comprises αPTHF containing 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the liposome comprises αPTHF containing 4 glutamyl groups. In some embodiments, the liposome comprises αPTHF containing 5 glutamyl groups. In some embodiments, the liposome comprises αPTHF containing 6 glutamyl groups. In some embodiments, the αPTHF contains 1, 2, 3, or more than 4 glutamyl groups with gamma linkages.

[0082] In further embodiments, the present disclosure provides methods of treating cancer, comprising administering to a subject having or at risk of having cancer an effective amount of a delivery vehicle (e.g., an antibody immunoconjugate or liposome) comprising polyglutamylated alphatetrahydrofolate. In some embodiments, the delivery vehicle is an antibody-containing immunoconjugate (e.g., comprising a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery vehicle is a liposome (e.g., Lp-αPTHF, such as PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is not pegylated. In further embodiments, the administered delivery vehicle comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a cancer cell. In further embodiments, the administered delivery vehicle comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a cancer cell.In further embodiments, the delivery vehicle is selected from the group consisting of GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-delta), 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 ectodomain 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, C D11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD 44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 and a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of an EphA receptor, an EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, an 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 vehicle comprises a targeting moiety that specifically binds to a cell surface antigen, such as a neoantigen, determined to be derived from or expressed by a particular target cancer (tumor). In some embodiments, the targeting moiety specifically binds to a cell surface antigen, such as a neoantigen, determined to be derived from or expressed by a particular target tumor. In some embodiments, the targeting moiety is an antibody or antigen-binding antibody fragment. In some embodiments, the delivery vehicle administered comprises αPTHF containing 4, 5, 2-10, 4-6, or more than 6 -glutamyl groups. In some embodiments, the delivery vehicle administered comprises αPTHF containing 4 glutamyl groups. In some embodiments, the delivery vehicle administered comprises αPTHF containing 5 glutamyl groups. In some embodiments, the delivery vehicle administered comprises αPTHF containing 6 glutamyl groups. In some embodiments, the αPTHF is selected from the group consisting of: (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., (e) polyglutamated tetrahydrofolic acid THF (e.g., polyglutamated [6S]-tetrahydrofolic acid THF); (f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In some embodiments, the αPTHF is polyglutamated 5,10-methylene-THF. In further embodiments, the αPTHF is polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the αPTHF is polyglutamated [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, 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, bile duct cancer, 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, myeloma, and other plasma cell dysplasias or cachexia. In some embodiments, the cancer is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, and villous adenoma, non-leukemic meningitis, soft tissue sarcoma (demenoid tumor, aggressive fibromatosis), bladder cancer, and 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 cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In certain embodiments, the cancer is colorectal cancer.

[0083] In further embodiments, the present disclosure provides methods of treating cancer, comprising administering to a subject having or at risk of having cancer an effective amount of a liposome comprising polyglutamylated alphatetrahydrofolate (e.g., Lp-αPTHF, e.g., PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF), or the like. In some embodiments, the liposome is PEGylated. In some embodiments, the liposome is not PEGylated. In further embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a cancer cell.In further embodiments, the liposomes are selected from the group consisting of GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a 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 ectodomain 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, C D11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD 44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 and a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of an EphA receptor, an EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, an 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, and CD15. In some embodiments, the liposomes contain a targeting moiety that has specific affinity for an epitope of a cell surface antigen, such as a neoantigen, that has been shown to be derived from or expressed on a particular target tumor. In some embodiments, the targeting moiety is an antibody or antigen-binding antibody fragment. In some embodiments, the liposomes contain αPTHF containing 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the administered liposomes are selected from the group consisting of: (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., , polyglutaminated [6S]-5-methyl-THF; (e) polyglutamated tetrahydrofolic acid THF (e.g., polyglutamated [6S]-tetrahydrofolic acid THF); (f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In some embodiments, the administered liposome comprises polyglutamated 5,10-methylene-THF. In further embodiments, the administered liposome comprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, the liposomes administered comprise polyglutamylated 5-methyl-THF. In further embodiments, the liposomes administered comprise [6S]-5-methyl-THF. In other embodiments, the liposomes administered comprise [6R,S]-5-methyl-THF.In some embodiments, the liposomes administered comprise polyglutamylated 5-formyl-THF. In further embodiments, the liposomes administered comprise polyglutamylated [6S]-5-formyl-THF. In other embodiments, the liposomes administered comprise polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposome composition administered comprises tetraglutamylated αPTHF. In some embodiments, the liposome composition administered comprises pentaglutamylated αPTHF. In some embodiments, the liposome composition administered comprises hexaglutamylated αPTHF. In some embodiments, the liposomes of the liposome composition administered comprise αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the liposomes of the liposome composition administered comprise αPTHF containing 1, 2, 3, or 4 or more glutamyl groups with gamma linkages. In some embodiments, the cancer is selected from the group consisting of lung (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, and hematological malignancies (e.g., leukemia or lymphoma). In further embodiments, the cancer cells are primary cells or cells 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 from a cell line obtained / derived from colorectal cancer.

[0084] 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 an effective amount of a liposome composition comprising a liposome comprising polyglutamylated alphatetrahydrofolate and a targeting moiety having specific affinity for an epitope of an antigen on the surface of the cancer.In some embodiments, the liposomes are 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 ectodomain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelial receptors, 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, C D44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 and a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of an EphA receptor, an EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, an 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 liposomes comprise a targeting moiety that has specific affinity for an epitope of a cell surface antigen, e.g., a neoantigen, that has been shown to originate from or be expressed on a particular target cancer (tumor). In some embodiments, the targeting moiety is an antibody or antigen-binding antibody fragment. In some embodiments, the liposomes comprise αPTHF containing 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the administered liposomes comprise αPTHF containing 4 glutamyl groups. In some embodiments, the administered liposomes comprise αPTHF containing 5 glutamyl groups. In some embodiments, the administered liposomes comprise αPTHF containing 6 glutamyl groups. In some embodiments, the administered liposomes are selected from the group consisting of: (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., , polyglutaminated [6S]-5-methyl-THF; (e) polyglutamated tetrahydrofolic acid THF (e.g., polyglutamated [6S]-tetrahydrofolic acid THF); (f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In some embodiments, the administered liposome comprises polyglutamated 5,10-methylene-THF. In further embodiments, the administered liposome comprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, the liposomes administered comprise polyglutamylated 5-methyl-THF.In further embodiments, the liposomes administered comprise [6S]-5-methyl-THF. In other embodiments, the liposomes administered comprise [6R,S]-5-methyl-THF. In some embodiments, the liposomes administered comprise polyglutamylated 5-formyl-THF. In further embodiments, the liposomes administered comprise polyglutamylated [6S]-5-formyl-THF. In other embodiments, the liposomes administered comprise polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposomes administered comprise αPTHF containing one, two, three, or four or more glutamyl groups with gamma linkages.

[0085] In some embodiments, the liposome composition administered comprises PEGylated liposomes (e.g., TPLp-αPTHF). In some embodiments, the liposome composition administered comprises non-PEGylated liposomes. In some embodiments, the liposomes of the liposome composition administered comprise αPTHF containing 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the liposomes administered comprise αPTHF containing 4 glutamyl groups. In some embodiments, the liposomes administered comprise αPTHF containing 5 glutamyl groups. In some embodiments, the liposomes administered comprise αPTHF containing 6 glutamyl groups. In some embodiments, the administered liposomes are selected from the group consisting of: (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., , polyglutaminated [6S]-5-methyl-THF; (e) polyglutamated tetrahydrofolic acid THF (e.g., polyglutamated [6S]-tetrahydrofolic acid THF); (f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In some embodiments, the administered liposome comprises polyglutamated 5,10-methylene-THF. In further embodiments, the administered liposome comprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, the liposomes administered comprise polyglutamylated 5-methyl-THF. In further embodiments, the liposomes administered comprise [6S]-5-methyl-THF.In other embodiments, the liposomes administered comprise [6R,S]-5-methyl-THF. In some embodiments, the liposomes administered comprise polyglutamylated 5-formyl-THF. In further embodiments, the liposomes administered comprise polyglutamylated [6S]-5-formyl-THF. In other embodiments, the liposomes administered comprise polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposomes of the liposome composition administered comprise αPTHF containing one, two, three, or four or more glutamyl groups with gamma linkages. In some embodiments, the liposome composition is administered to treat a 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, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, myeloma, leukemia, and lymphoma. In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of 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 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 for treating cancer, comprising administering an effective amount of a liposome composition to a subject having or at risk of having a cancer that expresses a folate receptor on the cell surface, wherein the liposome composition comprises a liposome comprising (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 liposome composition administered comprises PEGylated liposomes (e.g., TPLp-αPTHF). In some embodiments, the liposome composition administered comprises non-PEGylated liposomes. In some embodiments, the liposomes of the liposome composition administered comprise αPTHF containing 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the liposomes administered comprise αPTHF containing 4 glutamyl groups. In some embodiments, the liposomes administered comprise αPTHF containing 5 glutamyl groups. In some embodiments, the liposomes administered comprise αPTHF containing 6 glutamyl groups.In some embodiments, the administered liposomes are selected from the group consisting of: (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., , polyglutaminated [6S]-5-methyl-THF; (e) polyglutamated tetrahydrofolic acid THF (e.g., polyglutamated [6S]-tetrahydrofolic acid THF); (f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In some embodiments, the administered liposome comprises polyglutamated 5,10-methylene-THF. In further embodiments, the administered liposome comprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, the liposomes administered comprise polyglutamylated 5-methyl-THF. In further embodiments, the liposomes administered comprise [6S]-5-methyl-THF. In other embodiments, the liposomes administered comprise [6R,S]-5-methyl-THF. In some embodiments, the liposomes administered comprise polyglutamylated 5-formyl-THF. In further embodiments, the liposomes administered comprise polyglutamylated [6S]-5-formyl-THF. In other embodiments, the liposomes administered comprise polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposomes of the liposome composition administered comprise one, two, three, or four or more glutamyl groups containing gamma linkages.In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of non-hematologic malignancies, 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, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain tumors, central nervous system cancers, and melanoma; and hematologic malignancies, such as, for example, leukemia, lymphoma, and other B-cell malignancies, myeloma, and other plasma cell dysplasias or cachexia. In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma and villous adenoma, non-leukemic meningeal carcinoma, soft tissue sarcoma (demenoid tumour, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of 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 adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma.

[0087] In further embodiments, the present disclosure provides a method for cancer maintenance therapy, comprising administering to a subject undergoing or who has undergone cancer treatment an effective amount of a liposome composition comprising liposomes comprising polyglutamylated alpha-tetrahydrofolate (Lp-αPTHF). In some embodiments, the liposome composition administered is PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF. In some embodiments, the liposome composition administered comprises PEGylated liposomes (e.g., PLp-αPTHF, NTPLp-αPTHF, or TPLp-αPTHF). In some embodiments, the liposome composition administered comprises targeted liposomes (e.g., TLp-αPTHF or TPLp-αPTHF). In some embodiments, the liposome composition administered comprises PEGylated liposomes and comprises a targeting moiety (e.g., TPLp-αPTHF). In some embodiments, the liposomes of the liposome composition administered comprise polyglutamylated alphatetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the liposomes administered comprise αPTHF containing 4 glutamyl groups. In some embodiments, the liposomes administered comprise αPTHF containing 5 glutamyl groups. In some embodiments, the liposomes administered comprise αPTHF containing 6 glutamyl groups.In some embodiments, the administered liposomes are selected from the group consisting of: (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., , polyglutaminated [6S]-5-methyl-THF; (e) polyglutamated tetrahydrofolic acid THF (e.g., polyglutamated [6S]-tetrahydrofolic acid THF); (f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In some embodiments, the administered liposome comprises polyglutamated 5,10-methylene-THF. In further embodiments, the administered liposome comprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, the liposomes administered comprise polyglutamylated 5-methyl-THF. In further embodiments, the liposomes administered comprise [6S]-5-methyl-THF. In other embodiments, the liposomes administered comprise [6R,S]-5-methyl-THF. In some embodiments, the liposomes administered comprise polyglutamylated 5-formyl-THF. In further embodiments, the liposomes administered comprise polyglutamylated [6S]-5-formyl-THF. In other embodiments, the liposomes administered comprise polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposomes of the liposome composition administered comprise one, two, three, or four or more glutamyl groups containing gamma linkages.

[0088] In further embodiments, the present disclosure provides methods of treating an immune system disorder, comprising administering to a subject having or at risk of having an immune system disorder an effective amount of a liposome composition comprising liposomes comprising polyglutamylated alphatetrahydrofolate (e.g., Lp-αPTHF, such as PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF). In some embodiments, the liposome composition is administered to treat an autoimmune disease. In a further embodiment, the liposome composition is administered to treat rheumatoid arthritis. In another embodiment, the liposome composition is administered to treat inflammation. In some embodiments, the administered liposome composition comprises a pegylated liposome (e.g., PLp-αPTHF, NTPLp-αPTHF, or TPLp-αPTHF). In some embodiments, the liposome composition administered comprises a targeted liposome (e.g., TLp-αPTHF or TPLp-αPTHF) comprising a targeting moiety that has specific affinity for a surface antigen on a target cell (e.g., an immune cell) of interest. In further embodiments, the liposome composition administered comprises a liposome (e.g., TPLp-αPTHF) that is pegylated and comprises a targeting moiety. In some embodiments, the liposome of the liposome composition administered comprises a polyglutamylated alpha-tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the liposome composition administered comprises αPTHF containing 4 glutamyl groups. In some embodiments, the liposome administered comprises αPTHF containing 5 glutamyl groups. In some embodiments, the liposome administered comprises αPTHF containing 6 glutamyl groups.In some embodiments, the administered liposomes are selected from the group consisting of: (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., , polyglutaminated [6S]-5-methyl-THF; (e) polyglutamated tetrahydrofolic acid THF (e.g., polyglutamated [6S]-tetrahydrofolic acid THF); (f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In some embodiments, the administered liposome comprises polyglutamated 5,10-methylene-THF. In further embodiments, the administered liposome comprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, the liposomes administered comprise polyglutamylated 5-methyl-THF. In further embodiments, the liposomes administered comprise [6S]-5-methyl-THF. In other embodiments, the liposomes administered comprise [6R,S]-5-methyl-THF. In some embodiments, the liposomes administered comprise polyglutamylated 5-formyl-THF. In further embodiments, the liposomes administered comprise polyglutamylated [6S]-5-formyl-THF. In other embodiments, the liposomes administered comprise polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposomes of the liposome composition administered comprise one, two, three, or four or more glutamyl groups containing gamma linkages.

[0089] The present disclosure also provides a method for delivering polyglutamated alpha-tetrahydrofolate to tumor and / or cancer cells, comprising administering to a subject having a tumor a composition comprising polyglutamated alpha-tetrahydrofolate (L-αPTHF) and a targeting moiety having specific binding affinity for an epitope on a surface antigen of a tumor or cancer cell. In some embodiments, the administered targeting moiety is conjugated to a delivery vehicle. In some embodiments, the delivery vehicle is an antibody or an antigen-binding fragment of an antibody. In further embodiments, the delivery vehicle is a liposome. In some embodiments, the antibody, antigen-binding antibody fragment, or liposome is pegylated. In some embodiments, the administered composition comprises polyglutamated alpha-tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the administered composition comprises tetraglutamated alpha-tetrahydrofolate. In some embodiments, the administered composition comprises pentaglutamated alpha-tetrahydrofolate. In other embodiments, the composition administered comprises hexaglutamated alphatetrahydrofolic acid. In some embodiments, the composition administered comprises (a) polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF); (b) polyglutamated 10-formyl-THF (e.g., polyglutamated [6R]-10-formyl-THF); (c) polyglutamated 5,10-methenyl-THF (e.g., polyglutamated [6R]-5,10-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g., (e) polyglutamated [6S]-5-methyl-THF; (f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF).In some embodiments, the composition administered comprises polyglutamylated 5,10-methylene-THF. In further embodiments, the composition administered comprises polyglutamylated [6R]-5,10-methylene-THF. In other embodiments, the composition administered comprises polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the composition administered comprises polyglutamylated 5-methyl-THF. In further embodiments, the composition administered comprises [6S]-5-methyl-THF. In other embodiments, the composition administered comprises [6R,S]-5-methyl-THF. In some embodiments, the composition administered comprises polyglutamylated 5-formyl-THF. In further embodiments, the composition administered comprises polyglutamylated [6S]-5-formyl-THF. In other embodiments, the composition administered comprises polyglutamylated [6R,S]-5-formyl-THF.

[0090] In further embodiments, the present disclosure provides methods for preparing liposomal compositions comprising liposomal polyglutamated alpha-tetrahydrofolate (αPTHF) compositions, the methods comprising: forming a mixture in a solution comprising liposome components and α-polyglutamated tetrahydrofolate; homogenizing the mixture in the solution to form liposomes; and treating the mixture to form liposomes comprising polyglutamated tetrahydrofolate. In some embodiments, the polyglutamated alpha-tetrahydrofolate comprises 4, 5, 2-10, 4-6, or more than 6 glutamyl groups. In some embodiments, the αPTHF composition comprises pentaglutamated alpha-tetrahydrofolate. In some embodiments, the αPTHF composition comprises tetraglutamated alpha-tetrahydrofolate. In other embodiments, the αPTHF composition comprises hexaglutamated alpha-tetrahydrofolate. In some embodiments, polyglutamylated alpha tetrahydrofolate contains 1, 2, 3, or 4 or more glutamyl groups containing gamma linkages. In some embodiments, αPTHF compositions contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more glutamyl groups in the D-form. In some embodiments, αPTHF compositions contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more glutamyl groups in the L-form. In some embodiments, αPTHF compositions contain 2, 3, 4, 5, or 6 or more glutamyl groups in the L-form and 1, 2, 3, 4, 5, or 6 or more glutamyl groups in the D-form.In some embodiments, the composition comprises: (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., poly (e) polyglutamated tetrahydrofolic acid THF (e.g., polyglutamated [6S]-tetrahydrofolic acid THF); (f) polyglutamated 5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutamated 5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In some embodiments, the composition comprises polyglutamated 5,10-methylene-THF. In further embodiments, the composition comprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the composition comprises polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises polyglutamated 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 administered comprises polyglutamylated [6R,S]-5-formyl-THF.

[0091] In one embodiment, the present disclosure provides a kit comprising a polyglutamylated alphatetrahydrofolate composition and / or an αPTHF delivery vehicle, such as a liposome comprising αPTHF or an αPTHF immunoconjugate (e.g., an ADC) described herein. [Brief explanation of the drawings]

[0092] [Figures 1A-1N] Alpha tetrahydrofolate: alpha 5-10 methylene THF (Figure 1A), alpha 5-10 methylene THF diglutamate (Figure 1B), alpha 5-10 methylene THF triglutamate (Figures 1C and 1D), alpha 5-10 methylene THF tetraglutamate (Figures 1E and 1F), alpha 5-10 methylene THF pentaglutamate (Figures 1G and 1H), alpha 5-10 methylene THF hexaglutamate (Figures 1I and 1J), Figures 1K and 1L show representative chemical formulas of alpha 5-10 methylene THF heptaglutamic acid (Figures 1K and 1L), alpha 5-10 methylene THF octaglutamic acid (Figures 1M and 1N), and representative alpha tetrahydrofolate polyglutamic acid derivatives, tetrahydrofolate THF, 10 formyl THF, 5 formyl THF, 5-methyl THF, 5 formimino THF, 5,10 methenyl THF, and 5,10 methylene THF (Figures 1O-1Q). 1R-1U show depictions of representative branched 5-10 methylene THF polyglutamic acid structures, including a branched polyglutamic acid having an alpha-glutamyl backbone and gamma-glutamyl branches (FIG. 1S), a branched polyglutamic acid having a gamma-glutamyl backbone and alpha-glutamyl branches (FIG. 1T), and a branched polyglutamic acid having an alpha-glutamyl backbone and both gamma-glutamyl and alpha-glutamyl branches (FIG. 1U). [Figure 2] Figure 1 shows the relative efficacy of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6) and its enantiomer liposomal alpha-D hexaglutamate (liposomal aDG6) compared to pemetrexed after 48 hours of exposure to the cancer cell lines SW620 (CRC), HT-29 (colon cancer), H1806 (triple-negative breast cancer), OAW28 (ovarian cancer), H292 (NSCLC, adenocarcinoma subtype), and H2342 (NSCLC, adenocarcinoma subtype). [Figure 3]An example of the dose-response relationship, expressed as the percentage of viable cells after 48 hours of treatment, is shown for free pemetrexed L-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6), pemetrexed, and the folate receptor alpha-targeted antibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6-FR1Ab) in NCI H2342 non-small cell lung cancer (NSCLC), adenocarcinoma subtype. Folate receptor alpha-targeted liposomes containing alpha polyglutamated pemetrexed are predicted to successfully target and reduce the viability of NCI H2342 NSCLC cells. [Figure 4] An example of the dose-response relationship over 48 hours in HT-29 (colon cancer) cells is shown for free pemetrexed L-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6), pemetrexed, and the folate receptor alpha-targeted antibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6-FR1Ab). Folate receptor alpha-targeted liposomes containing alpha polyglutamated pemetrexed are also predicted to successfully target and reduce viability of HT-29 (colon cancer) cells. [Figure 5] 1 shows the therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (Lps Hexa aG6), liposomal pemetrexed alpha-D hexaglutamate (Lps Hexa aDG6), and pemetrexed on HCC1806 triple-negative breast cancer cells after 48 hours of exposure. [Figure 6] 1 shows the therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (LPS Hexa aG6), liposomal pemetrexed alpha-D hexaglutamate (LPS Hexa aDG6), and pemetrexed on OAW28 ovarian cancer cells after 48 hours of exposure. [Figure 7]The therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (Lps Hexa aG6) and liposomal pemetrexed alpha-D hexaglutamate (Lps Hexa aDG6) on H292 non-small cell lung cancer cells over a 48-hour period are shown in comparison to pemetrexed. [Figure 8] This figure shows the therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and pemetrexed at various dose levels ranging from 16 to 128 nM on H292 non-small cell lung cancer cells after 48 hours of exposure. Within each dose range tested, the liposomal pemetrexed aG6 formulation exhibited superior inhibition of H292 non-small cell lung cancer cells compared to pemetrexed. [Figure 9] This figure shows the therapeutic efficacy of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and pemetrexed at various dose levels ranging from 16 to 128 nM against HCC1806 triple-negative breast cancer cells after 48 hours of exposure. At each dose range tested, the liposomal pemetrexed aG6 formulation was superior to pemetrexed in inhibiting HCC1806 triple-negative breast cancer cells. [Figure 10] This figure shows the therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and pemetrexed at a range of concentrations on OAW28 ovarian cancer cells after 48 hours of exposure. At a dose of 128 nM, pemetrexed appears to be more effective than the liposomal pemetrexed aG6 liposomal formulation; however, at doses of 32 nM and 64 nM, the liposomal formulations have superior therapeutic effects to pemetrexed; and at 16 nM, the therapeutic effect of liposomal pemetrexed aG6 is comparable to that of pemetrexed. [Figure 11]Figure 1 shows the toxicity of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and pemetrexed at 64 nM, 128 nM, and 264 nM toward differentiating human neutrophils. The figure shows that liposomal pemetrexed aG6 is significantly less toxic than pemetrexed toward differentiating human neutrophils. [Figure 12] Shown are the effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and the corresponding pemetrexed agents at various dose levels ranging from 16 to 128 nM on neutrophils (differentiated from CD34+ cells) after 48 hours of exposure. [Figure 13]

[0033] Figure 1 shows the effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and the corresponding pemetrexed at 16 nM, 32 nM, 64 nM, and 128 nM for 48 hours on AML12 liver cells. Notably, none of the liposomal agents tested at any dose level appeared to be toxic to AML12 liver cells after treatment with liposomal pemetrexed aG6. In contrast, pemetrexed treatment resulted in an approximately 40% reduction in AML12 liver cell count at all doses tested. [Figure 14]

[0023] Figure 1 shows the effects on CCD841 colonic epithelial cells after 48 hours of exposure to liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and the corresponding pemetrexed at 16 nM, 32 nM, 64 nM, and 128 nM. At each concentration tested, treatment with each liposomal composition tested resulted in a reduction in CCD841 colonic epithelial cell count of about 20% or less, while pemetrexed resulted in a reduction in cell count of about 50% or more. [Figure 15]The structures of the polyglutamated antifolate, cisplatin (CDDP), and two possible aG6-cisplatin complexes are shown. The pH-dependent formation of inter- and / or intra-chain coordination between the carboxyl groups of the polyglutamated antifolate and cisplatin may lead to its degradation into separate molecules of aG6 and cisplatin upon encountering the acidic pH of the lysosome (pH 4-5) and in the presence of intracellular chloride ions. [Figure 16] Hematological parameters: Effect of liposomal aG6 treatment in mice at 40 mg / kg and 80 mg / kg once weekly for 4 weeks on white blood cell (WBC) count, neutrophil count, and platelet count. No significant decrease in mean neutrophil, mean white blood cell, or mean platelet count was observed. [Figure 17] The effects of weekly treatment of mice with liposomal aG6 at 40 mg / kg and 80 mg / kg for 4 weeks on hemoglobin and reticulocyte count indices are shown. The higher dose level results in the smallest decrease in mean hemoglobin concentration, accompanied by a slight increase in mean reticulocyte count indices. [Figure 18] The effect of treatment of mice with liposomal aG6 at 40 mg / kg and 80 mg / kg once weekly for 4 weeks on liver markers including serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) in addition to serum albumin is shown. No significant increase in mean AST or ALT levels of the liver aminotransferases was observed, and no change in mean albumin levels was observed. [Figure 19] Relative tumor volumes in immunocompromised female Nu / J mice (6-8 weeks old) inoculated with NCI-H292 (non-small cell lung cancer) cells and treated with control, pemetrexed, and 167 mg / kg of liposomal aG6 intravenously once every three weeks are shown. As can be seen from these preliminary data, liposomal aG6 results in lower tumor control compared to pemetrexed. [Figures 20A-20F]Liposomal pemetrexed alpha-L triglutamate (LPT) was administered for 48 hours to H2342 (NSCLC, adenocarcinoma subtype) (Figure 20A), H292 (NSCLC, adenocarcinoma subtype) (Figure 20B), HT-29 (colon cancer) (Figure 20C), HCC1806 (triple-negative breast cancer) (Figure 20D), MCF7 (ER+ breast cancer) (Figure 20E), and OAW28 (ovarian cancer) (Figure 20F). Figure 1 shows the dose-response relationships for liposomal pemetrexed alpha-L pentaglutamate (liposome aG3), liposomal pemetrexed alpha-L octaglutamate (liposome aG7), and the combination of liposomal pemetrexed alpha-L hexaglutamate (liposome aG6) and alpha-L dodecaglutamate (liposome aG12) (liposome aG6 and aG12). Cell viability was measured using the CellTiter-Glo® (CTG) luminescent cell viability assay essentially as described in Example 1. As can be seen in both cell lines, the potency of each polyglutamated pemetrexed liposome composition significantly exceeded that of the liposome vehicle and empty liposome controls. DETAILED DESCRIPTION OF THE INVENTION

[0093] The present disclosure generally relates to polyglutamated alpha-tetrahydrofolate compositions, which offer an advancement over previous treatments for hyperproliferative diseases such as cancer. Methods for the manufacture, delivery, and use of the polyglutamated alpha-tetrahydrofolate compositions are also provided. Polyglutamated 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. Polyglutamated alpha compositions also have uses in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effectiveness of the therapeutic agent, or as "chemopreventive agents" (e.g., in combination with antifolates such as methotrexate) to reduce toxic side effects associated with the therapeutic agent.

[0094] I. Definition 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] Whenever an embodiment is described herein with the word "comprising," it is understood that other similar embodiments are also provided that are described with the terms "containing," "consisting of," and / or "consisting essentially of." However, when used as transitional phrases in the claims, each should be construed separately and in the appropriate legal and factual context (e.g., in the claims, the transitional phrase "comprising" is considered to be the more open phrase, the transitional phrase "consisting of" is more exclusive, and "consisting essentially of" is intermediate).

[0096] As used herein, the singular forms "a," "an," and "the" include plural referents unless specifically stated otherwise or it is clear from the context that plural reference is not 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 encompass 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, respectively.

[0098] Headings and subheadings are used for convenience and / or to comply with official rules only, do not limit the subject technology, and are not to be cited in connection with interpreting the description of the subject technology. Features described under one heading or one subheading of the subject disclosure may be combined with features described under other headings or subheadings in various embodiments. Furthermore, features under a single heading or subheading are not necessarily all used together in the embodiments.

[0099] The terms "tetrahydrofolic acid" and "THF" are used interchangeably to include salt, acid, and / or free base forms of tetrahydrofolic acid (e.g., disodium tetrahydrofolate). Unless otherwise specified or clearly evident from the context, "THF" and "tetrahydrofolic acid" include natural and unnatural forms of THF, including single-carbon substituted THF derivatives. In particular, unless otherwise specified or clearly evident from the context, "THF" and "tetrahydrofolic acid" include diasteromeric compositions having the [6R] configuration at the C-6 atom of the tetrahydropterin component of THF, diasteromeric compositions having the [6S] configuration at the C-6 atom, and / or mixtures (e.g., 1:1) of [6,R,S] diastereomers. Unless otherwise specified or undoubtedly apparent 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- (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 compositions 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]-tetrahydrofolate-THF; (f) [6R]-5,10-methylene-THF; and (g) [6S]-5-formimino-THF. In some embodiments, the present disclosure provides compositions comprising a THF diastereomeric mixture (e.g., the diastereomeric mixture [6R,S]-5-methyl-THF (1:1) and / or the diastereomeric mixture [6R,S]-5-CHO-THF (1:1). Compositions comprising THF salts can be prepared with a variety of cations, e.g., Na. + , Mg 2+ , K. + , NH4 + , and / or Ca 2+ In certain embodiments, the salt is a pharmaceutically acceptable salt. In further particular embodiments, the THF salt is Na + Tetrahydrofolic acid contains one L-gamma glutamyl group and is therefore considered monoglutamated for the purposes of this 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]formamido}pentanedioic acid. "Tetrahydrofolic acid THF" is sometimes referred to herein as a type of tetrahydrofolic acid (THF).

[0101] The terms "polyglutamic acid," "polyglutamation," or variations thereof refer to a composition comprising at least one chain of two or more linked glutamyl groups. The polyglutamic acid chain can be linear or branched. A linear polyglutamic acid chain can include, for example, glutamyl groups with alpha or gamma carboxylic linkages. A branched polyglutamic acid chain can include, for example, one or more glutamyl groups with both alpha and gamma carboxylic linkages to other glutamyl groups, thereby creating branching points for the polyglutamic acid. Representative branched polyglutamic acids are shown in Figures 1R-1U. A polyglutamic acid chain includes an N-terminal glutamyl group and one or more C-terminal glutamyl groups. The N-terminal glutamyl group of a polyglutamic acid chain is not linked to another glutamyl group through its amino group, but is linked to one or more glutamyl groups through its carboxylic acid group. In some embodiments, the N-terminal glutamyl group of a polyglutamated tetrahydrofolate is the glutamyl group of tetrahydrofolate, and the C-terminal glutamyl group of the polyglutamate chain is bonded to another glutamyl group through its amino group, but not to another glutamyl group through its carboxylic acid group.

[0102] The terms "polyglutamylated-tetrahydrofolate," "polyglutamylated-THF," "THF-PG," "PTHF," and iterations thereof, are used herein to refer to tetrahydrofolate compositions containing at least one glutamyl group in addition to the glutamyl groups of tetrahydrofolate (i.e., THF-PG). n, where n≧1). When referring herein to the number of glutamyl groups in αPTHF (αTHF-PG), the glutamyl groups of tetrahydrofolate are taken into account. For example, a THF-PG composition containing five glutamyl residues in addition to the glutamyl groups of THF is referred to herein as hexaglutamated tetrahydrofolate or tetrahydrofolate hexaglutamate. In some embodiments, the polyglutamated tetrahydrofolate is (a) polyglutamated 5-formyl-THF; (b) polyglutamated 10-formyl-THF; (c) polyglutamated 5,10-methenyl-THF; (d) polyglutamated 5-methyl-THF; (e) polyglutamated tetrahydrofolate ((2S)-2-{[4-({[2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl] (f) polyglutaminated 5,10-methylene-THF; and (g) polyglutaminated 5-formimino-THF. In a further embodiment, the polyglutamated tetrahydrofolate is selected from the group consisting of (a) polyglutamated [6S]-5-formyl-THF; (b) polyglutamated [6R]-10-formyl-THF; (c) polyglutamated [6R]-5,10-methenyl-THF; (d) polyglutamated [6S]-5-methyl-THF; (e) polyglutamated [6S]-tetrahydrofolate THF; and (f) polyglutamated [6R]-5,10-methyl-THF. and (g) polyglutamic acid [6S]-5-formimino-THF. In some embodiments, the polyglutamated-tetrahydrofolic acid is [6R]-5,10-methylene-THF. In some embodiments, the polyglutamated-tetrahydrofolic acid is [6S]-5-methyl-THF. In some embodiments, the polyglutamated-tetrahydrofolic acid is [6S]-5-formyl-THF.In other embodiments, the polyglutamated-tetrahydrofolic acid is a [6R,S]-5,10-methylene-THF diastereomeric mixture, a [6R,S]-5-methyl-THF diastereomeric mixture, or a [6R,S]-5-formyl-THF diastereomeric mixture (e.g., a 1:1 weight ratio).

[0103] The terms "alpha glutamyl group," "alpha glutamic acid," and "alpha linkage," when referring to the linkage of a glutamyl group, refer to a glutamyl group containing an alpha carboxyl linkage. In some embodiments, the alpha linkage is an amide bond between the alpha carboxyl group of one glutamyl group and a second glutamyl group. The alpha linkage can be a bond between a glutamyl group and a glutamyl group in tetrahydrofolic acid, or a bond between a glutamyl group and a second glutamyl group not present in tetrahydrofolic acid, such as a glutamyl group in a polyglutamic acid chain linked to tetrahydrofolic acid. In some embodiments, the "alpha glutamyl group" of a provided polyglutamylated alpha tetrahydrofolic acid has both an alpha carboxyl linkage and a gamma carboxyl linkage. In some embodiments, the alpha glutamyl group is in the L-form. In some embodiments, the alpha glutamyl group is in the D-form. In some embodiments, the glutamyl group is in the L-form. In some embodiments, one or more glutamyl groups in the polyglutamated alphatetrahydrofolate are in the L-form and one or more glutamyl groups in the polyglutamated alphatetrahydrofolate are in the D-form.

[0104] The terms "polyglutamated alpha tetrahydrofolate," "α-polyglutamated tetrahydrofolate," "αPTHF," "polyglutamated alpha-tetrahydrofolate," "polyglutamated alpha THF," "αTHF-PG," and iterations thereof, as used herein, refer to tetrahydrofolate compositions (e.g., THF-PG) containing at least one glutamyl group with an alpha carboxyl group linkage. nwhere n≧1 α-glutamyl group). When referring herein to the number of glutamyl groups in αPTHF (αTHF-PG), the glutamyl groups of tetrahydrofolate are taken into account. For example, an αTHF-PG composition containing five glutamyl groups in addition to the glutamyl groups of THF, at least one of which has an alpha carboxyl linkage, may be referred to herein as alpha hexaglutamated tetrahydrofolate, hexaglutamated alpha tetrahydrofolate, or alpha tetrahydrofolate hexaglutamate.

[0105] The terms "gamma glutamyl group," "gamma glutamic acid," and "gamma linkage," when they relate to the linkage of a glutamyl group, refer to a glutamyl group containing a gamma carboxyl group linkage. In some embodiments, a gamma linkage refers to an amide bond between the gamma carboxyl group of one glutamyl group and a second glutamyl group. A gamma linkage can be between a glutamyl group and a glutamyl group of a tetrahydrofolate, or between a glutamyl group present in a tetrahydrofolate and a second glutamyl group, such as a glutamyl group in a polyglutamic acid chain linked to a tetrahydrofolate. In some embodiments, one or more gamma-linked glutamyl groups in a polyglutamylated alpha-tetrahydrofolate are in the L-form. In some embodiments, one or more gamma-linked glutamyl groups in a polyglutamylated alpha-tetrahydrofolate are in the D-form. In some embodiments, one or more gamma-linked glutamyl groups in the polyglutamated alphatetrahydrofolate are in the L-form and one or more gamma-linked glutamyl groups in the polyglutamated alphatetrahydrofolate are in the D-form.

[0106] As used herein, the term "isolated" refers to a composition in a form not found in nature. Isolated polyglutamated alpha compositions include those that have been purified to the extent that they are no longer in a form found in nature. In some embodiments, isolated polyglutamated alpha tetrahydrofolate is substantially pure. Isolated compositions are free or substantially free of materials that naturally accompany them, such as other cellular components, such as proteins and nucleic acids, that may be found in nature or in the environment in which the composition is prepared (e.g., cell culture). Polyglutamated alpha compositions can be formulated with diluents or adjuvants and even isolated for practical purposes; for example, for use in diagnostic or therapeutic methods, polyglutamated alpha compositions are typically mixed with a pharmaceutically acceptable carrier or diluent. In some embodiments, isolated polyglutamated alpha compositions (e.g., alpha polyglutamates and delivery vehicles, such as liposomes, containing alpha polyglutamates) contain less than 1% or less than 0.1% undesired DNA or protein content. In some embodiments, the alpha polyglutamic acid composition (eg, alpha polyglutamic acid and a delivery vehicle, such as a liposome, comprising alpha polyglutamic acid) is "isolated."

[0107] As used herein, the term "targeting moiety" refers to a molecule that enhances affinity for a selected target, e.g., a cell, cell type, tissue, organ, body region, or compartment, e.g., a cell, tissue, or organ compartment. Targeting moieties 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 avimer, a receptor-binding ligand, a nucleic acid, a biotin-avidin binding pair, a peptide, a protein, a carbohydrate, a lipid, a vitamin, a toxin, a component of a microorganism, 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 binds," and "enhanced affinity" mean that a targeting moiety, such as an antibody or antigen-binding antibody fragment, reacts or binds to an epitope, protein, or target molecule more frequently, more rapidly, for a longer period of time, with higher affinity, or with some combination thereof, than to another substance, including a protein unrelated to the antigen containing the target epitope. Due to sequence identity between homologous proteins in different species, a particular affinity, in some embodiments, includes binding substances that recognize epitopes on proteins or target molecules in more than one species. Similarly, due to homology within specific regions of the polypeptide sequences of different proteins, the terms "specific affinity" or "specific binding" can include binding substances that recognize epitopes present on more than one protein and / or target molecule. It is understood that in certain embodiments, 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 exclusive binding, e.g., binding to an epitope on a single target (although it may include such binding). Thus, a targeting moiety may, in certain embodiments, specifically bind to an epitope present on more than one target, and in certain embodiments, multiple targets may be bound by the same targeting moiety that specifically binds to an epitope present on multiple targets.

[0109] The term "epitope" refers to a portion of an antigen that can be recognized and specifically bound by a targeting moiety (i.e., binding moiety) such as an antibody. When the antigen is a polypeptide, epitopes can be formed from both contiguous and non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained when the protein is denatured, whereas epitopes formed by tertiary folding are typically lost when the protein is denatured. Epitopes typically contain at least three amino acids, more commonly at least five or eight to ten amino acids, in a unique spatial conformation.

[0110] Expressions known in the art such as " target binding affinity ", " target binding ", " enhanced affinity " and similar expressions refer to the property of targeting moiety, which can be directly measured by determining affinity constant, for example, the amount of targeting moiety that binds and dissociates at a given antigen concentration.Other methods can be used to characterize intermolecular interactions, including but not limited to, competitive analysis, equilibrium analysis, and microcalorimetry, and real-time interaction analysis based on surface plasmon resonance interaction (for example, using Biacore® device).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 refers to any composition that serves to assist, promote, or facilitate the entry of polyglutamated alphatetrahydrofolate into cells. Such delivery vehicles are known in the art and include, but are not limited to, liposomes, lipospheres, polymers (e.g., polymer conjugates), peptides, proteins such as antibodies (e.g., immunoconjugates such as antibody-drug conjugates (ADCs), and antigen-binding antibody fragments and derivatives thereof), cellular components, cyclic oligosaccharides (e.g., cyclodextrins), micelles, microparticles (e.g., microspheres), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), hydrogels, lipoprotein particles, viral sequences, viral agents, or lipid or liposomal formulations, and combinations thereof. The delivery vehicle can be directly or indirectly conjugated to a targeting moiety. In some examples, the targeting moiety is selected from a macromolecule, a protein, a peptide, a monoclonal antibody, or a fatty acid lipid.

[0112] "Subject" means a human or a vertebrate mammal, including, but not limited to, a dog, a cat, a horse, a goat, and a primate, e.g., a monkey. Thus, the present invention can also be used to treat a disease or condition in a non-human subject. For example, cancer is one of the leading causes of death in companion animals (e.g., cats and dogs). In some embodiments of the present invention, the subject is a human. In this disclosure, the terms "subject" and "patient" are used interchangeably and have the same meaning. Generally, it is preferable to use a maximum dosage, i.e., the highest safe dosage according to sound medical judgment.

[0113] As used herein, an "effective amount" refers to a drug dosage sufficient to achieve a medically desirable result. An effective amount may vary depending on the desired outcome, the specific condition being treated or prevented, the age and health of the subject being treated, the severity of the condition, the duration of treatment, the nature of concurrent or concomitant therapy (if any), the specific route of administration, and similar factors within the knowledge and expertise of a medical professional. An "effective amount" can be determined empirically and routinely in conjunction with the stated purpose. In the case of cancer, an effective amount of a drug is one that can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into surrounding organs; inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder. To the extent a drug can prevent the growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer treatment, in vivo efficacy can be measured, for example, by assessing survival time, progression-free survival (PFS), response rate (RR), duration of response, and / or quality of life.

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

[0115] "Cancer," "tumor," or "malignant tumor" are used interchangeably and refer to any of numerous cell types or diseases characterized by uncontrolled and abnormal growth of cells, the ability of affected cells to spread (metastasize) locally or via the bloodstream and lymphatic system to other parts of the body, and / or any characteristic structural and / or molecular features known to be associated with the cell type or disease. As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. A "cancerous tumor," or "malignant cell," is understood to be a cell that has specific structural characteristics, lacks differentiation, and is capable of invasion and metastasis. Cancers that can be treated using the αPTHF compositions provided herein include, but are not limited to, non-hematological tumors, 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, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors, such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. In some embodiments, the cancer is selected from the group consisting of 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 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 described herein or known in the art. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0117] Terms such as "treating," "treatment," or "treat" refer to both (a) therapeutic measures that cure, suppress, alleviate symptoms, and / or halt progression of a diagnosed condition or disorder, and (b) prophylactic or preventative measures that prevent and / or slow the onset of the targeted disease or condition. Thus, subjects in need of treatment include those already with cancer, a disorder, or disease, those at risk of having cancer or a condition, and those in whom an infection or condition is to be prevented. A subject has been identified, using well-known medical and diagnostic techniques, as being "at risk of having" cancer, an infectious disease, an immune system disorder, a hyperproliferative disease, or another disease or disorder referred to herein. In certain embodiments, a subject has been successfully "treated" by the methods provided herein if the subject experiences, for example, complete, partial, or temporary remission or elimination of symptoms associated with the disease or condition (e.g., cancer, inflammation, and rheumatoid arthritis). In certain embodiments, the term "treating" or "treatment" or "treat" refers to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as tumor growth, not necessarily discernible by the patient. In other embodiments, the term "treating" or "treatment" or "treat" refers to the inhibition of progression of a proliferative disorder, either physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physical parameter, or both. In other embodiments, the term "treating" or "treatment" or "treat" refers to the reduction or stabilization of tumor size, tumor cell growth or survival, or cancer cell number. Treatment can be with the αPTHF composition alone or in combination with an additional therapeutic agent.

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

[0119] "Treatment of a proliferative disorder" is used herein to encompass maintaining or reducing tumor size, inducing tumor regression (partial or complete), inhibiting tumor growth, and / or extending the lifespan of 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, bile duct cancer, 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 dysplasias or cachexia.

[0120] The term "autoimmune disease" as used herein is defined as a disorder caused by an autoimmune response. Autoimmune diseases are the result of an inappropriate or excessive response to 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 mellitus (type 1), 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, leukoplakia, myxedema, pernicious anemia, and ulcerative colitis.

[0121] The term "therapeutic agent" is used to mean a drug or derivative thereof that can interact with hyperproliferative cells, such as cancer cells or immune cells, thereby reducing the proliferative state of the cells and / or killing the cells. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, cytotoxic drugs, platinum-based drugs (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 derivatives thereof), antitumor antibiotics (e.g., mitomycin, doxorubicin), and plant-derived antitumor agents (e.g., vincristine, vindesine, Taxol®). Such agents further include, but are not limited to, the anticancer agents trimetrexate, temozolomide, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benziguanidine (6-BG), bis-chloronitrosourea (BCNU), and camptothecin™, or any therapeutic derivative thereof. Additional examples of therapeutic agents that may be suitable for use with the methods of the present disclosure include, but are not limited to, antirestenotic agents, pro- or antiproliferative agents, anti-inflammatory agents, antineoplastic agents, antimitotic agents, antiplatelet agents, anticoagulants, antifibrinogens, antithrombin agents, cytostatic agents, antibiotics and other anti-infective agents, antienzymes, antimetabolites, angiogenic agents, cytoprotective agents, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, and / or cardioprotective agents. "Therapeutic agent" also refers to salt, acid, and free base forms of the above agents.

[0122] As used herein, the term "chemotherapeutic agent," when used in the context of cancer therapy, refers to 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 polyglutamated alphatetrahydrofolate 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 "anti-metabolite" as used herein refers to a therapeutic agent that inhibits the utilization of a metabolite or its prodrug. Examples of anti-metabolites include 5-FU and 5-FU metabolites and / or prodrugs, such as 5-FUMP, 5-FUDP, 5-FdUMP, capecitabine, tegafur 5-fluorodeoxyuridine monophosphate, and the like; and cytarabine and cytarabine prodrugs such as nelarabine, 5-azacytidine, gemcitabine, mercaptopurine, thioguanine, azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, and cladribine. Anti-metabolites useful in practicing the methods of the present disclosure include nucleoside analogs, including purine or pyrimidine analogs. In some embodiments, the polyglutamated alpha tetrahydrofolate composition is used in combination with an antimetabolite selected from the group consisting of fluoropyrimidine, 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-azacytidine, capecitabine, N4-octadecylcytarabine, elaidic acid cytarabine, fludarabine, cladribine, clofarabine, nelarabine, forodesine, and pentostatin, or a derivative thereof. In one example, the nucleoside analog is a substrate for a nucleoside deaminase that is an adenosine deaminase or a cytidine deaminase. In some examples, the nucleoside analog is selected from fludarabine, cytarabine, gemcitabine, decitabine, and azacitidine or a derivative thereof.In some examples, the nucleoside analogs include 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-fluoro-cytidine and 5'-deoxy-5-fluorouridine, 1-hexylcarbamoyl-5-fluorouracil, B-3839, The antimetabolite is selected from 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 (Japanese Patent Publication No. 55059173), N'-(2-furanidyl)-5-fluorouracil (Japanese Patent Publication No. 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, "taxane" refers to an anti-cancer drug that interferes with or disrupts the stability, formation, and / or function of microtubules. Taxane drugs include paclitaxel and docetaxel, and derivatives thereof, which function on microtubules in the same manner as the taxane from which they are derived. In certain embodiments, the taxane is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the taxane is paclitaxel (Taxol®), docetaxel (Taxotere®), albumin-bound paclitaxel (nab-paclitaxel; Abraxane®), DHA-paclitaxel, or PG-paclitaxel.

[0125] The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a 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 and compatible for administration to humans or other subjects.

[0126] The present disclosure generally relates to polyglutamated alpha tetrahydrofolate (αPTHF) compositions and methods of making and using the compositions to treat diseases, including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria. Gamma polyglutamated compositions also find use in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effectiveness of the therapeutic agent, or as "chemopreventive agents" (e.g., in combination with antifolates such as methotrexate) to reduce toxic side effects associated with the therapeutic agent.

[0127] In some embodiments, the present disclosure provides: [1] A composition containing polyglutamylated alpha-tetrahydrofolic acid. [2] Polyglutamylated alpha-tetrahydrofolate (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-tetrahydrofolate contains 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups with alpha carboxyl group bonds. [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 polyglutamated alpha-tetrahydrofolate is pentaglutamated alpha-tetrahydrofolate (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 polyglutamated alpha-tetrahydrofolic acid is hexaglutamated 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 having an alpha carboxyl group bond; (b) each glutamyl group other than the glutamyl group of tetrahydrofolic acid has an alpha carboxyl linkage; or (c) two or more glutamyl groups have gamma carboxyl linkages; The composition according to any one of items [1] to [6]. [8] (a) the C-terminal glutamyl group and the glutamyl group other than the glutamyl group of tetrahydrofolate each have an alpha carboxyl group bond; or (b) each glutamyl group other than the C-terminal glutamyl group has 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 the alpha-polyglutamylated tetrahydrofolate are in the L-configuration; (b) each glutamyl group of the alpha-polyglutamylated tetrahydrofolate is in the L-configuration; (c) at least one of the glutamyl groups of the alpha polyglutamylated tetrahydrofolate is in the D-form; (d) each glutamyl group of the alpha-polyglutamylated tetrahydrofolate other than the glutamyl group of the tetrahydrofolate is in the D-form, or (e) alpha polyglutamylated tetrahydrofolate wherein at least two of the glutamyl groups are in the L-configuration and at least one of the glutamyl groups is in the D-configuration; The composition according to any one of items [1] to [9];

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

[10] , wherein the polyglutamic acid is linear;

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

[10] , wherein the polyglutamic acid is branched;

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

[12] ;

[14] The LαPP composition according to item

[13] , wherein the alpha polyglutamylated tetrahydrofolate contains an L-type glutamyl group having an alpha carboxyl group bond;

[15] The Lp-αPTHF composition according to item

[13] or

[14] , wherein each glutamyl group of the alpha polyglutamylated tetrahydrofolate is in the L-form;

[16] The Lp-αPTHF composition according to item

[13] or

[14] , wherein at least one of the glutamyl groups of the alpha polyglutamylated tetrahydrofolate 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 tetrahydrofolate 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 the alpha polyglutamylated tetrahydrofolate 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] , comprising 2, 3, 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups having both alpha carboxyl group bonds and gamma carboxyl group bonds;

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

[13] to

[20] , wherein the liposome contains alpha-polyglutamated tetrahydrofolic acid, including alpha-tetraglutamated tetrahydrofolic acid, alpha-pentaglutamated tetrahydrofolic acid, or alpha-hexaglutamated 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-polyglutamated tetrahydrofolic acid, or wherein in the process of preparing Lp-αPTHF, at least 1% of the starting material of alpha-polyglutamated THF is encapsulated (encapsulated) in 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 a liposome component;

[28] The Lp-αPTHF composition according to item

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

[29] The Lp-αPTHF composition according to item

[27] or

[28] , wherein the liposome component comprises 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 component comprises 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 comprise 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); phosphatidylpolyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymers; oligoglycerin, polyethylene glycol and polypropylene oxide-containing copolymers, poloxamer 188, and polyvinyl alcohol;

[33] The Lp-αPTHF composition according to item

[32] , wherein the steric stabilizer is PEG, and the 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 polyglutamylated alpha-tetrahydrofolic acid and an aqueous pharmaceutically acceptable carrier;

[40] The Lp-αPTHF composition according to item

[39] , wherein the pharmaceutically acceptable carrier comprises an isotonicity agent such as dextrose, mannitol, glycerol, potassium chloride, or sodium chloride 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 comprises 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 inner space of the liposome contains 5% dextrose suspended in a HEPES buffer solution;

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

[39] to

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

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

[13] to

[46] , wherein the inner space of the liposome has a pH of 5 to 8, or a pH of 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 alpha polyglutamated tetrahydrofolate;

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

[13] to

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

[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 a target cell of interest;

[51] The Lp-αPTHF composition according to item

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

[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 has a nucleotide sequence of 0.5 x 10 as measured using BIACORE® analysis. -10 ~10×10 -6 the Lp-αPTHF composition according to any one of items

[50] to

[53] , which binds to a surface antigen with an equilibrium dissociation constant (Kd) in the range of

[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 contains 1 to 1,000 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 immunostimulatory agent, a detectable marker, and a maleimide, wherein the immunostimulatory agent, the detectable marker, or the 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] Immunostimulants include fluorescein, fluorescein isothiocyanate (FITC), DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan; resolvins (e.g., D n-6DPA Or D n-3DPA the Lp-αPTHF composition according to item

[58] or

[59] , wherein the Lp-αPTHF composition is at least one selected from the group consisting of resolvin D, resolvin E, or T-series resolvins, and toll-like receptor (TLR) modulators, such as oxidized low-density lipoproteins (e.g., OXPAC, PGPC) and eritran lipids (e.g., E5564);

[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 cryoprotectant selected from the group consisting of mannitol; trehalose; sorbitol; and sucrose in the inner space, the outer space, or both inner spaces;

[65] A targeting 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-polyglutamated tetrahydrofolate composition according to any one of items

[13] to

[67] ;

[69] A pharmaceutical composition comprising the alpha polyglutamated 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 treating a disease;

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

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

[72] Use of any of the compositions [1]-

[70] in the manufacture of a medicament for the treatment of a disease and / or for use in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effect of the therapeutic agent(s), or as a "chemopreventive agent" (e.g., in combination with antifolates such as methotrexate) to reduce toxic side effects associated with the therapeutic agent(s);

[73] A method for treating or preventing a disease in a subject in need of such treatment or prevention, comprising administering to the subject any of the compositions of [1] to

[69] ;

[74] A method for treating or preventing a disease in a subject in need of such treatment or prevention, comprising administering to the subject the liposomal polyglutamated alpha-tetrahydrofolate composition according to any one of

[13] to

[69] ;

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

[69] ;

[76] A method for killing hyperproliferative cells, comprising contacting the hyperproliferative cells with the liposomal polyglutamated alpha-tetrahydrofolate composition according to any one of

[13] to

[69] ;

[77] The method according to item

[75] or

[76] , wherein the hyperproliferative 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 or at risk of having cancer;

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

[13] to

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

[80] The method according to item

[78] or

[79] , for treating or preventing cancer, wherein the cancer is selected from the group consisting of non-hematological tumors, 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, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors, such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias;

[81] The method according to item

[78] or

[79] , 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;

[82] The method according to item

[78] or

[79] , 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;

[83] The method according to item

[78] or

[79] , 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;

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

[50] to

[66] to a subject having or at risk of having cancer cells that express a folate receptor on their surface to which a targeting moiety binds;

[85] A maintenance therapy for a subject undergoing or having undergone cancer treatment, comprising administering an effective amount of the composition according to any one of items [1] to

[69] to the subject undergoing or having undergone cancer treatment;

[86] A maintenance therapy for a subject undergoing or having undergone cancer treatment, comprising administering to the subject undergoing or having undergone cancer treatment an effective amount of the liposomal polyglutamated alpha-tetrahydrofolate composition according to any one of items

[13] to

[69] ;

[87] 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, wherein 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, Takayasu's disease, and psoriasis;

[88] A method for treating an immune system disorder, comprising administering an effective amount of the liposomal polyglutamated alphatetrahydrofolate composition according to any one of items

[13] to

[69] to a subject having or at risk of having an immune system disorder, wherein 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, Takayasu's disease, and psoriasis;

[89] Treatment with: (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 or 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, wherein the inflammation is, optionally, 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-polyglutamated tetrahydrofolate 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 polyglutamated alpha-tetrahydrofolate to a tumor expressing a folate receptor on its surface, comprising administering to a subject having a tumor the Lp-αPTHF composition described in any of items [1] to

[69] in an amount sufficient to deliver a therapeutically effective amount of alpha-polyglutamated tetrahydrofolate to the tumor;

[92] A method for preparing an alpha polyglutamated tetrahydrofolate composition comprising the liposomal alpha polyglutamated tetrahydrofolate composition according to any one of items

[13] to

[69] , the method comprising: forming a mixture in a solution containing liposome components and a polyglutamated alpha folate anti-metabolite; homogenizing the mixture in the solution to form liposomes; and treating the mixture to form liposomes containing alpha polyglutamated tetrahydrofolate;

[93] A method for preparing alpha-polyglutamated tetrahydrofolate containing the liposomal alpha-polyglutamated tetrahydrofolate composition according to any one of items

[13] to

[69] , the method comprising: forming a mixture containing liposome components and alpha-polyglutamated tetrahydrofolate in a solution; and treating the mixture to form liposomes containing alpha-polyglutamated tetrahydrofolate.

[94] The method according to item

[93] , wherein treating the mixture includes 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 in a solution containing liposome components and alpha-polyglutamated tetrahydrofolate; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes in which the alpha-polyglutamated tetrahydrofolate is entrapped and / or encapsulated; and providing a targeting moiety on the surface of the liposome, 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 containing liposome components and alpha-polyglutamated tetrahydrofolate in a solution; treating the mixture to form liposomes in which the alpha-polyglutamated tetrahydrofolate is entrapped and / or encapsulated; and providing a targeting moiety on the surface of the liposome, 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 comprises one or more steps of thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw method, reverse phase evaporation, 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 comprises one or more steps of changing the size of the liposomes by one or more steps of extrusion, high-pressure microfluidization, and / or sonication; and / or

[0100] The method according to any one of items

[92] to

[99] , wherein at least 1% of the alpha polyglutamylated tetrahydrofolate starting material is encapsulated or entrapped in liposomes.

[0128] II. Polyglutamylated alpha-tetrahydrofolate (αPTHF) The present disclosure generally relates to polyglutamylated alpha-tetrahydrofolate (αPTHF) compositions. αPTHF compositions have at least one glutamyl group with an alpha carboxyl linkage. These compositions are structurally distinct from L-gamma polyglutamylated tetrahydrofolate (produced by the enzyme folylpoly-gamma-). In some embodiments, αPTHF compositions contain 2-20, 2-15, 2-10, 2-5, 2-6, or more than 6 glutamyl groups (including those in tetrahydrofolate). In some embodiments, each glutamyl group in αPTHF other than the glutamyl group in tetrahydrofolate has an alpha linkage. In some embodiments, each glutamyl group in αPTHF other than the C-terminal glutamyl group and in tetrahydrofolate has an alpha linkage. In some embodiments, each glutamyl group in αPTHF other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, two or more of the glutamyl groups in αPTHF have gamma linkages. In some embodiments, at least one glutamyl group of the alpha polyglutamated tetrahydrofolate has both an alpha carboxylic linkage and a gamma carboxylic linkage. In some embodiments, each glutamyl group in αPTHF is in the L-form. In some embodiments, the glutamyl groups in αPTHF other than the glutamyl group of the tetrahydrofolate are in the D-form. In some embodiments, αPTHF contains two or more glutamyl groups in the L-form and one or more glutamyl groups in the D-form. 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, the alpha polyglutamated tetrahydrofolate is diglutamated. That is, the alpha polyglutamated tetrahydrofolate contains one additional glutamyl group in addition to the glutamyl groups of tetrahydrofolate (αTHF-PG1), and the additional glutamyl group is linked to the glutamyl group in the tetrahydrofolate via an alpha bond. In some embodiments, each glutamyl group in the alpha diglutamated tetrahydrofolate is in the L-form. In other embodiments, the alpha diglutamated THF contains a glutamyl group in the D-form.

[0130] In some embodiments, the alpha polyglutamated tetrahydrofolate is triglutamated. That is, the alpha polyglutamated 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 linkage. In other embodiments, one of the two additional glutamyl groups has an alpha linkage and the other glutamyl group has a gamma linkage. In some embodiments, one of the two additional glutamyl groups has an alpha linkage. In some embodiments, one of the two additional glutamyl groups has a gamma linkage. In some embodiments, two of the three glutamyl groups have alpha linkages. In other embodiments, one of the three glutamyl groups has an alpha linkage and another glutamyl group has a gamma linkage. In some embodiments, one glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, each of the glutamyl groups of alpha triglutamated tetrahydrofolate is in the L-form. In other embodiments, the alpha triglutamated THF comprises a glutamyl group in the D-form. In further embodiments, each of the glutamyl groups in the alpha triglutamated tetrahydrofolate other than the glutamyl group in the tetrahydrofolate is in the D-form. In further embodiments, the triglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0131] In some embodiments, the alpha polyglutamated tetrahydrofolate is tetraglutamated and therefore contains three additional glutamyl groups in addition to the glutamyl groups in tetrahydrofolate (αTHF-PG3). In some embodiments, each of the three glutamyl groups has an alpha linkage. In other embodiments, one or two of the three additional glutamyl groups have an alpha linkage, and the remaining two or one glutamyl groups each have a gamma linkage. In some embodiments, two of the three additional glutamyl groups have an alpha linkage. In other embodiments, one of the three glutamyl groups has an alpha linkage, and another additional glutamyl group has a gamma linkage. In other embodiments, one of the three additional glutamyl groups has an alpha linkage and a gamma linkage. In other embodiments, three of the four glutamyl groups have alpha linkages. In some embodiments, at least one glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, the alpha tetraglutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alpha-tetraglutamated tetrahydrofolate is in the L-form. In other embodiments, the alpha-tetraglutamated THF comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group of the alpha-tetraglutamated tetrahydrofolate other than the glutamyl group of the tetrahydrofolate is in the D-form. In further embodiments, the tetraglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0132] In some embodiments, the polyglutamylated alpha tetrahydrofolate is pentaglutamylated (αTHF-PG4) and comprises a chain of four additional glutamyl groups linked to a glutamyl group in the tetrahydrofolate. In some embodiments, each of the four additional glutamyl groups in the chain has an alpha linkage. In some embodiments, each of the four additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha linkage. In other embodiments, one, two, or three of the four additional glutamyl groups have an alpha linkage, and the remaining three, two, or one glutamyl groups are linked to the glutamyl groups of the molecule via a gamma linkage. In other embodiments, one or two of the four additional glutamyl groups have an alpha linkage, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via a gamma linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the five glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the five glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, the alpha-pentaglutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group in the alpha-pentaglutamated tetrahydrofolic acid is in the L-form. In other embodiments, the alpha-pentaglutamated THF contains a glutamyl group in the D-form. In further embodiments, each glutamyl group in the alpha-pentaglutamated tetrahydrofolic acid other than the glutamyl group in tetrahydrofolic acid is in the D-form. In further embodiments, the pentaglutamated THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0133] In some embodiments, the alpha polyglutamated tetrahydrofolate is hexaglutamated (αTHF-PG5) and comprises a chain of five additional glutamyl groups linked to a glutamyl group in the tetrahydrofolate. In some embodiments, each of the five additional glutamyl groups in the chain has an alpha linkage. In some embodiments, each of the five additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, four of the five additional glutamyl groups in the chain have alpha linkages. In other embodiments, one, two, three, or four of the five additional glutamyl groups are linked to a glutamyl group in the molecule via an alpha linkage, and the remaining four, three, two, or one glutamyl groups are each linked to a glutamyl group in the molecule via a gamma linkage. In other embodiments, one, two, three, or four of the five additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the six glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the six glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, five of the six glutamyl groups have alpha linkages. In some embodiments, the alpha hexaglutamylated THF contains two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups in the alpha hexaglutamylated tetrahydrofolic acid is in the L-form. In some embodiments, the alpha hexaglutamylated THF contains a glutamyl group in the D-form. In further embodiments, each glutamyl group of the alpha hexaglutamated tetrahydrofolate other than the glutamyl group of the tetrahydrofolate is D-type. In further embodiments, the hexaglutamated THF contains a glutamyl group of D-type and two or more glutamyl groups of L-type. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0134] In some embodiments, the alpha polyglutamated tetrahydrofolate is heptaglutamated (αTHF-PG6), thus comprising a chain of six additional glutamyl groups linked to the glutamyl groups of the tetrahydrofolate. In some embodiments, each of the six additional glutamyl groups has an alpha linkage. In some embodiments, each of the six additional glutamyl groups in the chain, other than the C-terminal glutamyl group, has an alpha linkage. In some embodiments, five of the six additional glutamyl groups in the chain have an alpha linkage. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have an alpha linkage, and the remaining five, four, three, two, or one glutamyl groups each have a gamma linkage. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have an alpha linkage, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via a gamma linkage. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the seven glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the seven glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, six of the seven glutamyl groups have alpha linkages. In some embodiments, the alphaheptaglutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group in the alphaheptaglutamated tetrahydrofolic acid is in the L-form. In other embodiments, the alphaheptaglutamated THF contains a glutamyl group in the D-form. In further embodiments, each glutamyl group in the alphaheptaglutamated tetrahydrofolic acid other than the glutamyl group in tetrahydrofolic acid is in the D-form. In further embodiments, the heptaglutamated THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

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

[0136] In some embodiments, the alpha polyglutamated tetrahydrofolate is nonaglutamated (αTHF-PG8) and comprises a chain of eight additional glutamyl groups linked to the glutamyl groups of the tetrahydrofolate. In some embodiments, each of the eight additional glutamyl groups in the chain, other than the C-terminal glutamyl group, has an alpha linkage. In some embodiments, seven of the eight additional glutamyl groups in the chain have an alpha linkage. In some embodiments, each of the eight additional glutamyl groups has an alpha linkage. In other embodiments, one, two, three, four, five, six, or seven of the eight additional glutamyl groups have an alpha linkage, and the remaining seven, six, five, four, three, two, or one glutamyl groups each have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, or 7 of the 8 additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 9 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 9 glutamyl groups other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 8 of the 9 glutamyl groups have alpha linkages. In some embodiments, the alpha nonaglutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alpha nonaglutamated tetrahydrofolate is in the L-form. In other embodiments, the alpha nonaglutamated THF contains a glutamyl group in the D-form. In further embodiments, each glutamyl group of the alpha-nonaglutamated tetrahydrofolate other than the glutamyl group of the tetrahydrofolate is in the D-form. In further embodiments, the nonaglutamated THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0137] In some embodiments, the alpha polyglutamated tetrahydrofolate is deca-glutamated (αTHF-PG9) (i.e., comprises a chain of nine additional glutamyl groups linked to a glutamyl group of tetrahydrofolate). In some embodiments, each of the nine additional glutamyl groups has an alpha linkage. In some embodiments, each of the nine additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, eight of the nine additional glutamyl groups in the chain have an alpha linkage. In other embodiments, one, two, three, four, five, six, seven, or eight of the nine additional glutamyl groups have an alpha linkage, and the remaining eight, seven, six, five, four, three, two, or one glutamyl groups each have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 of the 9 additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 10 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 10 glutamyl groups other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 9 of the 10 glutamyl groups have alpha linkages. In some embodiments, the alpha-decaglutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the alpha-decaglutamated tetrahydrofolic acid is in the L-form. In other embodiments, the alpha-decaglutamated THF contains a glutamyl group in the D-form. In further embodiments, each glutamyl group of the alpha-decaglutamated tetrahydrofolate other than the glutamyl group of the tetrahydrofolate is in the D-form. In further embodiments, the deca-glutamated THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0138] In some embodiments, the alpha polyglutamated tetrahydrofolate is undecaglutamated (αTHF-PG 10In some embodiments, each of the 10 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 10 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 9 of the 10 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an alpha linkage, and the remaining 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an alpha linkage, and the remaining non-C-terminal glutamyl groups are connected to the glutamyl groups of the molecule via a gamma linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 11 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 11 glutamyl groups other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 10 of the 11 glutamyl groups have an alpha linkage. In some embodiments, the alpha-undeca-glutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups in the alpha-undeca-glutamated tetrahydrofolic acid is in the L-form. In other embodiments, the alpha-undeca-glutamated THF contains a glutamyl group in the D-form. In further embodiments, each of the glutamyl groups in the alpha-undeca-glutamated tetrahydrofolic acid other than the glutamyl group in tetrahydrofolic acid is in the D-form. In further embodiments, the undecaglutamated THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

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

[0140] In some embodiments, the alpha polyglutamated tetrahydrofolate is triskite-decaglutamated (αTHF-PG 12In some embodiments, each of the 12 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 12 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 11 of the 12 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alpha linkage, and the remaining 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 13 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 13 glutamyl groups other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 12 of the 13 glutamyl groups have alpha linkages. In some embodiments, the alpha-Triskaidecaglutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups in the alpha-Triskaidecaglutamated tetrahydrofolic acid is in the L-form. In other embodiments, the alpha-Triskaidecaglutamated THF contains glutamyl groups in the D-form. In further embodiments, each glutamyl group of the alpha-triskaidecaglutamated tetrahydrofolate other than the glutamyl group of the tetrahydrofolate is in the D-form. In further embodiments, the triskaidecaglutamated THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0141] In some embodiments, the alpha polyglutamated tetrahydrofolate is tetradecaglutamated (αTHF-PG 13In some embodiments, each of the 13 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 13 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 12 of the 13 additional glutamyl groups in the chain have an alpha linkage. 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 linkage, and the remaining 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 14 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 14 glutamyl groups other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 13 of the 14 glutamyl groups have alpha linkages. In some embodiments, the alpha-tetradecaglutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the alpha-tetradecaglutamated tetrahydrofolate is in the L-form. In other embodiments, the alphatetradecaglutamated THF comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group in the alphatetradecaglutamated tetrahydrofolate other than the glutamyl group in tetrahydrofolate is in the D-form. In further embodiments, the tetradecaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

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

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

[0144] In some embodiments, the alpha polyglutamated tetrahydrofolate is heptadecaglutamated (αTHF-PG 16In some embodiments, each of the 16 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 16 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 15 of the 16 additional glutamyl groups in the chain have an alpha linkage. 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 linkage, 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 linkage. 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 alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 17 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 17 glutamyl groups other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 16 of the 17 glutamyl groups have alpha linkages. In some embodiments, the alphaheptadecaglutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the alphaheptadecaglutamated tetrahydrofolic acid is in the L-form. In other embodiments, the alphaheptadecaglutamated THF comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group in the alphaheptadecaglutamated tetrahydrofolic acid other than the glutamyl group in tetrahydrofolic acid is in the D-form. In further embodiments, the heptadecaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0145] In some embodiments, the alpha polyglutamated tetrahydrofolate is octadeca-glutamated (αTHF-PG 17In some embodiments, each of the 17 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 17 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 16 of the 17 additional glutamyl groups in the chain have an alpha linkage. 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 linkage, 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 linkage. 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 alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 18 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 18 glutamyl groups other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 17 of the 18 glutamyl groups have alpha linkages. In some embodiments, the alphaoctadecaglutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the alphaoctadecaglutamated tetrahydrofolic acid is in the L-form. In other embodiments, the alpha-octadeca-glutamated THF comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group in the alpha-octadeca-glutamated tetrahydrofolic acid other than the glutamyl group in tetrahydrofolic acid is in the D-form. In further embodiments, the octadeca-glutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0146] In some embodiments, the alpha polyglutamated tetrahydrofolate is eniadecadecaglutamated (αTHF-PG 18In some embodiments, each of the 18 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 18 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 17 of the 18 additional glutamyl groups in the chain have an alpha linkage. 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 linkage, 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 linkage. 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 alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 19 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 19 glutamyl groups other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 18 of the 19 glutamyl groups have alpha linkages. In some embodiments, the alphaeniadecaglutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alphaeniadecaglutamated tetrahydrofolic acid is in the L-form. In other embodiments, the alphaeniadecaglutamated THF comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group of the alphaeniadecaglutamated tetrahydrofolic acid other than the glutamyl group of the tetrahydrofolic acid is in the D-form. In further embodiments, the eniadecaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain is linear.In another embodiment, the polyglutamic acid chain is branched.

[0147] In some embodiments, the alpha polyglutamated tetrahydrofolate is eicosiglutamated (αTHF-PG 19In some embodiments, each of the 19 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 19 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 18 of the 19 additional glutamyl groups in the chain have an alpha linkage. 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 linkage, 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 linkage. 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 alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 20 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 20 glutamyl groups other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 19 of the 20 glutamyl groups have alpha linkages. In some embodiments, the alpha-icosiglutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alpha-icosiglutamated tetrahydrofolic acid is in the L-form. In other embodiments, the alpha-icosiglutamated THF comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group of the alpha-icosiglutamated tetrahydrofolic acid other than the glutamyl group of the tetrahydrofolic acid is in the D-form. In further embodiments, the alpha-icosiglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamic acid chain is linear.In another embodiment, the polyglutamic acid chain is branched.

[0148] In some embodiments, the alpha polyglutamated tetrahydrofolate is hydroxylated (αTHF-PG 20In some embodiments, each of the 20 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 20 additional glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 19 of the 20 additional glutamyl groups in the chain have an alpha linkage. 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 linkage, 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 linkage. 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 alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 21 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 21 glutamyl groups other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, 20 of the 21 glutamyl groups have alpha linkages. In some embodiments, the alpha-cosmic glutamate-modified THF contains two or more glutamyl groups in the L-form. In a further embodiment, each glutamyl group of the alpha-coshikaihenaglutamated tetrahydrofolic acid is in the L-form. In another embodiment, the alpha-coshikaihenaglutamated THF contains a glutamyl group in the D-form. In a further embodiment, each glutamyl group of the alpha-coshikaihenaglutamated tetrahydrofolic acid other than the glutamyl group of tetrahydrofolic acid is in the D-form. In a further embodiment, the alpha-coshikaihenaglutamated THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form.In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0149] In some embodiments, the alpha polyglutamated tetrahydrofolate comprises 4 to 7 glutamyl groups linked to tetrahydrofolate (i.e., αTHF-PGn, n=4-7), and each of the 4 to 7 linked glutamyl groups has an alpha linkage. In some embodiments, the alpha polyglutamated tetrahydrofolate comprises 4 to 7 glutamyl groups linked to tetrahydrofolate (i.e., αTHF-PGn, n=4-7), and each of the 4 to 7 linked glutamyl groups other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, each of the 4 to 7 linked glutamyl groups is in the L-configuration. In other embodiments, each of the 4 to 7 linked glutamyl groups is in the D-configuration. In other embodiments, the 4 to 7 linked glutamyl groups are in both the L- and D-configuration. In some embodiments, the polyglutamic acid chain is linear. In other embodiments, the polyglutamic acid chain is branched.

[0150] In one embodiment, the alpha polyglutamated tetrahydrofolate is tetraglutamated, and each of the three glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate comprises an alpha linkage. In one embodiment, the alpha polyglutamated tetrahydrofolate is tetraglutamated, and each of the three glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate, other than the C-terminal glutamyl group, comprises an alpha linkage. In some embodiments, each of the four glutamyl groups is L-type. In some embodiments, each of the glutamyl groups in the alpha tetraglutamated tetrahydrofolate, other than the glutamyl group of the tetrahydrofolate, is D-type. In other embodiments, at least two glutamyl groups in the alpha tetraglutamated tetrahydrofolate are L-type and at least one glutamyl group is D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0151] In one embodiment, the alpha polyglutamated tetrahydrofolate is pentaglutamated, and each of the four glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate comprises an alpha linkage. In one embodiment, the alpha polyglutamated tetrahydrofolate is pentaglutamated, and each of the four glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate, other than the C-terminal glutamyl group, comprises an alpha linkage. In some embodiments, each of the four glutamyl groups is L-type. In some embodiments, each of the glutamyl groups in the alpha pentaglutamated tetrahydrofolate, other than the glutamyl group in the tetrahydrofolate, is D-type. In other embodiments, at least two glutamyl groups in the alpha pentaglutamated tetrahydrofolate are L-type and at least one glutamyl group is D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0152] In one embodiment, the alpha polyglutamated tetrahydrofolate is hexaglutamated, and each of the five glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate comprises an alpha linkage. In one embodiment, the alpha polyglutamated tetrahydrofolate is hexaglutamated, and each of the five glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate, other than the C-terminal glutamyl group, comprises an alpha linkage. In some embodiments, each of the five glutamyl groups is L-type. In some embodiments, each of the glutamyl groups in the alpha hexaglutamated tetrahydrofolate, other than the glutamyl group in the tetrahydrofolate, is D-type. In other embodiments, at least two glutamyl groups in the alpha hexaglutamated tetrahydrofolate are L-type and at least one glutamyl group is D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0153] In another embodiment, the alpha polyglutamated tetrahydrofolate is heptaglutamated, and each of the six glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate comprises an alpha linkage. In another embodiment, the alpha polyglutamated tetrahydrofolate is heptaglutamated, and each of the six glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate, other than the C-terminal glutamyl group, comprises an alpha linkage. In some embodiments, each of the six glutamyl groups is L-type. In some embodiments, each of the glutamyl groups in the alpha heptaglutamated tetrahydrofolate, other than the glutamyl group in the tetrahydrofolate, is D-type. In other embodiments, at least two glutamyl groups in the alpha heptaglutamated tetrahydrofolate are L-type and at least one glutamyl group is D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

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

[0155] In some embodiments, alpha polyglutamylated tetrahydrofolate (αPTHF) contains a total of 2 to 20, 2 to 15, 2 to 10, 2 to 5, or any range therebetween, including the glutamyl groups of tetrahydrofolate. In some embodiments, each glutamyl group in αPTHF other than the glutamyl groups of tetrahydrofolate has an alpha linkage. In some embodiments, each glutamyl group in αPTHF other than the C-terminal glutamyl group and the glutamyl group of tetrahydrofolate has an alpha linkage. In some embodiments, each glutamyl group in αPTHF other than the C-terminal glutamyl group has an alpha linkage. 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 linkage. In some embodiments, αPTHF contains two or more glutamyl groups with gamma linkages. In further embodiments, 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 groups of tetrahydrofolic acid have alpha linkages, 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 gamma linkages. In some embodiments, each glutamyl group in αPTHF is in the L-form. In some embodiments, each glutamyl group in αPTHF other than the glutamyl groups of tetrahydrofolic acid is in the D-form. 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 in the L-form. 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 in the D-form.

[0156] In some embodiments, the alpha polyglutamated 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 groups of tetrahydrofolate. In further embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additional glutamyl groups have alpha linkages. In further embodiments, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group in the alpha polyglutamated tetrahydrofolate has gamma linkages. In some embodiments, at least one glutamyl group has both alpha and gamma linkages. In some embodiments, the glutamyl group in the tetrahydrofolic acid has an alpha linkage. In some embodiments, the glutamyl group in the tetrahydrofolic acid has both an alpha linkage and a gamma linkage.

[0157] In some embodiments, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in the alpha polyglutamated tetrahydrofolate are in the L-form, the D-form, or the L- and D-form. In some embodiments, each glutamyl group in the polyglutamated alpha tetrahydrofolate is in the L-form. In other embodiments, each glutamyl group in the polyglutamated alpha tetrahydrofolate other than the glutamyl group in the tetrahydrofolate is in the D-form. In alternative embodiments, at least two of the glutamyl groups in the polyglutamated alpha tetrahydrofolate are in the L-form and at least one of the glutamyl groups in the polyglutamated alpha tetrahydrofolate is in the D-form. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 glutamyl groups in the polyglutamated alphatetrahydrofolate are in the L-form. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 glutamyl groups in the polyglutamated alphatetrahydrofolate are in the D-form. In some embodiments, at least one glutamyl group has both an alpha linkage and a gamma linkage.

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

[0159] In further embodiments, provided compositions comprise αPTHF that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups having an alpha linkage. In some embodiments, αPTHF contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups in the L-form. In some embodiments, αPTHF contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups in the D-form. In some embodiments, αPTHF contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups in the L-form and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups in the D-form. In other embodiments, the polyglutamated alphatetrahydrofolate contains at least one glutamyl group with both alpha and gamma linkages. In some embodiments, the polyglutamated alphatetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 10 or more glutamyl groups with both alpha and gamma linkages.

[0160] In some embodiments, the alpha polyglutamated tetrahydrofolate comprises at least one glutamyl group with an alpha linkage and two, three, four, five, six, seven, eight, nine, one to ten, one to twenty, or more glutamyl groups with gamma linkages. For example, in some embodiments, the polyglutamated alpha tetrahydrofolate comprises one, two, three, four, five, six, seven, eight, nine, one to ten L-alpha glutamyl group linkages and one, two, three, four, five, six, seven, eight, nine, or one to ten L-gamma glutamyl group linkages. In some further embodiments, the polyglutamated alpha tetrahydrofolate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 L-alpha glutamyl group linkages and further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma glutamyl group linkages. In even further embodiments, the polyglutamated alpha tetrahydrofolate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-alpha glutamyl group linkages and further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma glutamyl group linkages. In other further embodiments, the polyglutamated alpha tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma glutamyl group linkages and 1, 2, 3, 4, 5, 6, or 1-10 L-gamma glutamyl group linkages. In other embodiments, the polyglutamated alpha tetrahydrofolate contains at least one glutamyl group with both alpha and gamma linkages. In some embodiments, the polyglutamated alpha tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 10 or more glutamyl groups with both alpha and gamma linkages.

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

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

[0163] In some embodiments, the alpha polyglutamated tetrahydrofolate compositions provided herein have greater cytotoxicity against hyperproliferative cells than tetrahydrofolic acid. In some embodiments, the hyperproliferative cells are cancer cells. In some embodiments, the hyperproliferative 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 polyglutamated tetrahydrofolate is hexaglutamated tetrahydrofolate.

[0164] In some embodiments, the alpha polyglutamated tetrahydrofolate compositions provided herein have fewer toxic side effects than tetrahydrofolic acid. In some embodiments, the alpha polyglutamated tetrahydrofolic acid compositions provided herein are less toxic to non-hyperproliferative cells than tetrahydrofolic acid. In some embodiments, the alpha polyglutamated tetrahydrofolic acid compositions provided herein are less toxic to neutrophils, liver cells, or colonic epithelial cells than tetrahydrofolic acid. 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 colonic epithelial cells are CCD841 colonic epithelial cells. In some embodiments, toxicity is measured by an in vitro assay. In some embodiments, the alpha polyglutamated tetrahydrofolic acid is hexaglutamated tetrahydrofolic acid.

[0165] In some embodiments, the alpha polyglutamated tetrahydrofolate compositions provided herein have fewer toxic side effects than tetrahydrofolic acid. In some embodiments, the alpha polyglutamated tetrahydrofolic acid compositions provided herein have fewer or less severe toxic side effects than tetrahydrofolic acid in in vivo assays. In some embodiments, the in vivo assays are performed in an in vivo mouse model. In some embodiments, the alpha polyglutamated tetrahydrofolic acid compositions provided herein have fewer or less severe hematologic or liver toxic side effects than tetrahydrofolic acid. In some embodiments, hematologic side effects are assessed by mean neutrophil, mean white blood cell, or mean platelet counts. In some embodiments, liver toxic side effects are assessed by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the in vivo assays involve administering 40 mg / kg or 80 mg / kg of the alpha polyglutamated tetrahydrofolic acid composition once weekly for 4 weeks. In some embodiments, the alpha polyglutamated tetrahydrofolate is hexaglutamated tetrahydrofolate.

[0166] In some embodiments, treatment with the alpha polyglutamated tetrahydrofolate compositions provided herein does not induce significant hematologic or hepatotoxic side effects in an in vivo mouse model. In some embodiments, hematologic side effects are assessed by mean neutrophil, mean white blood cell, or mean platelet counts. In some embodiments, hepatotoxic side effects are assessed by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the alpha polyglutamated tetrahydrofolate compositions provided herein do not significantly reduce mean neutrophil, mean white blood cell, or mean platelet counts. In some embodiments, the alpha polyglutamated tetrahydrofolate compositions provided herein do not significantly increase serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. In some embodiments, the alpha polyglutamated tetrahydrofolate compositions provided herein do not significantly reduce serum albumin levels. In some embodiments, the in vivo assay involves administering 40 mg / kg or 80 mg / kg of a polyglutamated alpha tetrahydrofolate composition once a week for four weeks. In some embodiments, the alpha polyglutamated tetrahydrofolate is hexaglutamated tetrahydrofolate.

[0167] In some embodiments, the αPTHF composition does not contain fluorine atoms. In some embodiments, the αPTHF composition does not contain 4-fluoroglutamyl groups.

[0168] Polyglutamylated alphatetrahydrofolate (αPTHF) compositions and uses thereof are further disclosed in U.S. Patent Application Nos. 62 / 374,458, 62 / 583,432, 62 / 627,741, 62 / 630,820, and 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 contents of each of the foregoing applications are incorporated herein by reference in their entirety.

[0169] A. Polyglutamated Tetrahydrofolate Analogs and Derivatives The present disclosure also encompasses αPTHF derivatives and analogs. It is believed that the compositions and methods disclosed herein apply to any and all known derivatives or analogs of oxidized tetrahydrofolate. In some embodiments, the polyglutamated tetrahydrofolate analog or derivative compositions prepared and used according to the compositions and methods of the present disclosure are those illustrated in Figures 1I-1J. In some embodiments, the analog corresponds to a modified form of tetrahydrofolate in which the glutamyl group of the tetrahydrofolate is not linked to the remainder of the tetrahydrofolate molecule via a gamma peptide bond. In some embodiments, the analog is a variant of tetrahydrofolate in which the glutamyl group of the tetrahydrofolate is in the D-form. In some embodiments, the polyglutamated tetrahydrofolate or polyglutamated tetrahydrofolate analog or derivative is not fluorinated.

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

[0171] B.THF-PG synthesis The tetrahydrofolate polyglutamic acid compositions provided herein can be obtained by the following synthetic method using available reagents and synthetic intermediates. The addition of glutamyl residues to the glutamyl residues of tetrahydrofolate can be carried out using synthetic methods known in the art. In some embodiments, glutamyl residues are added sequentially to the glutamyl residues of tetrahydrofolate. In further embodiments, polyglutamates are added to the glutamyl residues of tetrahydrofolate using "click chemistry" methods or other bioconjugate chemistries known to those skilled in the art. Alternatively, a peptide of the desired length of glutamyl residues can be generated and added to a precursor lacking the glutamyl residues of tetrahydrofolate. Peptides can be generated using methods known in the art. In some embodiments, the first glutamyl residue is attached to Wang resin, and additional glutamyl residues are added sequentially by solid-phase peptide synthesis using F-moc chemistry. After the final glutamyl residue is added, the tetrahydrofolate precursor is coupled to the peptide, and the molecule is cleaved from the resin.

[0172] The addition of glutamyl residues to the glutamyl residue of tetrahydrofolate can be achieved using synthetic methods known in the art. In some embodiments, glutamyl residues are added sequentially to the glutamyl residue of tetrahydrofolate. In further embodiments, polyglutamates are added to the glutamyl residue of tetrahydrofolate using "click chemistry" methods and other bioconjugate chemistries known to those skilled in the art. Alternatively, a peptide of the desired length of glutamyl residues can be generated and added to a precursor of tetrahydrofolate that lacks glutamyl residues. Peptides can be generated using synthetic methods known in the art. In some embodiments, the first glutamyl residue is attached to Wang resin, and additional glutamyl residues are added sequentially by solid-phase peptide synthesis using F-moc chemistry. After the final glutamyl residue is added, the tetrahydrofolate precursor is coupled to the peptide, and the molecule is cleaved from the resin.

[0173] C. Tetrahydrofolate-PG complex The present inventors have surprisingly discovered that polyglutamated antifolates, which share substantially the same structural and chemical characteristics as tetrahydrofolic acid (αPTHF), can be complexed with other compositions containing therapeutic agents, including cytotoxic compounds such as platinum-based compounds. Thus, in some embodiments, the present disclosure provides complexes of αPTHF (e.g., the 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 cetaxel). In other embodiments, the αPTHF / complex comprises a cyclodextrin. In further embodiments, the αPTHF / complex is encapsulated in a liposome.

[0175] In some embodiments, the present disclosure provides compositions 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 containing 2-150, 2-100, 2-75, 2-50, 2-24, 2-30, 2-20, 2-19, 2-15, 2-10, or 2-5 glutamyl groups. In some embodiments, the αPTHF / therapeutic agent complex comprises one or more αPTHFs containing 3-10, 3-9, 3-8, or 3-7 glutamyl groups, or any range therebetween. In other embodiments, the αPTHF / therapeutic agent complex comprises one or more αPTHFs containing 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5 glutamyl groups, or any range therebetween. In certain embodiments, the complex comprises one or more αPTHFs containing 3 to 10 glutamyl groups. In further embodiments, the αPTHF / therapeutic agent complex comprises one or more αPTHFs containing 3 to 7 glutamyl groups. In another embodiment, the αPTHF / therapeutic agent complex comprises one or more αPTHFs containing 5 glutamyl groups. In another embodiment, the αPTHF / therapeutic agent complex comprises one or more αPTHFs containing 6 glutamyl groups. In some embodiments, the therapeutic agent is a cytotoxic compound, or a salt or acid thereof. In a further embodiment, 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 to therapeutic agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of αPTHF to therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of αPTHF to therapeutic agent in the complex is 1:1-20, 1:1-10, or 1:2-8, or any range therebetween.In some embodiments, the molar ratio of αPTHF / 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 greater than 1:>50. In some embodiments, the αPTHF / therapeutic agent complex is encapsulated in a liposome (e.g., as described herein or otherwise 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) to cyclodextrin in the complex is in the range of 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPTHF to cyclodextrin in the complex is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of αPTHF to cyclodextrin in the complex is in the range of 2 to 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 is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of 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 a liposome (e.g., as described herein or by other methods known in the art).

[0177] In some embodiments, the present disclosure provides compositions comprising an αPTHF / platinum-based chemotherapeutic agent conjugate. In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or a salt or acid thereof. In other embodiments, the αPTHF / platinum-based chemotherapeutic agent conjugate comprises an analog of cisplatin, carboplatin, or oxaliplatin, or a salt or acid thereof. In some embodiments, the molar ratio of αPTHF / platinum-based agent in the conjugate is in the range of 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPTHF / platinum-based agent in the conjugate is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of αPTHF / platinum-based agent in the conjugate is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPTHF to platinum-based drug 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 to platinum-based drug in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of αPTHF / platinum-based drug 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 / platinum-based drug complex is encapsulated in a liposome (e.g., as described herein or by other methods known in the art).

[0178] In further embodiments, the αPTHF / platinum-based chemotherapeutic agent conjugate comprises an analog of cisplatin, carboplatin, oxaliplatin, or a salt or acid thereof. In some embodiments, the molar ratio of αPTHF / platinum-based analog in the conjugate is in the range of 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPTHF / platinum-based analog in the conjugate is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of αPTHF / platinum-based agent in the conjugate is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPTHF to platinum-based analog in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of αPTHF to platinum-based analog in the complex is 1:1-20, 1:1-10, or 1:2-8, 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:(21-50), or 1:>50. In some embodiments, the αPTHF / platinum-based analog complex is encapsulated in a liposome (e.g., as described herein or by other methods known in the art).

[0179] In further embodiments, the present disclosure provides a complex comprising αPTHF and cisplatin, or a salt or acid thereof. In some embodiments, the molar ratio of αPTHF / cisplatin (or a cisplatin salt or acid) in the complex is in the range of 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPTHF / cisplatin (or a cisplatin salt or acid) in the complex is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of αPTHF / cisplatin (or a cisplatin salt or acid) in the complex is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPTHF to cisplatin (or cisplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of αPTHF to cisplatin (or cisplatin salt or acid) in the complex is 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of αPTHF / cisplatin (or cisplatin salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPTHF / cisplatin (or cisplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0180] In another embodiment, the present disclosure provides a complex comprising αPTHF and carboplatin, or a salt or acid thereof. In some embodiments, the molar ratio of αPTHF / carboplatin (or a carboplatin salt or acid) in the complex is in the range of 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of αPTHF / carboplatin (or a carboplatin salt or acid) in the complex is in the range of 1 to 10:1, or any range therebetween. In furth...

Claims

1. A liposome composition comprising a PEGylated liposome encapsulating an alpha polyglutamylated tetrahydrofolate, wherein 2 to 10 glutamyl groups of the alpha polyglutamylated tetrahydrofolate have an alpha carboxyl group bond; The alpha polyglutamylated tetrahydrofolate is (a) alpha polyglutamylated 5-formyl-THF (e.g., alpha polyglutamylated [6S]-5-formyl-THF); (b) alpha polyglutamylated 10-formyl-THF (e.g., alpha polyglutamylated [6R]-10-formyl-THF); (c) alpha polyglutamylated 5,10-methenyl-THF (e.g., alpha polyglutamylated [6R]-5,10-methenyl-THF); (d) alpha polyglutamylated 5-methyl-THF (e.g., alpha polyglutamylated [6S]-5-methyl-THF); (e) alpha polyglutamylated tetrahydrofolate (e.g., alpha polyglutamylated [6S]-tetrahydrofolate THF); (f) alpha polyglutamylated 5,10-methylene-THF (e.g., alpha polyglutamylated [6R]-5,10-methylene-THF); and (g) alpha polyglutamylated 5-formimino-THF (e.g., alpha polyglutamylated [6S]-5-formimino-THF) is selected from the group consisting of The liposomes further encapsulate one or more non-polyglutamylatable or non-polyglutamylatable antifolates. Liposomal compositions.

2. The alpha polyglutamylated tetrahydrofolate is (a) alpha polyglutamylated [6R]-5,10-methenyl-THF; (b) alpha polyglutamylated [6S]-5-methyl-THF; and (c) alpha polyglutamylated [6R]-5,10-methylene-THF The liposome composition of claim 1 , selected from the group consisting of:

3. the non-polyglutamylated polyglutamylatable antifolate is selected from the group consisting of methotrexate (MTX), pemetrexed (PMX), lometrexol (LMX), raltitrexed (RTX), pralatrexate, AG2034, GW1843, aminopterin, and LY309887; or The non-polyglutamylatable antifolate is selected from the group consisting of trimetrexate (TMQ), piritrexim (BW301U), tarotrexin (PT523) nolatrexed (AG337), previtrexed (ZD9331, BGC9331), and BGC945 (ONX0801); The liposome composition of claim 1.

4. 2. The liposome composition of claim 1, comprising alpha tetraglutamylated tetrahydrofolic acid, alpha pentaglutamylated tetrahydrofolic acid or alpha hexaglutamylated tetrahydrofolic acid.

5. (a) two or more glutamyl groups of the alpha polyglutamylated tetrahydrofolate have an alpha carboxyl group bond; (b) the glutamyl groups of the alpha polyglutamylated tetrahydrofolate each have an alpha carboxyl group bond; (c) two or more glutamyl groups of the alpha polyglutamylated tetrahydrofolate have a gamma carboxyl linkage; (d) at least one glutamyl group of the alpha polyglutamylated tetrahydrofolate has both an alpha carboxyl group linkage and a gamma carboxyl group linkage; (e) at least two glutamyl groups of the alpha polyglutamylated tetrahydrofolate are in the L-configuration; (f) each glutamyl group of the alpha polyglutamylated tetrahydrofolate is in the L-configuration; (g) at least one glutamyl group of said alpha polyglutamylated tetrahydrofolate is in the D-form; (h) each glutamyl group of said alpha polyglutamylated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form; or (i) at least two glutamyl groups of the alpha polyglutamylated tetrahydrofolate are in the L-form and at least one glutamyl group is in the D-form; The liposome composition of claim 1.

6. The liposome composition of claim 1 , wherein the polyglutamic acid is linear or branched.

7. 2. The liposome composition of claim 1, wherein the liposomes have a diameter in the range of 20 nm to 500 nm, or 20 nm to 200 nm, or 80 nm to 120 nm.

8. The liposomes are formed from liposome components including at least one of anionic and neutral lipids, DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide, and optionally 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, and optionally one or more of the liposome components are selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside 2. The liposome composition of claim 1, further comprising a steric stabilizer optionally selected from the group consisting of: (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidylpolyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid based polymers; oligoglycerin, polyethylene glycol and polypropylene oxide containing copolymers, poloxamer 188, and polyvinyl alcohol, optionally wherein the steric stabilizer is PEG, the PEG having a number average molecular weight (Mn) of 200 to 5000 Daltons.

9. The liposome is anionic. the liposome is cationic; The liposome is neutral. the liposome has a zeta potential of zero or less; the liposome has a zeta potential of 0 to -150 mV; or The liposome has a zeta potential of -30 to -50 mV. The liposome composition of claim 1.

10. 2. The liposome composition of claim 1, wherein the liposome has an interior space comprising alpha polyglutamated tetrahydrofolate and an aqueous pharma- ceutically acceptable carrier, optionally comprising an isotonicity agent such as dextrose, mannitol, glycerol, potassium chloride, sodium chloride at a concentration greater than 1%, 1%-50% trehalose, 5% dextrose suspended in HEPES buffer, or sodium acetate and calcium acetate at a total concentration of 50 mM-500 mM, optionally the liposome interior space has a pH of 5-8 or 6-7, or any range therebetween, and optionally the liposome comprises less than 500,000 molecules or less than 200,000 molecules of the alpha polyglutamated tetrahydrofolate, or 10-100,000 molecules or any range therebetween.

11. and further comprising a targeting moiety, said targeting moiety having specific affinity for a surface antigen on a target cell of interest, optionally wherein the targeting moiety is covalently attached to one or both of the PEG and the exterior of said liposome, and optionally wherein said targeting moiety has a specific affinity for a surface antigen on a target cell of interest of at least 0.5×10 －１０ ~10x10 －６ 10. The liposome composition of claim 1, wherein the PEGylated liposome binds to a surface antigen with an equilibrium dissociation constant (Kd) in the range of 0.1 to 0.5; and optionally 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); and optionally each PEGylated liposome comprises 1 to 1000 or 30 to 200 targeting moieties; and optionally 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.

12. The liposome further comprises one or more of an immunostimulant, a detectable marker, and a maleimide, wherein the immunostimulant, the detectable marker, or the maleimide is attached to PEG or the outside of the liposome, and the immunostimulant is selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), DNP, beta glucan, beta-1,3-glucan, beta-1,6-glucan, resolvin D (e.g., D ｎ－６ＤＰＡ Or D ｎ－３ＤＰＡ , resolvin E, or T-series resolvins), and oxidized low density lipoproteins (e.g., OXPAC, PGPC), and Toll-like receptor (TLR) modulators such as erythropoietin lipids (e.g., E5564), optionally further comprising at least one cryoprotectant selected from the group consisting of mannitol, trehalose, sorbitol, and sucrose, or further comprising carboplatin and / or pembrolizumab.

13. A pharmaceutical composition comprising the liposome composition of claim 1.

14. The liposomal composition according to any of claims 1 to 12 or the pharmaceutical composition according to claim 13 for use in the treatment of a disease or chemical-induced toxicity, optionally wherein the disease is cancer, a disorder of the immune system or an infectious disease, and optionally wherein the disease is leukopenia, an autoimmune disease, rheumatoid arthritis or an inflammatory condition.

15. 13. A method for preparing a liposome composition according to any one of claims 1 to 12, comprising forming a mixture comprising liposome components and alpha-polyglutamated tetrahydrofolate in a solution; homogenizing the mixture in the solution to form liposomes; and processing the mixture to form liposomes comprising alpha-polyglutamated tetrahydrofolate, optionally wherein the processing step comprises one or more of the following steps: thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw method, reverse phase evaporation, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, freeze-dried double emulsion, 3D printing, membrane contactor method, and stirring, or the processing step comprises applying a taconazole to the surface of the liposomes.

13. A method for treating liposomes comprising: providing a liposome-targeting moiety having specific affinity for at least one of the folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ); and optionally, the treating step comprises one or more steps of thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw technique, reverse phase evaporation, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, lyophilized double emulsion, 3D printing, membrane contactor, and agitation, or the treating step comprises one or more steps of modifying the size of the liposomes by one or more steps of extrusion, high pressure microfluidization, and / or sonication.

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