Gamma polyglutamylated tetrahydrofolate and uses thereof

Abstract

To provide a γ polyglutamated tetrahydrofolate composition for treating hyperproliferative disorders (e.g., cancer) and immune system disorders (e.g., inflammatory and autoimmune diseases).SOLUTION: Provided is a composition comprising a liposome encapsulating γ- polyglutamated tetrahydrofolate, the γ- polyglutamated tetrahydrofolate containing two or more glutamyl groups linked by γ-carboxyl group linkages and being selected from: (a) γ-polyglutamated 5-formyl-THF; (b) γ-polyglutamated 10-formyl-THF; (c) γ-polyglutamated 5,10-methenyl-THF; (d) γ-polyglutamated 5-methyl-THF; (e) γ-polyglutamated tetrahydrofolate; (f) γ-polyglutamated 5,10-methylene-THF; and (g) γ-polyglutamine oxidized 5-formimino-THF, and the liposome composition being pegylated and comprising a targeting moiety.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Folate is an essential cofactor mediating the transfer of one-carbon units involved in 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, and folate monoglutamate is the only form of folate transported across cell membranes, as is the monoglutamate form of tetrahydrofolate. Upon entry into cells, intracellular tetrahydrofolate is polyglutamated by the enzyme folylpoly-gamma-glutamate synthetase (FPGS). Polyglutamation of tetrahydrofolate by FPGS serves at least two primary therapeutic purposes: (1) it greatly enhances tetrahydrofolate affinity for DHFR; and (2) it promotes the accumulation of polyglutamated tetrahydrofolate, which, unlike tetrahydrofolate (monoglutamate), is not readily transported out of cells by cellular efflux pumps.

[0003] The gamma polyglutamated tetrahydrofolate compositions provided offer a strategy for improving the therapeutic efficacy of tetrahydrofolate. Summary of the Invention

[0004] The present disclosure generally relates to gamma polyglutamated tetrahydrofolate (THF) compositions and methods of making and using the compositions to treat diseases, including hyperproliferative diseases such as cancer, disorders of the immune system such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria. The alpha polyglutamated tetrahydrofolate compositions also have uses in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the efficacy of the therapeutic agent(s), or as "chemoprotectants" (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 comprising gamma-polyglutamylated tetrahydrofolic acid; [2] The composition of [1], wherein the gamma polyglutamylated tetrahydrofolate 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., polyglutamylated [6S]-5-methyl-THF); (e) polyglutamylated tetrahydrofolate (e.g., polyglutamylated [6S]-tetrahydrofolate); (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); [3] The gamma-polyglutamated tetrahydrofolate of the composition of [1] or [2] contains 4, 5, 2-10, 4-6, or more than 5 glutamyl groups with gamma carboxyl linkages; [4] The composition according to any one of [1]-[3], wherein the gamma-polyglutamated tetrahydrofolic acid is gamma-tetraglutamated tetrahydrofolic acid; [5] The composition according to any one of [1]-[3], wherein the gamma-polyglutamated tetrahydrofolate is gamma-pentaglutamated tetrahydrofolate; [6] The composition according to any one of [1]-[3], wherein the gamma-polyglutamated tetrahydrofolate is gamma-hexaglutamated tetrahydrofolate; [7] A composition according to any one of [1]-[6] below: (a) gamma-polyglutamated tetrahydrofolate contains two or more gamma-carboxyl-linked L-glutamyl groups; (b) each of the glutamyl groups of the gamma-polyglutamylated tetrahydrofolate is in the L-configuration and has a gamma-carboxyl linkage; (c) at least one of the glutamyl groups of the gamma polyglutamylated tetrahydrofolate is in the D-form and has a gamma carboxyl group bond; (d) each of the glutamyl groups of the gamma-polyglutamated tetrahydrofolate other than the glutamyl groups of the tetrahydrofolate is in the D-form and has a gamma carboxyl linkage; or (e) gamma-polyglutamated tetrahydrofolate contains two or more L-glutamyl groups and at least one D-glutamyl group with a gamma carboxyl group bond; [8] The composition according to [4], (a) each of the glutamyl groups is in the L-configuration and has a gamma carboxyl linkage; or (b) each of the glutamyl groups other than the glutamyl group of tetrahydrofolic acid is in the D-configuration and each of the glutamyl groups has a gamma carboxyl linkage; [9] The composition of [5], (a) each of the glutamyl groups is in the L-configuration and has a gamma carboxyl linkage; or (b) each of the glutamyl groups other than the glutamyl group of tetrahydrofolic acid is in the D-configuration and each of the glutamyl groups has a gamma carboxyl linkage;

[10] The composition of [6], (a) each of the glutamyl groups is in the L-configuration and has a gamma carboxyl linkage; or (b) each of the glutamyl groups other than the glutamyl group of tetrahydrofolic acid is in the D-configuration and each of the glutamyl groups has a gamma carboxyl linkage;

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

[10] , wherein gamma-polyglutamated tetrahydrofolate can be polyglutamated by FGPS under physiological conditions, and / or polyglutamated THF has a lower hepatocyte uptake rate (<30%) than THF;

[12] [1]-

[11] liposome composition containing gamma-polyglutamic tetrahydrofolate (Lp-γPTHF);

[13] The Lp-γPTHF composition according to

[12] , in which γ-polyglutamic tetrahydrofolate contains two or more L-glutamyl groups;

[14] Lp-γPTHF composition according to

[12] or

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

[15] The Lp-γPTHF composition of

[12] or

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

[16] The liposome contains 1-10 gamma-polyglutamic tetrahydrofolic acid containing glutamyl groups with gamma-carboxyl group bonds, and the Lp-γPTHF composition according to any of

[12] -

[15] ;

[17] Liposomes containing gamma polyglutamic tetrahydrofolic acid containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups, Lp-γPTHF compositions according to any of

[12] -

[16] ;

[18] The liposome contains gamma-tetraglutamylated tetrahydrofolic acid; Lp-γPTHF composition according to any of

[12] -

[17] ;

[19] Liposomes containing gamma pentaglutamated tetrahydrofolic acid; Lp-γPTHF composition according to any of

[12] -

[17] ;

[20] The liposome contains gamma hexaglutamated tetrahydrofolic acid, and the Lp-γPTHF composition is one of

[12] -

[17] ;

[21] Liposomes are not PEGylated (PγLp-γPTHF), Lp-γPTHF compositions according to either

[12] -

[20] ;

[22] The liposomes are PEGylated (PγLp-γPTHF), Lp-γPTHF compositions according to any of

[12] -

[20] ;

[23] An Lp-γPTHF composition according to any of

[12] -

[22] , wherein the liposomes contain at least 1% weight / weight (w / w) of γ-polyglutamated tetrahydrofolic acid, or wherein during the process of preparing Lp-γPTHF, at least 1% of the starting material of γ-polyglutamated THF is encapsulated (trapped) within the Lp-γPTHF;

[24] The liposomes have a diameter ranging from 20 nm to 500 nm; Lp-γPTHF compositions according to any of

[12] -

[23] ;

[25] The liposomes have a diameter ranging from 20 nm to 200 nm; Lp-γPTHF compositions according to any of

[12] -

[24] ;

[26] The liposomes have a diameter ranging from 80 nm to 120 nm. Lp-γPTHF compositions according to any of

[12] -

[25] ;

[27] Liposomes are formed from liposome components, and Lp-γPTHF compositions are prepared according to any of

[12] -

[26] ;

[28] The Lp-γPTHF composition according to

[27] , wherein the liposome component includes at least one of anionic lipids and neutral lipids;

[29] The Lp-γPTHF composition according to

[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-γPTHF composition according to any one of

[27] -

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

[27] -

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

[32] The Lp-γPTHF composition according to

[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-γPTHF composition according to

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

[34] Lp-γPTHF compositions according to either

[12] -

[33] , in which the liposomes are anionic or neutral;

[35] The liposomes have a zeta potential of less than zero. Lp-γPTHF compositions according to any of

[12] -

[33] ;

[36] The liposomes have a zeta potential of 0 to -150 mV. Lp-γPTHF compositions according to any of

[12] -

[33] ;

[37] The liposomes have a zeta potential of -30 to -50 mV. Lp-γPTHF compositions according to any of

[12] -

[33] ;

[38] The liposomes are cationic, and the Lp-γPTHF composition is one of

[12] -

[33] ;

[39] A liposome having an internal space containing gamma-polyglutamated tetrahydrofolic acid and an aqueous, pharmaceutically acceptable carrier, comprising an Lp-γPTHF composition according to any of

[12] -

[38] ;

[40] The pharmaceutically acceptable carrier includes an isotonic agent such as dextrose, mannitol, glycerin, potassium chloride, or sodium chloride at a concentration of more than 1% in the Lp-γPTHF composition of

[39] ;

[41] The aqueous Lp-γPTHF composition of

[39] , in which the pharmaceutically acceptable carrier is trehalose;

[42] The Lp-γPTHF composition of

[41] , wherein the pharmaceutically acceptable carrier contains 1% to 50% trehalose;

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

[39] -

[42] , wherein the pharmaceutically acceptable carrier comprises 1% to 50% dextrose solution;

[44] The inner space of the liposome contains 5% dextrose suspended in HEPES buffer. Lp-γPTHF compositions according to any of

[39] -

[43] ;

[45] Pharmaceutically acceptable carriers include buffers such as HEPES-buffered saline (HBS) or similar, at a concentration of 1-200 mM and a pH of 2-8, for Lp-γPTHF compositions according to any of

[39] -

[44] ;

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

[39] -

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

[47] Lp-γPTHF compositions according to any of

[12] -

[46] , in which the interior space of the liposome has a pH of 5-8 or 6-7, or any range therebetween;

[48] ​​Liposomes containing less than 500,000 or less than 200,000 molecules of gamma-polyglutamylated tetrahydrofolate, Lp-γPTHF compositions according to any of

[12] -

[47] ;

[49] Liposomes containing 10 to 100,000 molecules of gamma-polyglutamated tetrahydrofolate, or any range therebetween, comprise any of the Lp-γPTHF compositions according to

[12] -

[48] ;

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

[12] -

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

[51] The Lp-γPTHF composition according to

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

[52] The Lp-γPTHF composition of

[50] or

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

[53] Lp-γPTHF compositions according to any of

[50] -

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

[54] The targeting moiety has a 0.5x10 -10 ~10x10 -6 Lp-γPTHF compositions that bind to surface antigens with an equilibrium dissociation constant (Kd) in the range of

[50] -

[53] ;

[55] 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-γPTHF composition according to any of

[50] -

[55] , 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] Lp-γPTHF compositions according to any of

[50] -

[56] , in which each PEGylated liposome contains 1-1000 or 30-200 targeting moieties;

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

[39] -

[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 attached to the outer surface of the PEG or liposome;

[59] The Lp-γPTHF composition of

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

[60] The Lp-γPTHF composition of

[58] or

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

[61] Lp-γPTHF compositions using any of

[58] -

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

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

[58] -

[61] , further comprising a hapten;

[63] The Lp-γPTHF composition of

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

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

[12] -

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

[65] [1]-

[64] . A targeting composition comprising a composition according to any one of the above. a non-targeted composition comprising a composition according to any of

[66] [1]-

[49] ;

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

[12] -

[66] , further comprising carboplatin and / or pembrolizumab; a pharmaceutical composition comprising a liposomal gamma-polyglutamated tetrahydrofolate composition according to any one of

[68]

[12] -

[67] ;

[69] [1]-[7] A pharmaceutical composition comprising a gamma polyglutamic acid tetrahydrofolate composition;

[70] Any of the compositions [1]-

[69] for use in treating a disease;

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

[70] in the manufacture of a medicament for the treatment of a disease and / or in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effectiveness of the therapeutic agent(s), or as a "chemoprotectant" (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 (e.g., treating or preventing) a disease or chemical-induced toxicity in a subject in need of such treatment or prevention, comprising administering to the subject any of the compositions of [1]-

[70] ;

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

[12] -

[69] ;

[74] A method for killing hyperproliferative cells, comprising contacting the hyperproliferative cells with any of the compositions of [1]-

[69] ;

[75] A method for killing hyperproliferative cells, comprising contacting the hyperproliferative cells with any of the liposomal gamma-polyglutamated tetrahydrofolate compositions of

[12] -

[69] ;

[76] The method of

[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 any of the compositions of [1]-

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

[78] A method for treating (e.g., treating or preventing) cancer, comprising administering an effective amount of any of the liposomal gamma-polyglutamated tetrahydrofolate compositions

[12] -

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

[79] The method of

[77] or

[78] , wherein the composition is administered to treat or prevent cancer, wherein the cancer is 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;

[80] The method of

[77] or

[78] , wherein the composition is administered to treat or prevent cancer, and 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 of

[77] or

[78] , wherein the composition is administered to treat or prevent cancer, and the cancer is a member 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;

[82] The method of

[77] or

[78] , wherein the composition is administered to treat or prevent colorectal cancer;

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

[50] -

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

[84] A maintenance therapy comprising administering an effective amount of any of the compositions of [1]-

[69] to a subject undergoing or having undergone cancer therapy;

[85] A maintenance therapy comprising administering an effective amount of any of the liposomal gamma-polyglutamated tetrahydrofolate compositions

[12] -

[69] to a subject undergoing or having undergone cancer therapy;

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

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

[87] A method for treating (e.g., treating or preventing) an immune system disorder, comprising administering an effective amount of any of the liposomal gamma-polyglutamated tetrahydrofolate compositions of [8]-

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

[88] A method for treating (e.g., treating or preventing) the following: (a) a method for treating (e.g., treating or preventing) leukopenia, comprising administering an effective amount of a composition according to any of [1]-

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

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

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

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

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

[59] 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 (e.g., treating or preventing) a skin disease, comprising administering an effective amount of a composition according to any of [1]-

[59] to a subject having or at risk of having a skin disease, wherein optionally the skin disease is psoriasis;

[89] A method for treating (e.g., treating or preventing) an infectious disease, comprising administering an effective amount of a liposomal gamma-polyglutamated tetrahydrofolate composition according to any one of

[12] -

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

[90] A method for delivering gamma polyglutamated tetrahydrofolate to a tumor expressing a folate receptor on its surface, comprising: administering any of the Lp-gamma PTHF compositions [1]-

[69] to a subject having a tumor in an amount sufficient to deliver a therapeutically effective amount of gamma polyglutamated tetrahydrofolate to the tumor; A method for preparing a gamma-polyglutamated tetrahydrofolate composition, including the liposomal gamma-polyglutamated tetrahydrofolate composition of any of

[91]

[12] -

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

[92] A method for preparing any of the compositions of

[12] -

[69] , comprising the steps of: forming a mixture in a solution containing liposome components and gamma-polyglutamated tetrahydrofolate; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes that entrap and / or encapsulate gamma-polyglutamated tetrahydrofolate; and providing a targeting moiety on the surface of the liposome, the targeting moiety having 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 processing step includes one or more of the following steps: thin film hydration, extrusion, in-line mixing, ethanol injection techniques, freeze-thaw techniques, reverse phase evaporation, dynamic high-pressure microfluidization, microfluidic mixing, double emulsion, freeze-dried double emulsion, 3D printing, membrane contactor techniques, and stirring; and / or

[94] The method according to

[92] , wherein the processing 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.

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

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

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

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

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

[0011] In some embodiments, the present disclosure provides for the use of a composition containing a delivery vehicle, such as a liposome, loaded with (e.g., encapsulating) and / or otherwise associated with gamma-polyglutamated tetrahydrofolate to enhance the efficacy of a therapeutic agent(s) in combination therapy with one or more therapeutic agents, such as a chemotherapeutic agent (e.g., 5-fluorouracil), or to reduce toxic side effects associated with a therapeutic agent(s) as a "chemoprotectant" (e.g., in combination with an antifolate such as methotrexate). In some embodiments, the gamma-polyglutamated tetrahydrofolate in DV-αPTHF contains 2-20, 2-15, 2-10, 2-5, more than 5, or more than 20 glutamyl groups (including the glutamyl groups in tetrahydrofolate). The DV-αPTHF loaded / bound delivery vehicle composition provides improved efficacy and safety of delivering tetrahydrofolate to cancer cells by providing preferential delivery of a more cytotoxic payload (e.g., polyglutamylated tetrahydrofolate) compared to the cytotoxicity of tetrahydrofolate administered in its monoglutamate form (THF).

[0012] In additional embodiments, the present disclosure provides compositions comprising gamma polyglutamylated tetrahydrofolate (γPTHF).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0036] In one embodiment, the composition comprises gamma polyglutamated 5-formimino-THF (i.e., hexaglutamated 5-formimino-THF) containing a chain of five glutamyl groups attached to the glutamyl group of a tetrahydrofolic acid. In some embodiments, the composition comprises gamma hexaglutamated [6S]-5-formimino-THF. In some embodiments, the composition comprises gamma hexaglutamated [6R,S]-5-formimino-THF. In some embodiments, the composition comprises gamma hexaglutamated [6R]5-formimino-THF. In some embodiments, the gamma hexaglutamated 5-formimino-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the gamma hexaglutamated 5-formimino-THF comprises a glutamyl group in the D-form. In a further embodiment, the gamma hexaglutamated 5-formimino-THF contains a D-glutamyl group and two or more L-glutamyl groups.

[0037] In additional embodiments, the present disclosure provides compositions comprising liposomes encapsulating (loaded with) gamma polyglutamated tetrahydrofolate (Lp-γPTHF).

[0038] In some embodiments, the present disclosure provides a composition comprising a liposome encapsulating (loaded with) gamma-polyglutamylated 5-formyl-THF. In some embodiments, the liposome comprises gamma-polyglutamylated [6S]-5-formyl-THF. In some embodiments, the liposome comprises gamma-polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposome comprises gamma-polyglutamylated [6R]-5-formyl-THF. In some embodiments, the liposome contains gamma-polyglutamylated 5-formyl-THF having 2-20, 2-15, 2-10, 2-5, or more than 20 glutamyl groups (including the glutamyl group in 5-formyl-THF). In some embodiments, the liposome contains gamma-polyglutamylated 5-formyl-THF having two or more L-configured glutamyl groups. In other embodiments, the liposomes contain gamma polyglutamylated 5-formyl-THF having a glutamyl group in the D-form. In further embodiments, the liposomes contain gamma polyglutamylated 5-formyl-THF having a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

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

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

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

[0042] In some embodiments, the present disclosure provides a composition comprising a liposome encapsulating (loaded with) gamma-polyglutamylated 5,10-methenyl-THF. In some embodiments, the liposome comprises gamma-polyglutamylated [6R]-5,10-methenyl-THF. In some embodiments, the liposome comprises gamma-polyglutamylated [6R,S]-5,10-methenyl-THF. In some embodiments, the liposome comprises gamma-polyglutamylated [6S]-5,10-methenyl-THF. In some embodiments, the liposome contains gamma-polyglutamylated 5,10-methenyl-THF having 2-20, 2-15, 2-10, 2-5, or more than 20 glutamyl groups (including the glutamyl groups in 5,10-methenyl-THF). In some embodiments, the liposome contains gamma-polyglutamylated 5-formyl-THF having two or more L-type glutamyl groups. In other embodiments, the liposomes contain gamma polyglutamylated 5,10-methenyl-THF having a glutamyl group in the D-form. In further embodiments, the liposomes contain gamma polyglutamylated 5,10-methenyl-THF having a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

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

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

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

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

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

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

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

[0050] In some embodiments, the present disclosure provides compositions comprising liposomes encapsulating (loaded with) gamma-polyglutamylated THF. In some embodiments, the liposomes comprise gamma-polyglutamylated [6S]-THF. In some embodiments, the liposomes comprise gamma-polyglutamylated [6R,S]-THF. In some embodiments, the liposomes comprise gamma-polyglutamylated [6R]-THF. In some embodiments, the liposomes contain gamma-polyglutamylated THF having 2-20, 2-15, 2-10, 2-5, or more than 20 glutamyl groups (including glutamyl groups in THF). In some embodiments, the liposomes contain gamma-polyglutamylated THF having two or more L-type glutamyl groups. In other embodiments, the liposomes contain gamma-polyglutamylated THF having D-type glutamyl groups. In further embodiments, the liposomes contain gamma-polyglutamylated THF having D-type glutamyl groups and two or more L-type glutamyl groups.

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

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

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

[0054] In some embodiments, the present disclosure provides compositions comprising liposomes encapsulating (loaded with) gamma-polyglutamylated 5,10-methylene-THF. In some embodiments, the liposomes comprise gamma-polyglutamylated [6R]-5,10-methylene-THF. In some embodiments, the liposomes comprise gamma-polyglutamylated [6R,S]-5,10-methylene-THF. In some embodiments, the liposomes comprise gamma-polyglutamylated [6S]-5,10-methylene-THF. In some embodiments, the liposomes contain gamma-polyglutamylated 5,10-methylene-THF with 2-20, 2-15, 2-10, 2-5, or more than 20 glutamyl groups (including the glutamyl groups in 5,10-methylene-THF). In some embodiments, the liposomes contain gamma-polyglutamylated 5,10-methylene-THF with two or more L-type glutamyl groups. In another embodiment, the liposome contains gamma polyglutamylated 5,10-methylene-THF having a glutamyl group in the D-form. In a further embodiment, the liposome contains gamma polyglutamylated 5,10-methylene-THF having a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

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

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

[0057] In one embodiment, the Lp-γPTHF composition comprises gamma-polyglutamated 5,10-methylene-THF (i.e., hexaglutamated 5,10-methylene-THF), which contains a chain of five glutamyl groups attached to the glutamyl group of a tetrahydrofolic acid. In some embodiments, the liposome comprises gamma-hexaglutamated [6R]5,10-methylene-THF. In some embodiments, the liposome comprises gamma-hexaglutamated [6R]5,10-methylene-THF. In some embodiments, the liposome comprises gamma-hexaglutamated [6R,S]-5,10-methylene-THF. In some embodiments, the liposome comprises gamma-hexaglutamated [6S]-5,10-methylene-THF. In some embodiments, the gamma-hexaglutamated 5,10-methylene-THF comprises two or more L-glutamyl groups. In other embodiments, the gamma hexaglutamated 5,10-methylene-THF contains a glutamyl group in the D-configuration. In further embodiments, the gamma hexaglutamated 5,10-methylene-THF contains a glutamyl group in the D-configuration and two or more glutamyl groups in the L-configuration.

[0058] In some embodiments, the present disclosure provides a composition comprising a liposome encapsulating (loaded with) gamma-polyglutamylated 5-formimino-THF. In some embodiments, the liposome comprises gamma-polyglutamylated [6S]-5-formimino-THF. In some embodiments, the liposome comprises gamma-polyglutamylated [6R,S]-5-formimino-THF. In some embodiments, the liposome comprises gamma-polyglutamylated [6R]-5-formimino-THF. In some embodiments, the liposome contains gamma-polyglutamylated 5-formimino-THF having 2-20, 2-15, 2-10, 2-5, or more than 20 glutamyl groups (including the glutamyl groups in 5-formimino-THF). In some embodiments, the liposome contains gamma-polyglutamylated 5-formimino-THF having two or more L-configured glutamyl groups. In other embodiments, the liposome contains gamma polyglutamylated 5-formimino-THF having a glutamyl group in the D-form. In further embodiments, the liposome contains gamma polyglutamylated 5-formimino-THF having a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

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

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

[0061] In one embodiment, the Lp-γPTHF composition comprises gamma-polyglutamated-5-formimino-THF (i.e., hexaglutamated 5-formimino-THF), which contains a chain of five glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the liposome comprises gamma-hexaglutamated [6S]-5-formimino-THF. In some embodiments, the liposome comprises gamma-hexaglutamated [6R,S]-5-formimino-THF. In some embodiments, the liposome comprises gamma-hexaglutamated [6R]5-formimino-THF. In some embodiments, the gamma-hexaglutamated 5-formimino-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the gamma-hexaglutamated 5-formimino-THF comprises a glutamyl group in the D-form. In a further embodiment, the gamma hexaglutamated 5-formimino-THF contains a D-glutamyl group and two or more L-glutamyl groups.

[0062] In some embodiments, the Lp-γPTHF composition is cationic. In some embodiments, the Lp-γPTHF 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-γPTHF 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-γPTHF composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w gamma-polyglutamylated THF. In some embodiments, during the process of preparing Lp-γPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of gamma-polyglutamated THF is encapsulated (trapped) in the cationic Lp-γPTHF. In additional embodiments, the gamma-polyglutamated tetrahydrofolate encapsulated by the liposomes is present in a HEPES buffer solution within the liposomes.

[0063] In other embodiments, the Lp-γPTHF composition is anionic or neutral. In some embodiments, the Lp-γPTHF liposomes are anionic or neutral and have 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-γPTHF liposomes are anionic or neutral and the composition has a diameter ranging from 80 nm to 120 nm, or any range therebetween. In some embodiments, the Lp-γPTHF liposomes are anionic and have a diameter 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-γPTHF liposomes are anionic and the composition has a diameter ranging from 80 nm to 120 nm, or any range therebetween. In some embodiments, Lp-γPTHF 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-γPTHF compositions comprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or greater than 75% w / w gamma-polyglutamylated THF. In some embodiments, during the process of preparing Lp-γPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting gamma-polyglutamated THF material is encapsulated (trapped) in anionic or neutral Lp-γPTHF. In some embodiments, the anionic or neutral Lp-γPTHF composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of gamma-tetraglutamated THF.In some embodiments, anionic or neutral Lp-γPTHF 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 gamma-pentaglutamated THF. In some embodiments, anionic or neutral Lp-γPTHF 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 gamma-hexaglutamated THF. In additional embodiments, the gamma-polyglutamated tetrahydrofolate encapsulated by the liposomes is present in a HEPES buffer solution within the liposomes.

[0064] In additional embodiments, the liposomal gamma polyglutamated tetrahydrofolate composition is PEGylated (PLp-γPTHF).

[0065] In some embodiments, the liposomal gamma-polyglutamated tetrahydrofolate composition is non-targeted (NTLp-γPTHF). That is, the NTLp-γPTHF 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 gamma-polyglutamated tetrahydrofolate composition is pegylated (NTPLp-γPTHF).

[0066] In other embodiments, the liposomal gamma-polyglutamated tetrahydrofolate compositions are targeted (TLp-γPTHF). That is, the TLp-γPTHF compositions contain 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-γPTHF or TPLp-γPTHF is not attached to the liposome via a covalent bond. In other embodiments, the targeting moiety of TLp-γPTHF or TPLp-γPTHF is attached to one or both of PEG and the outer surface of the liposome. The targeted liposomal gamma-polyglutamated tetrahydrofolate compositions (TLp-γPTHF and TPLp-γPTHF) provide further improvements in the efficacy and safety profile of tetrahydrofolate by specifically delivering gamma-polyglutamated (e.g., gamma-pentaglutamated and / or gamma-hexaglutamated) tetrahydrofolate to target cells, such as cancer cells. In some embodiments, the targeted liposomal gamma-polyglutamated tetrahydrofolate composition is PEGylated (TPLp-γPTHF). In some embodiments, the targeting moiety of TLp-γPTHF or TPLp-γPTHF is attached to one or both of PEG and the outer surface of the liposome. In some embodiments, the targeting moiety of TLp-γPTHF or TPLp-γPTHF is attached to the liposome via a covalent bond. The function of the targeting moiety of TLp-γPTHF and / or TPLp-γPTHF composition includes, but is not limited to: targeting the liposome to a target cell of interest in vivo or in vitro; interacting with a surface antigen to which the targeting moiety has specific affinity; and delivering the liposomal payload (γPTHF) into the cell. Suitable targeting moieties are known in the art and include, but are not limited to, antibodies, antigen-binding antibody fragments, scaffold proteins, polypeptides, and peptides. In some embodiments, the targeting moiety is a polypeptide.In 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-γPTHF or TPLp-γPTHF 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-γPTHF or TPLp-γPTHF has specific affinity for an epitope that is selectively expressed on target cells, such as tumor cells, compared to normal or non-tumor cells. In some embodiments, the targeting moiety has specific affinity for an epitope on a tumor cell surface antigen that is present on tumor cells but absent or inaccessible on non-tumor cells. In some embodiments, the targeting moiety has a specific affinity of 0.5x10 as measured by BIACORE® analysis. -10 ~10x10 -6 It binds to the epitope of interest with an equilibrium dissociation constant (Kd) in the range of

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

[0069] In additional embodiments, the Lp-γPTHF composition comprises one or more of an immunostimulant, a detectable marker, and a maleimide disposed on at least one of the outer surfaces of the PEG and liposome. In some embodiments, the liposomal γPTHF composition (e.g., Lp-γPTHF, PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF, or TPLp-γPTHF) is cationic. In other embodiments, the liposomal γPTHF composition (e.g., Lp-γPTHF, PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF, or TPLp-γPTHF) is anionic or neutral. In additional embodiments, the liposomes of the liposomal γPTHF composition (e.g., Lp-γPTHF, PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF, or TPLp-γPTHF) have 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 γPTHF 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 γPTHF composition have diameters ranging from 80 nm to 120 nm, or any range therebetween. In some embodiments, the liposomal γPTHF composition is PEGylated (e.g., PLp-γPTHF, NTPLp-γPTHF, or TPLp-γPTHF). In some embodiments, the liposomal γPTHF composition comprises a targeting moiety (e.g., TLp-γPTHF or TPLp-γPTHF). In further embodiments, the liposomal γPTHF composition is pegylated and targeted (e.g., TPLp-γPTHF). In some embodiments, the liposomal γPTHF composition comprises a γ-polyglutamated tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomal γPTHF composition comprises a γ-tetraglutamated tetrahydrofolate. In some embodiments, the liposomal γPTHF composition comprises a γ-pentaglutamated tetrahydrofolate.In another embodiment, the liposomal gamma PTHF composition comprises gamma hexaglutamated tetrahydrofolic acid.

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

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

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

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

[0074] Also provided are liposome compositions comprising γPTHF-encapsulated liposomes. In some embodiments, the liposome composition comprises a PEGylated γPTHF composition. In some embodiments, the liposome composition comprises a γPTHF composition linked to or otherwise bound to a targeting moiety. In further embodiments, the liposome composition comprises a γPTHF composition that is PEGylated, linked to, or otherwise bound to a targeting moiety. In some embodiments, the liposome composition comprises a γPTHF composition containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome composition comprises γ-tetraglutamated tetrahydrofolate. In some embodiments, the liposome composition comprises γ-pentaglutamated tetrahydrofolate. In other embodiments, the liposome composition comprises γ-hexaglutamated tetrahydrofolate.

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

[0076] Also provided are compositions comprising gamma-polyglutamated tetrahydrofolate (γPTHF) with a delivery vehicle, such as liposomal γPTHF. In some embodiments, pharmaceutical compositions comprise pegylated γPTHF compositions. In some embodiments, pharmaceutical compositions comprise γPTHF compositions linked to or otherwise attached to a targeting moiety. In further embodiments, pharmaceutical compositions comprise γPTHF compositions that are pegylated and linked to or otherwise attached to a targeting moiety. In some embodiments, pharmaceutical compositions comprise γPTHF containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, pharmaceutical compositions comprise gamma-tetraglutamated tetrahydrofolate. In some embodiments, pharmaceutical compositions comprise gamma-pentaglutamated tetrahydrofolate. In other embodiments, pharmaceutical compositions comprise gamma-hexaglutamated tetrahydrofolate.

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

[0078] In additional embodiments, the present disclosure provides methods of killing cells comprising contacting the cells with a composition comprising a gamma polyglutamated tetrahydrofolate composition (γPTHF) (e.g., a γPTHF disclosed herein). In some embodiments, the contacted cells are mammalian cells. In further embodiments, the contacted cells are human cells. In some embodiments, the contacted cells are hyperproliferative cells. In further embodiments, the hyperproliferative cells are cancer cells. In further embodiments, the contacted cancer cells are primary cells or cell line-derived cells 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 contacted cancer cells are primary cells or cell line-derived cells 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 chorioadenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (demenoid tumor, aggressive fibromatosis, bladder cancer, and central nervous system (CNS) lymphoma. In yet another embodiment, the cancer cells are 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). In some embodiments, the γPTHF contains 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the γPTHF contains 5-formyl-THF, which contains 4 γ-glutamyl groups.In some embodiments, γPTHF contains 5-formyl-THF, which contains five γ-glutamyl groups. In some embodiments, γPTHF contains six γ-glutamyl groups. In some embodiments, γPTHF contains γ-glutamyl groups in the D form. In some embodiments, γPTHF contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D form. In some embodiments, γPTHF contains γ-glutamyl groups in the L form. In some embodiments, γPTHF contains γ-PTHF in both the L and D forms. In some embodiments, γPTHF contains 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D form. In some embodiments, the γPTHF composition comprises gamma-tetraglutamated tetrahydrofolic acid. In some embodiments, the γPTHF composition comprises gamma-pentaglutamated tetrahydrofolic acid. In other embodiments, the γPTHF composition comprises gamma-hexaglutamated tetrahydrofolic acid.

[0079] In additional embodiments, the present disclosure provides a method of killing a cell, comprising contacting the cell with a liposome containing gamma polyglutamylated tetrahydrofolate (e.g., Lp-γPTHF, such as PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF, or TPLp-γPTHF). 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 yet another embodiment, the contacted hyperproliferative cell is a cancer cell. In further embodiments, the cancer cells are primary cells or cell line-derived cells 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 contacted cancer cells are primary cells or cell line-derived cells 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 chorioadenoma, 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 selected from the group consisting of colorectal cancer, breast cancer, gastric cancer (e.g., gastric cancer), pancreatic cancer, liver cancer, lung cancer ( For example, primary cells or cell line-derived cells obtained / derived from a cancer selected from non-small cell lung cancer and / or adenocarcinoma, head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In certain embodiments, the cancer cells are primary cells or cell line-derived cells obtained / 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 liposome contains γPTHF containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups.In some embodiments, the liposomes contain gamma-tetraglutamated tetrahydrofolic acid. In some embodiments, the liposomes contain gamma-pentaglutamated tetrahydrofolic acid. In other embodiments, the liposomes contain gamma-hexaglutamated tetrahydrofolic acid.

[0080] In some embodiments, the liposomes comprise γPTHF containing 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the liposomes comprise γPTHF containing 4 γ-glutamyl groups. In some embodiments, the liposomes comprise γPTHF containing 5 γ-glutamyl groups. In some embodiments, the liposomes comprise γPTHF containing 6 γ-glutamyl groups. In some embodiments, the liposomes comprise γPTHF containing γ-glutamyl groups in the D form. In some embodiments, the liposomes comprise γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D form. In some embodiments, the liposomes comprise γPTHF containing γ-glutamyl groups in the L form. In some embodiments, the liposomes comprise γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L form. In some embodiments, the liposomes comprise γPTHF containing L- and D-type γ-glutamyl groups. In some embodiments, the liposomes comprise γPTHF containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the liposomes comprise γ-pentaglutamated tetrahydrofolate. In other embodiments, the liposomes comprise γ-hexaglutamated tetrahydrofolate.

[0081] In additional embodiments, the present disclosure provides methods for treating cancer comprising administering an effective amount of a delivery vehicle (e.g., an immunoconjugate or liposome) comprising gamma polyglutamated tetrahydrofolate to a subject having or at risk of having cancer. 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-γPTHF, such as PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF, or TPLp-γPTHF). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is non-pegylated. In additional embodiments, the administered delivery vehicle comprises a targeting moiety that has specific affinity for an epitope of an antigen on the surface of a cancer cell.In additional embodiments, the delivery vehicle comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, 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 extra domain B (ED-B), VEGFR2 (CD309). , tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 EphA receptors, EphB receptors, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrins (e.g., integrin αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRα, PDGFRβ, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery vehicle comprises a targeting moiety that specifically binds to a cell surface antigen(s) determined to be derived from or expressed on a particular subject's cancer (tumor), such as a neoantigen. In some embodiments, the targeting moiety has specific affinity for an epitope of a cell surface antigen(s) determined to be derived from or expressed on a particular subject's tumor, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or antigen-binding antibody fragment. In some embodiments, the administered delivery vehicle comprises γPTHF containing 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the administered delivery vehicle comprises γPTHF containing 4 γ-glutamyl groups. In some embodiments, the administered delivery vehicle comprises γPTHF containing 5 γ-glutamyl groups. In some embodiments, the administered delivery vehicle comprises γPTHF containing 6 γ-glutamyl groups. In some embodiments, the γPTHF is a member selected from: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamic acid (e) polyglutaminated tetrahydrofolic acid THF (e.g., polyglutaminated [6S]-tetrahydrofolic acid THF); (f) polyglutaminated 5,10-methylene-THF (e.g., polyglutamated [6R]-5,10-methylene-THF); and (g) polyglutaminated 5-formimino-THF (e.g., polyglutamated [6S]-5-formimino-THF). In some embodiments, the γPTHF is polyglutamated 5,10-methylene-THF. In further embodiments, the γPTHF is polyglutamated [6R]-5,10-methylene-THF.In other embodiments, γPTHF is polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, γPTHF is polyglutamated 5-methyl-THF. In further embodiments, γPTHF is [6S]-5-methyl-THF. In other embodiments, γPTHF is [6R,S]-5-methyl-THF. In some embodiments, γPTHF is polyglutamated 5-formyl-THF. In further embodiments, γPTHF is polyglutamated [6S]-5-formyl-THF. In other embodiments, γPTHF is polyglutamated [6R,S]-5-formyl-THF.

[0082] In some embodiments, the delivery vehicle comprises γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form. In some embodiments, the delivery vehicle comprises γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the delivery vehicle comprises γPTHF containing 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D-form. In some embodiments, the administered delivery vehicle comprises γ-tetraglutamylated tetrahydrofolic acid. In some embodiments, the administered delivery vehicle comprises γ-pentaglutamylated tetrahydrofolic acid. In other embodiments, the administered delivery vehicle comprises γ-hexaglutamylated tetrahydrofolic acid. In some embodiments, the administered delivery vehicle comprises a γPTHF selected from: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (e) polyglutaminated tetrahydrofolic acid THF (e.g., polyglutaminated [6S]-tetrahydrofolic acid THF); (f) polyglutaminated 5,10-methylene-THF (e.g., polyglutaminated [6R]-5,10-methylene-THF); and (g) polyglutaminated 5-formimino-THF (e.g., polyglutaminated [6S]-5-formimino-THF). In some embodiments, the administered delivery vehicle comprises polyglutamated 5,10-methylene-THF. In further embodiments, the administered delivery vehicle comprises polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the administered delivery vehicle comprises polyglutamylated [6R,S]-5,10-methylene-THF.In some embodiments, the administered delivery vehicle comprises polyglutamated 5-methyl-THF. In further embodiments, the administered delivery vehicle comprises [6S]-5-methyl-THF. In other embodiments, the administered delivery vehicle comprises [6R,S]-5-methyl-THF. In some embodiments, the administered delivery vehicle comprises polyglutamated 5-formyl-THF. In further embodiments, the administered delivery vehicle comprises polyglutamated [6S]-5-formyl-THF. In other embodiments, the administered delivery vehicle comprises polyglutamated [6R,S]-5-formyl-THF. In some embodiments, the administered delivery vehicle comprises Lγ polyglutamated tetrahydrofolate. In some embodiments, the administered delivery vehicle comprises Dγ polyglutamated tetrahydrofolate. In further embodiments, the administered delivery vehicle comprises L and Dγ polyglutamated tetrahydrofolate. In some embodiments, the cancer is 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, 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 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 chorioadenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (demenoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, breast cancer, gastric cancer (e.g., 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 additional embodiments, the present disclosure provides methods for treating cancer, comprising administering an effective amount of a liposome comprising gamma polyglutamylated tetrahydrofolate (e.g., Lp-γPTHF, such as PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF, or TPLp-γPTHF) to a subject having or at risk of having cancer. In some embodiments, the liposome is PEGylated. In some embodiments, the liposome is not PEGylated. In additional embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a cancer cell.In additional embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, 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 extra domain B (ED-B), VEGFR2 (CD309), Tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 EphA receptors, EphB receptors, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrins (e.g., integrin αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRα, PDGFRβ, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.This also includes the use of cancer stem cell targeting moieties, such as those targeting CD34, CD133 and CD44, CD138, and CD15. In some embodiments, the liposomes comprise a targeting moiety that has specific affinity for an epitope of a cell surface antigen(s) determined to be derived from or expressed on a particular subject's tumor, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or antigen-binding antibody fragment. In some embodiments, the liposomes comprise γPTHF containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered liposome comprises a γPTHF selected from: (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 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (e) polyglutaminated tetrahydrofolic acid THF (e.g., polyglutaminated [6S]-tetrahydrofolic acid THF); (f) polyglutaminated 5,10-methylene-THF (e.g., polyglutaminated [6R]-5,10-methylene-THF); and (g) polyglutaminated 5-formimino-THF (e.g., polyglutaminated [6S]-5-formimino-THF). In some embodiments, the administered liposomes comprise polyglutamated 5,10-methylene-THF. In further embodiments, the administered liposomes comprise polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the administered liposomes comprise polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, the administered liposomes comprise polyglutamylated 5-methyl-THF. In further embodiments, the administered liposomes comprise [6S]-5-methyl-THF. In other embodiments, the administered liposomes comprise [6R,S]-5-methyl-THF.In some embodiments, the administered liposomes comprise polyglutamylated 5-formyl-THF. In further embodiments, the administered liposomes comprise polyglutamylated [6S]-5-formyl-THF. In other embodiments, the administered liposomes comprise polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the administered liposomes of the liposome composition comprise γPTHF containing γ-glutamyl groups in the L-form. In some embodiments, the administered liposomes of the liposome composition comprise γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the administered liposomes of the liposome composition comprise γPTHF containing γ-glutamyl groups in both the L- and D-forms. In some embodiments, the liposomes of the administered liposome composition comprise γPTHF containing 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D-form. In some embodiments, the administered liposome composition comprises tetraglutamylated γPTHF. In some embodiments, the administered liposome composition comprises pentaglutamylated γPTHF. In some embodiments, the administered liposome composition comprises hexaglutamylated γPTHF. In some embodiments, the cancer is selected from the group consisting of lung (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, and blood tumors (e.g., leukemia or lymphoma). In yet another embodiment, the cancer cells are primary cells or cell line-derived cells 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 a specific embodiment, the cancer cells are primary cells or cell line-derived cells obtained / derived from colorectal cancer.

[0084] In an additional embodiment, the present disclosure provides a method for treating cancer comprising administering to a subject having or at risk of having cancer an effective amount of a liposome composition comprising a liposome comprising gamma polyglutamated tetrahydrofolate and a targeting moiety having specific affinity for an epitope of an antigen on the surface of the cancer.In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, 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 extra domain B (ED-B), VEGFR2 (CD309). , tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 EphA receptors, EphB receptors, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrins (e.g., integrin αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRα, PDGFRβ, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the liposome comprises a targeting moiety that has specific affinity for an epitope of a cell surface antigen(s) determined to be derived from or expressed on a particular subject's cancer (tumor), such as a neoantigen. In some embodiments, the targeting moiety is an antibody or antigen-binding antibody fragment. In some embodiments, the liposome comprises γPTHF containing 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the administered liposome comprises γPTHF containing 4 γ-glutamyl groups. In some embodiments, the administered liposome comprises γPTHF containing 5 γ-glutamyl groups. In some embodiments, the administered liposome comprises γPTHF containing 6 γ-glutamyl groups. In some embodiments, the administered liposome comprises a γPTHF selected from: (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 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (e) polyglutaminated tetrahydrofolic acid THF (e.g., polyglutaminated [6S]-tetrahydrofolic acid THF); (f) polyglutaminated 5,10-methylene-THF (e.g., polyglutaminated [6R]-5,10-methylene-THF); and (g) polyglutaminated 5-formimino-THF (e.g., polyglutaminated [6S]-5-formimino-THF). In some embodiments, the administered liposomes comprise polyglutamated 5,10-methylene-THF. In further embodiments, the administered liposomes comprise polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the administered liposomes comprise polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, the administered liposome comprises polyglutamylated 5-methyl-THF.In further embodiments, the administered liposomes comprise [6S]-5-methyl-THF. In other embodiments, the administered liposomes comprise [6R,S]-5-methyl-THF. In some embodiments, the administered liposomes comprise polyglutamylated 5-formyl-THF. In further embodiments, the administered liposomes comprise polyglutamylated [6S]-5-formyl-THF. In other embodiments, the administered liposomes comprise polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the liposomes comprise γPTHF containing a γ-glutamyl group in the L-form. In some embodiments, the liposomes comprise γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposomes comprise γPTHF containing a γ-glutamyl group in the D-form. In some embodiments, the liposome comprises γPTHF, which contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form. In some embodiments, the liposome comprises γ-tetraglutamated tetrahydrofolate. In some embodiments, the liposome comprises γ-pentaglutamated tetrahydrofolate. In some embodiments, the liposome comprises γ-hexaglutamated tetrahydrofolate.

[0085] In some embodiments, the administered liposome composition comprises PEGylated liposomes (e.g., TPLp-γPTHF). In some embodiments, the administered liposome composition comprises non-PEGylated liposomes. In some embodiments, the liposomes of the administered liposome composition comprise γPTHF containing 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the administered liposomes comprise γPTHF containing 4 γ-glutamyl groups. In some embodiments, the administered liposomes comprise γPTHF containing 5 γ-glutamyl groups. In some embodiments, the administered liposomes comprise γPTHF containing 6 γ-glutamyl groups. In some embodiments, the administered liposome comprises a γPTHF selected from: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (e) polyglutaminated tetrahydrofolic acid THF (e.g., polyglutaminated [6S]-tetrahydrofolic acid THF); (f) polyglutaminated 5,10-methylene-THF (e.g., polyglutaminated [6R]-5,10-methylene-THF); and (g) polyglutaminated 5-formimino-THF (e.g., polyglutaminated [6S]-5-formimino-THF). In some embodiments, the administered liposomes comprise polyglutamated 5,10-methylene-THF. In further embodiments, the administered liposomes comprise polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the administered liposomes comprise polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, the administered liposomes comprise polyglutamylated 5-methyl-THF. In further embodiments, the administered liposomes comprise [6S]-5-methyl-THF.In other embodiments, the administered liposomes comprise [6R,S]-5-methyl-THF. In some embodiments, the administered liposomes comprise polyglutamylated 5-formyl-THF. In further embodiments, the administered liposomes comprise polyglutamylated [6S]-5-formyl-THF. In other embodiments, the administered liposomes comprise polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the administered liposomes of the liposome composition comprise γPTHF containing a γ-glutamyl group in the D-form. In some embodiments, the administered liposomes of the liposome composition comprise γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form. In some embodiments, the administered liposomes of the liposome composition comprise γPTHF containing a γ-glutamyl group in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPTHF containing γ-glutamyl groups in both the L- and D-forms. In some embodiments, the liposomes of the administered liposome composition comprise γPTHF containing 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D-form. In some embodiments, the liposomes of the administered liposome composition comprise γ-tetraglutamated tetrahydrofolate. In some embodiments, the liposomes of the administered liposome composition comprise γ-pentaglutamated tetrahydrofolate. In other embodiments, the liposomes of the administered liposome composition comprise γ-hexaglutamated tetrahydrofolate. 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 additional embodiments, 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 its cell surface, the liposome composition comprising (a) gamma-polyglutamated tetrahydrofolate (γPTHF) and (b) a liposome comprising a targeting moiety having specific binding affinity for the folate receptor. In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α) and folate receptor beta (FR-β). In some embodiments, the administered liposome composition comprises a pegylated liposome (e.g., TPLp-γPTHF). In some embodiments, the administered liposome composition comprises non-PEGylated liposomes. In some embodiments, the liposomes of the administered liposome composition comprise γPTHF containing 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the administered liposomes comprise γPTHF containing 4 γ-glutamyl groups. In some embodiments, the administered liposomes comprise γPTHF containing 5 γ-glutamyl groups. In some embodiments, the administered liposomes comprise γPTHF containing 6 γ-glutamyl groups.In some embodiments, the administered liposome comprises a γPTHF selected from: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (e) polyglutaminated tetrahydrofolic acid THF (e.g., polyglutaminated [6S]-tetrahydrofolic acid THF); (f) polyglutaminated 5,10-methylene-THF (e.g., polyglutaminated [6R]-5,10-methylene-THF); and (g) polyglutaminated 5-formimino-THF (e.g., polyglutaminated [6S]-5-formimino-THF). In some embodiments, the administered liposomes comprise polyglutamated 5,10-methylene-THF. In further embodiments, the administered liposomes comprise polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the administered liposomes comprise polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, the administered liposomes comprise polyglutamylated 5-methyl-THF. In further embodiments, the administered liposomes comprise [6S]-5-methyl-THF. In other embodiments, the administered liposomes comprise [6R,S]-5-methyl-THF. In some embodiments, the administered liposomes comprise polyglutamylated 5-formyl-THF. In further embodiments, the administered liposomes comprise polyglutamylated [6S]-5-formyl-THF. In other embodiments, the administered liposomes comprise polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the administered liposomes of the liposome composition comprise γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form.In some embodiments, the liposomes of the administered liposome composition comprise γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPTHF containing 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D-form. In some embodiments, the liposomes of the administered liposome composition comprise γ-pentaglutamated tetrahydrofolate. In other embodiments, the liposomes of the administered liposome composition comprise γ-hexaglutamated tetrahydrofolate. In some embodiments, the liposome composition is administered to treat 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 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 lymphocytic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, and chorioadenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (demenoid tumor, 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 additional embodiments, the present disclosure provides a method for maintenance therapy of cancer, comprising administering an effective amount of a liposome composition comprising liposomes containing gamma-polyglutamated tetrahydrofolate (Lp-γPTHF) to a subject undergoing or who has undergone cancer therapy. In some embodiments, the administered liposome composition is PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF, or TPLp-γPTHF. In some embodiments, the administered liposome composition comprises PEGylated liposomes (e.g., PLp-γPTHF, NTPLp-γPTHF, or TPLp-γPTHF). In some embodiments, the administered liposome composition comprises targeted liposomes (e.g., TLp-γPTHF or TPLp-γPTHF). In some embodiments, the administered liposome composition comprises PEGylated liposomes containing a targeting moiety (e.g., TPLp-γPTHF). In some embodiments, the liposomes of the administered liposome composition comprise γ-polyglutamated tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the administered liposomes comprise γPTHF containing 4 γ-glutamyl groups. In some embodiments, the administered liposomes comprise γPTHF containing 5 γ-glutamyl groups. In some embodiments, the administered liposomes comprise γPTHF containing 6 γ-glutamyl groups.In some embodiments, the administered liposome comprises a γPTHF selected from: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (e) polyglutaminated tetrahydrofolic acid THF (e.g., polyglutaminated [6S]-tetrahydrofolic acid THF); (f) polyglutaminated 5,10-methylene-THF (e.g., polyglutaminated [6R]-5,10-methylene-THF); and (g) polyglutaminated 5-formimino-THF (e.g., polyglutaminated [6S]-5-formimino-THF). In some embodiments, the administered liposomes comprise polyglutamated 5,10-methylene-THF. In further embodiments, the administered liposomes comprise polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the administered liposomes comprise polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, the administered liposomes comprise polyglutamylated 5-methyl-THF. In further embodiments, the administered liposomes comprise [6S]-5-methyl-THF. In other embodiments, the administered liposomes comprise [6R,S]-5-methyl-THF. In some embodiments, the administered liposomes comprise polyglutamylated 5-formyl-THF. In further embodiments, the administered liposomes comprise polyglutamylated [6S]-5-formyl-THF. In other embodiments, the administered liposomes comprise polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the administered liposomes of the liposome composition comprise γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form.In some embodiments, the liposomes of the administered liposome composition comprise γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPTHF containing 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D-form. In some embodiments, the liposomes of the administered liposome composition comprise γ-tetraglutamated tetrahydrofolate. In some embodiments, the liposomes of the administered liposome composition comprise γ-pentaglutamated tetrahydrofolate. In other embodiments, the liposomes of the administered liposome composition comprise γ-hexaglutamated tetrahydrofolate.

[0088] In additional embodiments, the present disclosure provides methods for treating an immune system disorder, comprising administering an effective amount of a liposome composition comprising liposomes containing gamma-polyglutamated tetrahydrofolate (e.g., Lp-γPTHF, PLp-γPTHF, NTLp-γPTHF, NTPLp-γPTHF, TLp-γPTHF, or TPLp-γPTHF) to a subject having or at risk of having an immune system disorder. 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-γPTHF, NTPLp-γPTHF, or TPLp-γPTHF). In some embodiments, the administered liposome composition comprises a targeted liposome containing a targeting moiety with specific affinity for a surface antigen on a target cell (e.g., an immune cell) of interest (e.g., TLp-γPTHF or TPLp-γPTHF). In further embodiments, the administered liposome composition comprises a liposome that is pegylated and contains a targeting moiety (e.g., TPLp-γPTHF). In some embodiments, the administered liposome of the administered liposome composition comprises gamma-pentaglutamated tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 5 gamma-glutamyl groups. In some embodiments, the administered liposome comprises gamma-PTHF containing four gamma-glutamyl groups. In some embodiments, the administered liposome comprises gamma-PTHF containing five gamma-glutamyl groups. In some embodiments, the administered liposome comprises gamma-PTHF containing six gamma-glutamyl groups.In some embodiments, the administered liposome comprises a γPTHF selected from: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (e) polyglutaminated tetrahydrofolic acid THF (e.g., polyglutaminated [6S]-tetrahydrofolic acid THF); (f) polyglutaminated 5,10-methylene-THF (e.g., polyglutaminated [6R]-5,10-methylene-THF); and (g) polyglutaminated 5-formimino-THF (e.g., polyglutaminated [6S]-5-formimino-THF). In some embodiments, the administered liposomes comprise polyglutamated 5,10-methylene-THF. In further embodiments, the administered liposomes comprise polyglutamated [6R]-5,10-methylene-THF. In other embodiments, the administered liposomes comprise polyglutamated [6R,S]-5,10-methylene-THF. In some embodiments, the administered liposomes comprise polyglutamylated 5-methyl-THF. In further embodiments, the administered liposomes comprise [6S]-5-methyl-THF. In other embodiments, the administered liposomes comprise [6R,S]-5-methyl-THF. In some embodiments, the administered liposomes comprise polyglutamylated 5-formyl-THF. In further embodiments, the administered liposomes comprise polyglutamylated [6S]-5-formyl-THF. In other embodiments, the administered liposomes comprise polyglutamylated [6R,S]-5-formyl-THF. In some embodiments, the administered liposomes of the liposome composition comprise γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form.In some embodiments, the liposomes of the administered liposome composition comprise γPTHF containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPTHF containing 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D-form. In some embodiments, the liposomes of the administered liposome composition comprise γ-tetraglutamated tetrahydrofolate. In some embodiments, the liposomes of the administered liposome composition comprise γ-pentaglutamated tetrahydrofolate. In other embodiments, the liposomes of the administered liposome composition comprise γ-hexaglutamated tetrahydrofolate.

[0089] The present disclosure also provides a method for delivering gamma polyglutamated tetrahydrofolate to tumor and / or cancer cells, comprising administering to a subject having a tumor a composition comprising gamma polyglutamated tetrahydrofolate (L-γPTHF) and a targeting moiety having specific binding affinity for an epitope of a surface antigen on the 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 gamma polyglutamated tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered composition comprises gamma tetraglutamated tetrahydrofolate. In some embodiments, the administered composition comprises gamma pentaglutamated tetrahydrofolate. In other embodiments, the administered composition comprises gamma hexaglutamated tetrahydrofolate. In some embodiments, the composition administered comprises a γPTHF selected from: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-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 composition comprises polyglutamated 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 additional embodiments, the present disclosure provides methods for preparing liposomal compositions, including liposomal gamma-polyglutamated tetrahydrofolate (γPTHF) compositions, comprising: forming a mixture in solution containing liposome components and gamma-polyglutamated tetrahydrofolate; homogenizing the mixture in the solution to form liposomes; and treating the mixture to form liposomes containing polyglutamated tetrahydrofolate. In some embodiments, the gamma-polyglutamated tetrahydrofolate contains 4, 5, 2-10, 4-6, or more than 5 gamma-glutamyl groups. In some embodiments, the gamma-PTHF composition contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma-glutamyl groups in the D-form. In some embodiments, the gamma-PTHF composition contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma-glutamyl groups in the L-form. In some embodiments, the γPTHF composition contains 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D-form. In some embodiments, the γPTHF composition comprises γ-pentaglutamated tetrahydrofolate. In some embodiments, the γPTHF composition comprises γ-tetraglutamated tetrahydrofolate. In other embodiments, the γPTHF composition comprises γ-hexaglutamated tetrahydrofolate.In some embodiments, the composition comprises a γPTHF selected from: (a) polyglutamylated 5-formyl-THF (e.g., polyglutamylated [6S]-5-formyl-THF); (b) polyglutamylated 10-formyl-THF (e.g., polyglutamylated [6R]-10-formyl-THF); (c) polyglutamylated 5,10-methenyl-THF (e.g., polyglutamylated [6R]-5,10-methenyl-THF); (d) polyglutamic acid (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 administered composition comprises polyglutamylated [6R,S]-5-formyl-THF.

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

[0092] [Figures 1A-1L] Chemical formulas of exemplary 5-10 methylene THF derivatives are shown: 5-10 methylene THF (Figure 1A), γ5-10 methylene THF diglutamic acid (Figure 1B), γ5-10 methylene THF triglutamic acid (Figures 1C and 1D), γ5-10 methylene THF tetraglutamic acid (Figures 1E and 1F), γ5-10 methylene THF pentaglutamic acid (Figures 1G and 1H), γ5-10 methylene THF hexaglutamic acid (Figure 1 I and 1J), γ 5-10 methylene THF heptaglutamic acid (Figures 1K and 1L), γ 5-10 methylene THF octaglutamic acid (Figures 1M and 1N), and exemplary γ tetrahydrofolate polyglutamic acid derivatives of 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). [Figure 2] FIG. 1 shows an example dose-response relationship for free pemetrexed L-γ hexaglutamate (gG6), liposomal pemetrexed L-γ hexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha-targeting antibody (FR1Ab) liposomal pemetrexed L-γ hexaglutamate (liposomal gG6-FR1Ab) in NCI H2342 non-small cell lung cancer (NSCLC), adenocarcinoma subtype, shown as the percentage of viable cells after 48 hours of treatment. [Figure 3] FIG. 1 shows an example of the dose-response relationship of free pemetrexed L-γ hexaglutamate (gG6), liposomal pemetrexed L-γ hexaglutamate (liposomal gG6), pemetrexed, and folate receptor α-targeting antibody (FR1Ab) liposomal pemetrexed L-γ hexaglutamate (liposomal gG6-FR1Ab) in HT-29 (colon cancer) at 48 hours. [Figure 4]

[0023] Figure 1 shows the effects of free pemetrexed L-γ hexaglutamate (hexa gG6) and liposomal pemetrexed L-γ hexaglutamate (liposomal hexa gG6) on the proliferation of colon cancer SW260 cells after 48 hours of exposure to 256 nM of the corresponding agent. Non-targeted and targeted liposomal pemetrexed hexa gG6 can enter cells more efficiently than free pemetrexed hexa gG6 to inhibit the proliferation of colon cancer SW260 cells. [Figure 5] Figure 1 shows the relative efficacy of liposomal pemetrexed L-γ-hexaglutamate (liposomal gG6) and its mirror image, liposomal pemetrexed γ-D-hexaglutamate (liposomal gDG6), compared to pemetrexed after 48 hours of exposure in 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 6] 1 shows the therapeutic effects on HCC1806 triple-negative breast cancer cells after 48-hour exposure to liposomal pemetrexed gamma-L hexaglutamate (Lps Hexa gG6), liposomal pemetrexed gamma-D hexaglutamate (Lps Hexa gDG6), and pemetrexed. [Figure 7] 1 shows the therapeutic effect on OAW28 ovarian cancer cells after exposure to liposomal pemetrexed gamma-L hexaglutamate (Lps Hexa gG6), liposomal pemetrexed gamma-D hexaglutamate (Lps Hexa gDG6) compared to pemetrexed over a 48 hour period. [Figure 8] 1 shows the therapeutic effect on H292 non-small cell lung cancer cells after exposure to liposomal pemetrexed gamma-L hexaglutamate (Lps Hexa gG6), liposomal pemetrexed gamma-D hexaglutamate (Lps Hexa gDG6), and pemetrexed for 48 hours. [Figure 9]This figure shows the therapeutic effects on H292 non-small cell lung cancer cells after exposure to various dose levels ranging from 16 to 128 nM of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed for 48 hours. Within each dose range tested, the liposomal pemetrexed gG6 formulation exhibited superior inhibition of H292 non-small cell lung cancer cells compared to pemetrexed. [Figure 10] This figure shows the therapeutic efficacy of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed at various dose levels ranging from 16 to 128 nM against HCC1806 triple-negative breast cancer cells after exposure for 48 hours. At each tested dose, the liposomal pemetrexed gG6 formulation was superior to pemetrexed in inhibiting HCC1806 triple-negative breast cancer cells. [Figure 11] This figure shows the therapeutic effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed at various 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 gG6 liposomal formulation, but at doses of 32 nM and 64 nM, the liposomal formulation has superior therapeutic effects to pemetrexed; at 16 nM, the therapeutic effect of liposomal pemetrexed gG6 is similar to that of pemetrexed. [Figure 12] Figure 1 shows the toxicity of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed at 64 nM, 128 nM, and 264 nM to differentiated human neutrophils. The figure shows that liposomal pemetrexed gG6 is significantly less toxic than pemetrexed to differentiated human neutrophils. [Figure 13]Shown are the effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and the corresponding pemetrexed agents on neutrophils (differentiated from CD34+ cells) after 48 hours of exposure at various dose levels ranging from 16 to 128 nM. [Figure 14] This figure shows the effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and the corresponding pemetrexed at 16 nM, 32 nM, 64 nM, and 128 nM for 48 hours on AML12 liver cells. Remarkably, none of the liposomal agents tested at the dose levels of liposomal pemetrexed gG6 appeared to be toxic to AML12 liver cells. In contrast, pemetrexed treatment resulted in a reduction in AML12 liver cell counts by approximately 40% at all doses examined. [Figure 15]

[0033] Figure 1 shows the effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed at 16 nM, 32 nM, 64 nM, and 128 nM on CCD841 colonic epithelial cells after 48 hours of exposure. At all concentrations tested, pemetrexed results in a reduction of CCD841 colonic epithelial cell counts of about 50% or more, compared to a reduction of about 20% or less after treatment with each of the liposomal compositions tested. [Figure 16] The structures of the polyglutamate antifolate, cisplatin (CDDP), and two possible gG6-cisplatin complexes are shown. The pH-dependent formation of inter- and / or intra-chain coordination between the carboxyl groups of the polyglutamate antifolate and cisplatin may lead to its degradation into separate molecules of gG6 and cisplatin upon encountering the acidic pH of the lysosome (pH 3-5) and in the presence of intracellular chloride ions. [Figure 17]Hematological parameters: Effect of liposomal gG6 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 18] This figure shows the effect of liposomal gG6 treatment in mice at 40 mg / kg and 80 mg / kg once weekly for 4 weeks on hemoglobin and reticulocyte count indices. There is a minimal decrease in mean hemoglobin concentration at higher dose levels. Concomitantly, there is a slight increase in mean reticulocyte count indices. [Figure 19] The effects of liposomal gG6 treatment in mice 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, are shown. No apparent increase in liver aminotransferase mean AST or ALT levels was observed, and furthermore, no changes in mean albumin levels were observed. [Figure 20] 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 liposomal gG6 administered intravenously at 167 mg / kg once every three weeks are shown. These preliminary data show that liposomal gG6 results in reduced tumor control compared to pemetrexed. [Figure 21A-F]Liposomal pemetrexed γ-L triglyceride for 48 hours against H2342 (NSCLC, adenocarcinoma subtype) (Figure 21A), H292 (NSCLC, adenocarcinoma subtype) (Figure 21B), HT-29 (colon cancer) (Figure 21C), HCC1806 (triple-negative breast cancer) (Figure 21D), MCF7 (ER+ breast cancer) (Figure 21E), and OAW28 (ovarian cancer) (Figure 21F). Figure 1 shows the dose-response relationship for liposomal pemetrexed γ-L pentaglutamate (liposome aG3), liposomal pemetrexed γ-L pentaglutamate (liposome aG5), liposomal pemetrexed γ-L octaglutamate (liposome aG7), and the combination of liposomal pemetrexed γ-L hexaglutamate (aG6) and γ-L dodecaglutamate (aG12) (liposomes aG6 and aG12). Cell viability was measured using the CellTiter-Glo® (CTG) luminescent cell viability assay, essentially as described in Example 1. As shown in all cell lines, the potency of each polyglutamated pemetrexed liposome composition significantly exceeded that of the liposomal vehicle and empty liposome controls. DETAILED DESCRIPTION OF THE INVENTION

[0093] The present disclosure generally relates to gamma-polyglutamated tetrahydrofolate compositions. The compositions offer advancements over existing treatments for hyperproliferative diseases such as cancer. Methods for producing, delivering, and using the gamma-polyglutamated tetrahydrofolate compositions are also provided. The gamma-polyglutamated compositions have uses including, but not limited to, treating (e.g., treating and / or preventing) hyperproliferative diseases such as cancer, immune system disorders such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria. The gamma-polyglutamated compositions also have uses in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the efficacy of the therapeutic agent(s), or as "chemoprotectants" (e.g., in combination with antifolates such as methotrexate) to reduce toxic side effects associated with the therapeutic agent(s).

[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 term "comprising," it is understood that other similar embodiments described with the terms "containing," "consisting of," and / or "consisting essentially of" are also provided. 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 more open-ended, the transitional phrase "consisting of" is considered more exclusive, and "consisting essentially of" is considered intermediate).

[0096] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise stated or unless it is clearly 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" encompasses each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0098] Headings and subheadings are used for convenience and / or to comply with official regulations only, and are not intended to limit the subject technology, nor are they to be cited in connection with the interpretation of the description of the subject technology. Features described under one heading or one subheading of the subject disclosure may, in various embodiments, be combined with features described under other headings or subheadings. Furthermore, not all features under a single heading or subheading may necessarily be used together in some embodiments.

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

[0100] The term "tetrahydrofolate 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. "Tetrahydrofolate THF" is also referred to herein as a type of tetrahydrofolic acid (tetrathydrolate) (THF).

[0101] The terms "polyglutamylated-tetrahydrofolate," "polyglutamylated-THF," "THF-PG," "PTHF," and repeats thereof are used interchangeably 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). References herein to the number of glutamyl groups in γPTHF (THF-PG) refer to the glutamyl groups of tetrahydrofolic acid. For example, a THF-PG composition containing five glutamyl residues in addition to the glutamyl groups of THF is referred to herein as hexaglutamated tetrahydrofolic acid or tetrahydrofolic acid hexaglutamic acid. The polyglutamic acid chain comprises an N-terminal glutamyl group and one or more C-terminal glutamyl groups. The N-terminal glutamyl group of the polyglutamic acid chain is not bound to another glutamyl group through its amino group, but is bound to one or more glutamyl groups through its carboxylic acid group. In some embodiments, the N-terminal glutamyl group of the polyglutamic acid is the glutamyl group of tetrahydrofolic acid. The C-terminal glutamyl group(s) of the polyglutamic acid chain are bound to another glutamyl group through their amino group, but are not bound to another glutamyl group through their carboxylic acid group.

[0102] In some embodiments, the polyglutamated tetrahydrofolate is a member selected from: (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]methyl}amino)phenyl]formamido (f) polyglutaminated 5,10-methylene-THF; and (g) polyglutaminated 5-formimino-THF. In further embodiments, the polyglutamated tetrahydrofolate is a member selected from: (a) polyglutamated [6S] (b) polyglutamated [6R]-10-formyl-THF; (c) polyglutamated [6R]-5,10-methenyl-THF; (d) polyglutamated [6S]-5-methyl-THF; (e) polyglutamated [6S]-tetrahydrofolic acid; (f) polyglutamated [6R]-5,10-methylene-THF; and (g) polyglutamated [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., 1:1 w / w).

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

[0104] The terms "γ-polyglutamated tetrahydrofolate," "γ-polyglutamated tetrahydrofolate," "γPTHF," "γ-polyglutamated-tetrahydrofolate," "polyglutamated-THF," "γTHF-PG," and repeats thereof, are used interchangeably herein to refer to a tetrahydrofolate composition that contains at least one γ-glutamyl group with a γ-carboxyl group linkage in addition to the γ-glutamyl group of tetrahydrofolate (e.g., THF-PG). nwhere n≧1 γ-glutamyl group). As used herein, references to the number of glutamyl groups in γPTHF (γTHF-PG) take into account the glutamyl groups of tetrahydrofolate. For example, a γTHF-PG composition containing five γ-glutamyl groups in addition to the glutamyl groups of THF may be referred to herein as γ-hexaglutamated tetrahydrofolate or γ-tetrahydrofolate hexaglutamate.

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

[0106] As used herein, the term "isolated" refers to a composition in a form not found in nature. Isolated gamma-polyglutamated compositions include those that have been purified to the extent that they are no longer in the form found in nature. In some embodiments, isolated gamma-polyglutamated tetrahydrofolate is substantially pure. Isolated compositions are free or substantially free of naturally incorporated substances, such as proteins and other cellular components, such as nucleic acids, that may potentially be found in nature or in the environment in which they are produced (e.g., cell culture). Gamma-polyglutamated compositions can be formulated with a diluent or adjuvant and further isolated for practical purposes—for example, when used as a diagnostic or therapeutic agent, a gamma-polyglutamated composition is typically mixed with a pharmaceutically acceptable carrier or diluent. In some embodiments, isolated gamma polyglutamated compositions (e.g., gamma polyglutamic acid and delivery vehicles such as liposomes comprising gamma polyglutamic acid) contain less than 1% or less than 0.1% undesired DNA or protein content. In some embodiments, gamma polyglutamic acid compositions (e.g., gamma polyglutamic acid and delivery vehicles such as liposomes comprising gamma polyglutamic acid) are "isolated."

[0107] As used herein, the term "targeting moiety" refers to a molecule that confers enhanced affinity to 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, antigen-binding antibody fragment, bispecific antibody, or other antibody-based molecule or compound. In some embodiments, the targeting moiety is an aptamer, avimer, receptor-binding ligand, nucleic acid, biotin-avidin binding pair, peptide, protein, carbohydrate, lipid, vitamin, toxin, microbial component, hormone, 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 with or binds to an epitope, protein, or target molecule more frequently, more rapidly, for a longer period of time, with greater affinity, or 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 and / or target molecules in two or more species. Similarly, due to homology within specific regions of the polypeptide sequences of different proteins, the terms "specific affinity" or "specifically binds" can include binding substances that recognize epitopes present on two or more proteins and / or target molecules. 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 (although it can include) exclusive binding, e.g., binding to an epitope on only one target. Thus, a targeting moiety may, in certain embodiments, specifically bind epitopes present on more than one target. In certain embodiments, multiple targets may be bound by the same targeting moiety that specifically binds epitopes 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 upon protein denaturation, whereas epitopes formed by tertiary folding are typically lost upon protein denaturation. An epitope typically comprises 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 "binding affinity to target", "binding to target", "enhanced affinity" and similar expressions, refer to the affinity constant, a property of targeting moiety that can be directly measured by determining, for example, the amount of binding and dissociation of targeting moiety 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 a 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 acts to support, promote, or facilitate the entry of gamma-polyglutamated tetrahydrofolate 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 their derivatives), 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 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 preferred to use a maximum dose, i.e., the maximum safe dose according to sound medical judgment.

[0113] As used herein, "effective amount" refers to the administration of a drug sufficient to produce a medically desired result. An effective amount may vary depending on the desired outcome, the particular 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 professional opinion of a health practitioner. An "effective amount" can be determined empirically and routinely in connection with the stated purpose. In the case of cancer, an effective amount of a drug may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow to some extent, and preferably stop) the infiltration of cancer cells 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. Depending on the extent to which a drug may prevent and / or kill existing cancer cells, the drug may be cytostatic and / or cytotoxic. For cancer therapy, 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 and 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, but also other types of fibrosis, such as renal fibrosis), angiogenesis, psoriasis, smooth muscle proliferation in blood vessels, such as atherosclerosis, and post-angioplasty stenosis or restenosis.

[0115] "Cancer," "tumor," or "malignant tumor" are used interchangeably and refer to any of a number of cell types or diseases characterized by uncontrolled, abnormal proliferation of cells, the ability to spread infected cells locally or via the bloodstream and lymphatic system to other parts of the body (metastasis), and / or any of the characteristic structural and / or molecular features known to be associated with these cell types or diseases. As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all 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 γPTHF 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 γPTHF 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, slow, lessen the symptoms, and / or halt the progression of a diagnosed condition or disorder, and (b) prophylactic or preventative measures that prevent and / or delay the onset of the targeted disease or condition. Thus, subjects in need of treatment include those already with the cancer, disorder, or disease, those at risk of developing the cancer or condition, and those in whom an infection or condition is to be prevented. The 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, for example, the subject exhibits total, 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 improvement of at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which may or may not be 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, or 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 size, tumor cell growth or survival, or cancer cell number. Treatment can involve the use of γPTHF compositions alone or in combination with additional therapeutic agents.

[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 apes 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. In certain embodiments, the subject is a human.

[0119] As used herein, "treatment of a proliferative disorder" includes 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] As used herein, the term "autoimmune disease" is defined as a disorder resulting from 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] As used herein, the term "therapeutic agent" refers to 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, tetrahydrofolic acid, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benziguanidine (6-BG), bis-chloronitrosourea (BCNU), and camptothecin, or any therapeutic derivatives thereof. Further 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 anti-proliferative 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 disclosed gamma polyglutamated tetrahydrofolate compositions 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 fluorpyrimidine, such as 5-fluorouracil (5-FU)).

[0123] As used herein, the term "anti-metabolite" 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 cytarabine and cytarabine prodrugs, such as nelarabine, 5-azacytidine, gemcitabine, mercaptopurine, thioguanine, azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, and cladribine. Anti-metabolites useful for practicing the disclosed methods include nucleoside analogs, including purine or pyrimidine analogs. In some embodiments, the gamma polyglutamated 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 derivatives thereof. In one example, the nucleoside analog is a substrate of 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 embodiments, the nucleoside analog is selected from the following: N3-alkylated analogs of 5-fluorouracil, 5-fluorouracil derivatives having a 1,4-oxaheteroepane moiety, 5-fluorouracil and nucleoside analogs, cis- and trans-5-fluoro-5,6-dihydro-6-alkoxyuracil, cyclopentane 5-fluorouracil analogs, A-OT-fluorouracil, N4-trimethoxybenzoyl-5′-deoxy-5-fluoro-cytidine and 5′-deoxy-5-fluorouridine, 1-hexylcarbamoyl N-(2-furanidyl)-5-fluorouracil, B-3839, uracil-1-(2-tetrahydrofuryl)-5-fluorouracil, 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)-5-fluorouracil, doxifluridine, 5'-deoxy-5-fluorouridine, 1-acetyl-3-O-toluoyl-5-fluorouracil, 5-fluorouracil-m-formylbenzene-sulfonate (JP55059173), N'-(2-furanidyl)-5-fluorouracil (JP53149985), and 1-(2-tetrahydrofuryl)-5-fluorouracil or its derivatives. In certain embodiments, the antimetabolite is a pyrimidine analog or a pyrimidine analog prodrug (e.g., fluoropyrimidine). In certain embodiments, the antimetabolite is 5-fluorouracil.

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

[0125] The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the 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 compatible solid or liquid fillers, diluents, or encapsulating substances that are suitable for administration to humans or other subjects.

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

[0127] In some embodiments, the present disclosure provides: [1] A composition comprising γPTHF; [2] The composition of [1], wherein the gamma polyglutamylated tetrahydrofolate 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., polyglutamylated [6S]-5-methyl-THF); (e) polyglutamylated tetrahydrofolate (e.g., polyglutamylated [6S]-tetrahydrofolate); (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); [3] The composition of [1] or [2], wherein γPTHF contains 4, 5, 2-10, 4-6, or more than 5 glutamyl groups with γ-carboxyl group bonds; [4] γPTHF is γ-tetraglutamylated tetrahydrofolic acid; the composition according to any one of [1]-[3]; [5] γPTHF is γ-pentaglutamic tetrahydrofolic acid; any of the compositions according to [1]-[3]; [6] γPTHF is γ-hexaglutamated tetrahydrofolic acid; any of the compositions according to [1]-[3]; [7] (a) γPTHF contains two or more L-type glutamyl groups with γ-carboxyl group bonds; (b) each of the glutamyl groups of γPTHF is in the L-configuration and has a γ-carboxyl group bond; (c) at least one of the glutamyl groups of γPTHF is in the D-form and has a γ-carboxyl group bond; (d) each of the glutamyl groups of γPTHF other than the glutamyl group of tetrahydrofolic acid is in the D-form and has a γ-carboxyl linkage; or (e) a composition according to any one of [1]-[6], wherein γPTHF contains two or more L-glutamyl groups and at least one D-glutamyl group having a γ-carboxyl group bond; [8] The composition according to [4], wherein (a) each of the glutamyl groups is in the L-configuration and has a gamma carboxyl linkage, or (b) each of the glutamyl groups other than the glutamyl groups of tetrahydrofolic acid is in the D-configuration and each of the glutamyl groups has a gamma carboxyl linkage; [9] The composition of [5], wherein (a) each of the glutamyl groups is in the L-configuration and has a gamma carboxyl linkage, or (b) each of the glutamyl groups other than the glutamyl groups of tetrahydrofolic acid is in the D-configuration and each of the glutamyl groups has a gamma carboxyl linkage;

[10] The composition of [6], wherein (a) each of the glutamyl groups is in the L-configuration and has a gamma carboxyl linkage, or (b) each of the glutamyl groups other than the glutamyl groups of tetrahydrofolic acid is in the D-configuration and each of the glutamyl groups has a gamma carboxyl linkage;

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

[10] , wherein γ-PTHF can be polyglutamated by FGPS under physiological conditions, and / or the polyglutamated THF has a lower uptake rate by hepatocytes (<30%) than THF;

[12] [1]-

[11] A liposome composition containing γPTHF (Lp-γPTHF);

[13] The Lp-γPTHF composition according to

[12] , wherein γPTHF contains two or more L-type glutamyl groups;

[14] An Lp-γPTHF composition according to

[12] or

[13] , in which each of the glutamyl groups of γPTHF is in the L-form;

[15] The Lp-γPTHF composition of

[12] or

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

[16] The liposome contains γPTHF containing 1-10 glutamyl groups with γ-carboxyl group bonds, and the Lp-γPTHF composition according to any of

[12] -

[15] ;

[17] Liposomes containing γPTHF containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups, Lp-γPTHF compositions according to any of

[12] -

[16] ;

[18] The liposome contains gamma-tetraglutamylated tetrahydrofolic acid; Lp-γPTHF composition according to any of

[12] -

[17] ;

[19] Liposomes containing gamma pentaglutamated tetrahydrofolic acid; Lp-γPTHF composition according to any of

[12] -

[17] ;

[20] The liposome contains gamma hexaglutamated tetrahydrofolic acid, and the Lp-γPTHF composition is prepared according to any of

[12] -

[17] ;

[21] Liposomes are not PEGylated (PγLp-γPTHF), Lp-γPTHF compositions according to either

[12] -

[20] ;

[22] The liposomes are PEGylated (PγLp-γPTHF), Lp-γPTHF compositions according to any of

[12] -

[20] ;

[23] An Lp-γPTHF composition according to any of

[12] -

[22] , wherein the liposomes contain at least 1% weight / weight (w / w) γPTHF, or wherein during the process of preparing Lp-γPTHF, at least 1% of the starting material of γ-polyglutamylated THF is encapsulated (trapped) within the Lp-γPTHF;

[24] The liposomes have a diameter ranging from 20 nm to 500 nm; Lp-γPTHF compositions according to any of

[12] -

[23] ;

[25] The liposomes have a diameter ranging from 20 nm to 200 nm; Lp-γPTHF compositions according to any of

[12] -

[24] ;

[26] The liposomes have a diameter ranging from 80 nm to 120 nm. Lp-γPTHF compositions according to any of

[12] -

[25] ;

[27] Liposomes are formed from liposome components, and Lp-γPTHF compositions are prepared according to any of

[12] -

[26] ;

[28] The Lp-γPTHF composition according to

[27] , wherein the liposome component includes at least one of anionic lipids and neutral lipids;

[29] The Lp-γPTHF composition according to

[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-γPTHF composition according to any one of

[27] -

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

[27] -

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

[32] The Lp-γPTHF composition according to

[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-γPTHF composition according to

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

[34] Lp-γPTHF compositions according to either

[12] -

[33] , in which the liposomes are anionic or neutral;

[35] The liposomes have a zeta potential of less than zero. Lp-γPTHF compositions according to any of

[12] -

[33] ;

[36] The liposomes have a zeta potential of 0 to -150 mV. Lp-γPTHF compositions according to any of

[12] -

[33] ;

[37] The liposomes have a zeta potential of -30 to -50 mV. Lp-γPTHF compositions according to any of

[12] -

[33] ;

[38] The liposomes are cationic, and the Lp-γPTHF composition is one of

[12] -

[33] ;

[39] An Lp-γPTHF composition according to any of

[12] -

[38] , wherein the liposome has an internal space containing γPTHF and an aqueous, pharmaceutically acceptable carrier;

[40] The pharmaceutically acceptable carrier includes an isotonic agent such as dextrose, mannitol, glycerin, potassium chloride, or sodium chloride at a concentration of more than 1% in the Lp-γPTHF composition of

[39] ;

[41] The aqueous Lp-γPTHF composition of

[39] , in which the pharmaceutically acceptable carrier is trehalose;

[42] The Lp-γPTHF composition of

[41] , wherein the pharmaceutically acceptable carrier contains 1% to 50% trehalose;

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

[39] -

[42] , wherein the pharmaceutically acceptable carrier comprises 1% to 50% dextrose solution;

[44] The inner space of the liposome contains 5% dextrose suspended in HEPES buffer. Lp-γPTHF compositions according to any of

[39] -

[43] ;

[45] Pharmaceutically acceptable carriers include buffers such as HEPES-buffered saline (HBS) or similar, at a concentration of 1-200 mM and a pH of 2-8, for Lp-γPTHF compositions according to any of

[39] -

[44] ;

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

[39] -

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

[47] Lp-γPTHF compositions according to any of

[12] -

[46] , in which the interior space of the liposome has a pH of 5-8 or 6-7, or any range therebetween;

[48] ​​Lp-γPTHF compositions according to any of

[12] -

[47] , wherein the liposomes contain less than 500,000 or less than 200,000 γPTHF molecules;

[49] Lp-γPTHF compositions according to any of

[12] -

[48] , in which the liposomes contain 10 to 100,000 γPTHF molecules, or any range therebetween;

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

[12] -

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

[51] The Lp-γPTHF composition according to

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

[52] The Lp-γPTHF composition of

[50] or

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

[53] Lp-γPTHF compositions according to any of

[50] -

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

[54] The targeting moiety is 0.5x10 as measured by BIACORE® analysis. -10 ~10x10 -6 Lp-γPTHF compositions that bind to surface antigens with an equilibrium dissociation constant (Kd) in the range of

[50] -

[53] ;

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

[50] -

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

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

[50] -

[55] , 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] Lp-γPTHF compositions according to any of

[50] -

[56] , in which each PEGylated liposome contains 1-1000 or 30-200 targeting moieties;

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

[39] -

[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 attached to the outer surface of the PEG or the liposome;

[59] The Lp-γPTHF composition of

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

[60] The Lp-γPTHF composition of

[58] or

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

[61] Lp-γPTHF compositions using any of

[58] -

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

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

[58] -

[61] , further comprising a hapten;

[63] The Lp-γPTHF composition of

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

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

[12] -

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

[65] [1]-

[64] . A targeting composition comprising a composition according to any one of the above. a non-targeted composition comprising a composition according to any of

[66] [1]-

[49] ;

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

[12] -

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

[68]

[12] -

[67] . A pharmaceutical composition comprising a liposomal γPTHF composition.

[69] [1]-[7] A pharmaceutical composition comprising a γPTHF composition;

[70] Any of the compositions [1]-

[69] for use in treating a disease;

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

[70] in the manufacture of a medicament for the treatment of a disease and / or in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effectiveness of the therapeutic agent(s), or as a "chemoprotectant" (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 (e.g., treating or preventing) a disease or chemical-induced toxicity in a subject in need of such treatment or prevention, comprising administering to the subject any of the compositions of [1]-

[70] ;

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

[12] -

[69] ;

[74] A method for killing hyperproliferative cells, comprising contacting the hyperproliferative cells with any of the compositions of [1]-

[69] ;

[75] A method for killing hyperproliferative cells, comprising contacting the hyperproliferative cells with any of the liposomal γPTHF compositions of

[12] -

[69] ;

[76] The method of

[74] or

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

[77] A method for treating cancer, comprising delivering an effective amount of any of the compositions [1]-

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

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

[12] -

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

[79] The method of

[77] or

[78] , wherein the composition is administered to treat or prevent cancer, wherein the cancer is 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;

[80] The method of

[77] or

[78] , wherein the composition is administered to treat or prevent cancer, and 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 of

[77] or

[78] , wherein the composition is administered to treat or prevent cancer, and the cancer is a member 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;

[82] The method of

[77] or

[78] , wherein the composition is administered to treat or prevent colorectal cancer;

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

[50] -

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

[84] A maintenance therapy comprising administering an effective amount of any of the compositions of [1]-

[69] to a subject undergoing or having undergone cancer therapy;

[85] A maintenance therapy comprising administering an effective amount of any of the liposomal γPTHF compositions of

[12] -

[69] to a subject undergoing or having undergone cancer therapy;

[86] A method for treating an immune system disorder, comprising administering an effective amount of any of the compositions of [1]-

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

[87] A method for treating an immune system disorder, comprising administering an effective amount of any of the liposomal γPTHF compositions of [8]-

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

[88] A method for treating: (a) a method for treating leukopenia, comprising administering an effective amount of a composition according to any of [1]-

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

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

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

[59] 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 a composition according to any one of [1]-

[59] 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 a composition according to any of [1]-

[59] 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 a composition according to any of [1]-

[59] to a subject having or at risk of having a skin disease, wherein optionally the skin disease is psoriasis;

[89] A method for treating an infectious disease, comprising administering an effective amount of any of the liposomal γPTHF compositions of

[12] -

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

[90] A method for delivering γPTHF to a tumor expressing a folate receptor on its surface, comprising: administering any of the Lp-γPTHF compositions of [1]-

[69] to a subject having a tumor in an amount sufficient to deliver a therapeutically effective amount of γPTHF to the tumor; A method for preparing a gamma-polyglutamated tetrahydrofolate composition, including the liposomal gamma-polyglutamated tetrahydrofolate composition of any of

[91]

[12] -

[69] , comprising: forming a mixture in solution containing liposome components and a gamma-polyglutamated folate antimetabolite; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes containing gamma-polyglutamated tetrahydrofolate; A method for preparing a gamma-polyglutamated tetrahydrofolate composition, including the liposomal gamma-polyglutamated tetrahydrofolate composition of any of

[92]

[12] -

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

[93] The method of

[92] , wherein treating the mixture includes homogenizing the mixture in a solution to form liposomes. A method for preparing any of the compositions of

[94]

[50] -

[69] , comprising the steps of: forming a mixture in a solution containing liposome components and gamma-polyglutamated tetrahydrofolate; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes that entrap and / or encapsulate gamma-polyglutamated tetrahydrofolate; and providing a targeting moiety on the surface of the liposome, the targeting moiety having specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ); A method for preparing any of the compositions of

[95]

[50] -

[69] , comprising the steps of: forming a mixture in solution containing liposome components and gamma-polyglutamated tetrahydrofolate; treating the mixture to form liposomes that entrap and / or encapsulate gamma-polyglutamated tetrahydrofolate; and providing a targeting moiety on the surface of the liposome, the targeting moiety having specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ);

[96] The method of

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

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

[98] The method according to any of

[95] to

[97] , wherein the processing 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; and / or

[99] A method according to any of

[91] -

[98] , in which at least 1% of the gamma-polyglutamated tetrahydrofolate starting material is encapsulated or entrapped in liposomes.

[0128] II. γ-Polyglutamylated Tetrahydrofolate (γPTHF) Generally, the present disclosure relates to γPTHF (γPTHF) compositions that contain at least one glutamyl group with a γ-carboxyl linkage, which are structurally distinct from the L-γ polyglutamylated form of tetrahydrofolate (Lγ1PTHF) produced in cells by the enzyme folylpolyγglutamate synthetase (FPGS) during tetrahydrofolate therapy.

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

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

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

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

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

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

[0135] In some embodiments, γPTHF is heptaglutamated (γTHF-PG6), thus containing a chain of six γ-glutamyl groups attached to the glutamyl groups of tetrahydrofolate. In some embodiments, the γ-heptaglutamated THF contains two or more γ-glutamyl groups in the L-form. In further embodiments, each of the γ-glutamyl groups of the γ-heptaglutamated tetrahydrofolate is in the L-form. In other embodiments, the γ-heptaglutamated THF contains a γ-glutamyl group in the D-form. In some embodiments, the γ-tetraglutamated THF contains two, three, four, five, or six γ-glutamyl groups in the D-form. In further embodiments, each of the γ-glutamyl groups of the γ-heptaglutamated tetrahydrofolate other than the glutamyl group of the tetrahydrofolate is in the D-form. In additional embodiments, the heptaglutamated THF contains a γ-glutamyl group in the D-form and two or more γ-glutamyl groups in the L-form.

[0136] In some embodiments, γPTHF is octaglutamated (γTHF-PG7), thus containing a chain of seven γ-glutamyl groups attached to the glutamyl group of tetrahydrofolate. In some embodiments, γ-octaglutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of γ-octaglutamated tetrahydrofolate is in the L-form. In other embodiments, γ-octaglutamated THF contains a glutamyl group in the D-form. In some embodiments, γ-octaglutamated THF contains 2, 3, 4, 5, 6, or 7 γ-glutamyl groups in the D-form. In further embodiments, each of the glutamyl groups of γ-octaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form. In additional embodiments, octaglutamated THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

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

[0138] In some embodiments, γPTHF is deca-glutamated (γTHF-PG9) and contains a chain of nine γ-glutamyl groups attached to the glutamyl groups of tetrahydrofolate. In some embodiments, γ-decaglutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of γ-decaglutamated tetrahydrofolate is in the L-form. In other embodiments, γ-decaglutamated THF contains a glutamyl group in the D-form. In further embodiments, each of the glutamyl groups of γ-decaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form. In additional embodiments, deca-glutamated THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

[0139] In some embodiments, γPTHF is undecaglutamated (γTHF-PG 10), which contains a chain of 10 γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the γ-undeca-glutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the γ-undeca-glutamated tetrahydrofolic acid is in the L-form. In other embodiments, the γ-undeca-glutamated THF contains a D-glutamyl group. In further embodiments, each of the glutamyl groups of the γ-undeca-glutamated tetrahydrofolic acid, other than the glutamyl group of tetrahydrofolic acid, is in the D-form. In additional embodiments, the undecaglutamated THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

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

[0141] In some embodiments, γPTHF is triskite-decaglutamated (γTHF-PG 12), which contains a chain of 12 γ-glutamyl groups attached to the glutamyl group of tetrahydrofolate. In some embodiments, the γ-triskaidecaglutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the γ-triskaidecaglutamated tetrahydrofolate is in the L-form. In other embodiments, the γ-triskaidecaglutamated THF contains a glutamyl group in the D-form. In further embodiments, each of the glutamyl groups of the γ-triskaidecaglutamated tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, is in the D-form. In additional embodiments, the triskaidecaglutamated THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

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

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

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

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

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

[0147] In some embodiments, γPTHF is eneadecaglutamated (γTHF-PG 18), which contains a chain of 18 γ-glutamyl groups attached to the glutamyl group of tetrahydrofolic acid. In some embodiments, the γ-ene adeca glutamated THF contains two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the γ-ene adeca glutamated tetrahydrofolic acid is in the L-form. In other embodiments, the γ-ene adeca glutamated THF contains a D-glutamyl group. In further embodiments, each of the glutamyl groups of the γ-ene adeca glutamated tetrahydrofolic acid, other than the glutamyl group of tetrahydrofolic acid, is in the D-form. In additional embodiments, the ene adeca glutamated THF contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

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

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

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

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

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

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

[0154] In another embodiment, the γ-PTHF is heptaglutamated. In some embodiments, each of the six glutamyl groups is in the L-form. In some embodiments, each of the glutamyl groups in the γ-heptaglutamated tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, is in the D-form. In other embodiments, at least two glutamyl groups in the γ-heptaglutamated tetrahydrofolate are in the L-form and at least one glutamyl group is in the D-form.

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

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

[0157] In additional embodiments, provided compositions comprise γPTHF containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups (with γ-linkages). In some embodiments, γPTHF contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups in the L-form. In some embodiments, γPTHF 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, γPTHF 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.

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

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

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

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

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

[0163] B.THF-PG synthesis The tetrahydrofolate polyglutamic acid compositions provided herein can be obtained by the following synthetic methods using available reagents and synthetic intermediates. The addition of glutamyl residues to the glutamyl residues of tetrahydrofolate can be achieved using synthetic procedures known in the art. In some embodiments, glutamyl residues are sequentially added to the glutamyl residues of tetrahydrofolate. In additional embodiments, polyglutamic acid is 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 of tetrahydrofolate that does not have a glutamyl residue. The peptide can be produced using synthetic procedures known in the art. In some embodiments, the first glutamyl residue is coupled to a Wang resin, and additional glutamyl residues are sequentially added by solid-phase peptide synthesis using F-moc chemistry. After the final glutamyl residue is added, a tetrahydrofolate precursor is coupled to the peptide and the molecule is cleaved from the resin.

[0164] The addition of glutamyl residues to the glutamyl residues of tetrahydrofolate can be achieved using synthetic procedures known in the art. In some embodiments, glutamyl residues are added sequentially to the glutamyl residues of tetrahydrofolate. In additional embodiments, polyglutamic acid is 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 of tetrahydrofolate that does not have a glutamyl residue. Peptides can be produced using synthetic procedures known in the art. In some embodiments, the first glutamyl residue is attached to a Wang resin, and additional glutamyl residues are added sequentially using F-moc chemistry by solid-phase peptide synthesis. After the final glutamyl residue is added, a tetrahydrofolate precursor is coupled to the peptide, and the molecule is cleaved from the resin.

[0165] C. Tetrahydrofolate-PG complex Surprisingly, the inventors have discovered that polyglutamated antifolates, which share similar structural and chemical characteristics with tetrahydrofolic acid (γPTHF), can be complexed with other compositions, including therapeutic agents, including cytotoxic compounds, such as platinum-based compounds. Thus, in some embodiments, the present disclosure provides complexes of γPTHF (e.g., the γPTHF disclosed herein) with a therapeutic agent, or a salt or acid thereof.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0181] In additional embodiments, the present disclosure provides a complex of γPTHF (e.g., the γPTHF disclosed herein) and cyclodextrin. Cyclodextrins (CDs) are a group of cyclic oligosaccharides that have been shown to improve the physicochemical properties of many drugs through complexation. CDs are cyclic oligosaccharides composed of several D-glucose units linked by α-(1,4) bonds. This cyclic structure provides a hydrophobic interior cavity, giving CDs a shortened pyramidal shape. Many hydroxyl groups are located on the ends of the ring, making CDs both lipophilic and water-soluble. As a result, CDs can form complexes with a wide variety of hydrophobic drugs, thereby altering the physicochemical properties of these complexed drugs.

[0182] The terms "cyclodextrin" or "CD," unless otherwise specified, generally refer to parent or derivatized cyclic oligosaccharides capable of complexing with tetrahydrofolate-PG, containing a variable number of (α-1,4)-linked D-glucopyranoside units. Each cyclodextrin glucopyranoside subunit has secondary hydroxyl groups at the 2 and 3 positions and a primary hydroxyl group at the 6 position. The terms "parent," "underivatized," or "inactive" cyclodextrin refer to cyclodextrins of the basic formula CH, containing D-glucopyranoside units. 12This refers to a cyclodextrin with an O6 and glucose structure and no additional chemical substituents (e.g., α-cyclodextrin, consisting of six D-glucopyranoside units; β-cyclodextrin, consisting of seven D-glucopyranoside units; and γ-cyclodextrin, consisting of eight D-glucopyranoside units). The physical and chemical properties of the parent cyclodextrin can be modified by derivatizing the hydroxyl groups with other functional groups. Any substance located in the cyclodextrin internal phase is said to be "complexed" with the cyclodextrin or to form a complex (inclusion complex) with the cyclodextrin.

[0183] As used herein, there are no particular limitations on the cyclodextrin component of a γPTHF / cyclodextrin complex, so long as the cyclodextrin is capable of forming a complex with γPTHF. In certain embodiments, the cyclodextrin has been derivatized to have ionizable (e.g., weakly basic and / or weakly acidic) functional groups to facilitate complexation with γPTHF and / or liposome encapsulation.

[0184] Modification of cyclodextrin hydroxyl groups, such as those facing away from the cyclodextrin internal phase, with ionizable chemical groups is known to facilitate the addition of cyclodextrin and therapeutic agents complexed with the cyclodextrin. In some embodiments, the cyclodextrin in the γPTHF / cyclodextrin complex has at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 hydroxyl groups substituted with ionizable chemical groups. The term "charged cyclodextrin" refers to a cyclodextrin having hydroxyl groups substituted with one or more of its charged moieties. Such moieties may themselves be charged groups or may include organic moieties (e.g., C1-C6 alkyl or C1-C6 alkyl ether moieties) substituted with one or more charged moieties.

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

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

[0187] In another embodiment, the derivatized cyclodextrin is a "chargeable amphiphile." "Chargeable" means that the amphiphile has a pK in the pH range of 4-8 or 4-8.5. A chargeable amphiphile can therefore be a weak acid or base. "Amphoteric," as used herein, refers to a derivatized cyclodextrin having ionizable groups of both anionic and cationic character, where (a) at least one, and optionally both, cationic and anionic amphiphiles are chargeable and have at least one charged group with a pK between 4 and 8-8.5, (b) the cationic charge predominates at pH 4, and (c) the anionic charge predominates at pH 8-8.5.

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

[0189] Any one, some, or all of the hydroxyl groups of any one, some, or all of the α-D-glucopyranoside units of a cyclodextrin can be modified with an ionizable chemical group as described herein. Because each cyclodextrin hydroxyl group differs in chemical reactivity, reaction with the modifying moiety can produce an amorphous mixture of regio- and optical isomers. Alternatively, the pre-modified α-D-glucopyranoside units can be reacted to form a homogeneous product, depending on the specific chemistry.

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

[0191] In one embodiment, at least one hydroxyl moiety facing away from the interior of the cyclodextrin is substituted with an ionizable chemical group. For example, at least one γ-D-glucopyranoside unit among all three of the C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and C2-C3-C6 hydroxyls is substituted with an ionizable chemical group. Any such hydroxyl combination can be combined with any of the degrees of substitution described herein, as well as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and up to all α-D-glucopyranoside units in the modified cyclodextrin. One such derivative is sulfoalkyl ether cyclodextrin (SAE-CD). The sulfobutyl ether derivative of β-cyclodextrin (SBE-β-CD) has been shown to have significantly improved aqueous solubility compared to the parent cyclodextrin.

[0192] Additional cyclodextrin derivatives that can be complexed with therapeutic agents in the disclosed liposomal compositions include sugammadex or Org-25969, in which the 6-hydroxy group on γ-CD has been replaced with a carboxythioacetate ether linkage, and hydroxybutenyl-β-CD. Alternative forms of cyclodextrin include 2,6-di-O-methyl-β-CD (DIMEB), 2-hydroxypropyl-3-cyclodextrin (HP-β-CD), randommethylated-β-cyclodextrin (RAMEB), sulfobutyl ether Examples of suitable cyclodextrins include β-cyclodextrin (SBE-β-CD), sulfobutylether-γ-cyclodextrin (SBEγCD), sulfobutylated-β-cyclodextrin sodium salt, (2-hydroxypropyl)-γ-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, 2,6-di-O-methyl)-β-cyclodextrin (DIMEB-50 heptakis), 2,3,6-tri-O-methyl)-β-cyclodextrin (TRIMEB heptakis), methyl-β-cyclodextrin, octakis(6-deoxy-6-iodo)-γ-cyclodextrin, and octakis(6-deoxy-6-bromo)-γ-cyclodextrin.

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

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

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

[0196] In some embodiments, the cyclodextrin of the γPTHF / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is derivatized. In further embodiments, the cyclodextrin derivative of the complex has Formula I: [ka] wherein n is 4, 5, or 6; R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently -H, a linear or branched C1-C8 alkylene group, or an optionally substituted linear or branched C1-C6 group, and at least one of R1, R2, R3, R4, R5, R6, R7, R8, and R9 is a linear or branched C1-C8 alkylene (e.g., C1-C8-(alkylene)-SO3 - (base).

[0197] In some embodiments, the cyclodextrin derivative of the γPTHF / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex has Formula II: [ka] wherein n is 4, 5, or 6; R1, R2, R3, R4, R5, R6, R7, R8, and R9 each independently represent -O- or -O-(C2-C6 alkylene)-SO3 - group; and at least one of R1 and R2 is independently -O-(C2-C6 alkylene)-SO3 - groups; and S1, S2, S3, S4, S5, S6, S7, S8, and S9 are each independently a pharmaceutically acceptable cation. In a further embodiment, the pharmaceutically acceptable cation is Li + , Na + , or K + Alkali metals such as Ca 2+ , or Mg 2+ and amine cations such as ammonium ions and cations of (C-C)-alkylamines, piperidines, pyrazines, (C-C)-alkanolamines, and (C-C)-cycloalkanolamines. In some embodiments, at least one of R and R is independently selected from -O-(CH). man SO3- group, -O-(C2-C6 alkylene)-SO3- group, where m is 2 to 6, preferably 2 to 4 (e.g., -O-CH2CH2CH2SO3- or -O-CH2CH2CH2CH2SO3-); and S1, S2, S3, S4, S5, S6, S7, S8, and S9 are each independently H or a pharmaceutically acceptable cation, including, for example, an alkali metal (e.g., Li + , Na + , K. + ), alkaline earth metals (e.g., Ca 2+ , Mg 2+ ), ammonium ions and amine cations such as the cations of (C1-C6)-alkylamines, piperidines, pyrazines, (C1-C6)-alkanolamines and (C4-C8)-cycloalkanolamines.

[0198] In some embodiments, the cyclodextrin derivative of the γPTHF / cyclodextrin complex and / or cyclodextrin / therapeutic agent complex is a cyclodextrin disclosed in U.S. Patent Nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, 6,869,939; and WO 02005 / 117911, the contents of each of which are expressly incorporated herein by reference.

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

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

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

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

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

[0204] In some embodiments, provided liposomes further comprise an immunostimulatory agent, a detectable marker, or both, disposed on the exterior surface of the liposome. The immunostimulatory agent or detectable marker can be ionically or covalently bound to the exterior surface of the liposome, which may optionally include binding to a steric stabilizing component of the liposome.

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

[0206] In some embodiments, the liposome comprises a detectable marker, which may include, for example, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, an enzyme, a dye, an ink, a magnetic compound, a biocatalyst, or a pigment that is detectable by any suitable means known in the art, including, for example, at least, magnetic resonance imaging (MRI), optical imaging, fluorescence / luminescence imaging, and / or nuclear imaging techniques.

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

[0208] In some embodiments, the liposome further comprises an agent that increases uptake of the liposome into a desired intracellular compartment, including the cytosol.

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

[0210] In some embodiments, the liposomes in the provided liposome compositions comprise a mitochondrial penetrant selected from a guanidine-rich peptoid, a tetraguanidinium, a triguanidinium, a diguanidinium, a monoguanidinium, a guanidine-rich polycarbamate, a beta-oligoarginine, a proline-rich dendrimer, and a phosphonium salt (e.g., methyltriphenylphosphonium and / or tetraphenylphosphonium).

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

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

[0213] As discussed above, liposomes may contain a steric stabilizer that can extend their lifespan in the circulation. For those embodiments incorporating a steric stabilizer, the steric stabilizer may be at least one member selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); 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. In some embodiments, the steric stabilizer or stabilizers is PEG. In one embodiment, the steric stabilizer is PEG. In a further embodiment, the PEG has a number-average molecular weight (Mn) of 200 to 5,000 daltons. These PEGs can be of any configuration, such as linear, branched, star or comb, and are commercially available.

[0214] In some embodiments, the liposome composition comprises PEGylated liposomes (PLp-γPTHF). In some embodiments, the liposomes in the PEGylated liposome composition comprise γPTHF containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the γPTHF in Lp-γPTHF comprises two or more L-glutamyl groups. In other embodiments, the γPTHF in Lp-γPTHF comprises a D-glutamyl group. In further embodiments, the γPTHF in Lp-γPTHF comprises a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the liposome composition comprises PEGylated liposomes comprising gamma-pentaglutamated THF. In further embodiments, the liposomes comprise L-γ-pentaglutamated THF, D-γ-pentaglutamated THF, or L- and D-γ-pentaglutamated THF. In some embodiments, the liposome composition comprises PEGylated liposomes comprising gamma-hexaglutamated THF. In further embodiments, the liposomes comprise L-γ hexaglutamated THF, D-γ hexaglutamated THF, or L- and D-γ hexaglutamated THF. In some embodiments, the liposome composition comprises PEGylated liposomes that are anionic or neutral. In some embodiments, the liposome composition comprises PEGylated liposomes that are cationic. In some embodiments, the PLp-γPTHF composition is non-targeted (NTPLp-γPTHF). In other embodiments, the PLp-γPTHF composition is targeted (TPLp-γPTHF). In some embodiments, the liposome composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w γPTHF. In some embodiments, the liposome composition comprises PEGylated liposomes comprising at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w γPTHF encapsulated (entrapped) in PLp-γPTHF during the process of preparing the liposomes.In some embodiments, the liposome composition comprises PEGylated liposomes having a diameter in the range of 20 nm to 500 nm. In some embodiments, the liposome composition comprises PEGylated liposomes having a diameter in the range of 20 nm to 400 nm. In some embodiments, the liposome composition comprises PEGylated liposomes having a diameter in the range of 20 nm to 300 nm. In some embodiments, the liposome composition comprises PEGylated liposomes having a diameter in the range of 20 nm to 200 nm. In further embodiments, the liposome composition comprises PEGylated liposomes having a diameter in the range of 80 nm to 120 nm.

[0215] In some embodiments, more than 70%, 80%, or 90% of the polyglutamated tetrahydrofolate in a provided liposome composition is pentaglutamated. In some embodiments, more than 70%, 80%, or 90% of the polyglutamated tetrahydrofolate in a provided composition is hexaglutamated. In some embodiments, more than 70%, 80%, or 90% of the polyglutamated tetrahydrofolate in a composition has 4-10, 4-6, or more than 5 γ-glutamyl groups.

[0216] In some embodiments, the γPTHF composition (e.g., a delivery vehicle such as polyglutamic acid and liposomes containing polyglutamic acid) is in an aqueous solution. In some embodiments, the γPTHF composition is in a liposomal composition and is administered at a dose of 1000 mg / m 2 per square meter (m 3 ). 2 In a further embodiment, the γPTHF composition is administered in a liposomal composition at a dose of 0.1 to 1000 mg of γPTHF per square meter of body surface area, or any range therebetween.

[0217] (1) Liposome composition Lipids and other components of liposomes contained in the liposome composition can be any lipid, lipid combination and ratio, or combination of lipids and other liposome components and their respective ratios known in the art. However, those skilled in the art will understand that liposomal encapsulation of any particular drug, such as, but not limited to, gamma-polyglutamated THF discussed herein, can involve substantial routine experimentation to obtain a useful and functional liposome formulation. Generally, the provided liposomes can have any liposome structure, for example, a structure with an internal space isolated from the external medium by one or more lipid bilayers, or any microcapsule structure with a semipermeable membrane with a lipophilic central portion that separates the interior. The lipid bilayer can be any structure of amphiphilic molecules characterized by hydrophilic and hydrophobic portions. Typically, the amphiphilic molecules in the bilayer are arranged in a two-dimensional sheet, with the hydrophobic portion facing the inside of the sheet, while the hydrophilic portion faces outward. The amphiphilic molecules forming the provided liposomes can be any known or yet to be discovered amphiphilic molecules (e.g., synthetic or naturally occurring lipids or biocompatible lipids). Liposomes can be formed from amphiphilic polymers and surfactants, such as polymersomes and niosomes. In the present disclosure, these liposome-forming materials are also referred to as "lipids," although this disclosure is not limited thereto.

[0218] The liposome composition formulations provided herein can be in liquid or dry form, such as a dry powder or dry cake. The dry powder or dry cake can undergo primary drying, for example, under lyophilization conditions, or can undergo primary drying only or both primary and secondary drying. In dry form, the powder or cake can have, for example, 1% to 6% moisture, e.g., 2% to 5% moisture, or 2% to 4% moisture. One example of a drying method is lyophilization (also called freeze-drying or cryodesication). Any of the disclosed compositions and methods can include liposomes, lyophilized liposomes, or liposomes reconstituted from lyophilized liposomes. In some embodiments, the disclosed compositions and methods include one or more lyoprotectants or cryoprotectants. These protectants are typically sugars (mono-, di-, and polysaccharides), polyhydric alcohols and their derivatives, glycerol or polyhydroxy compounds such as polyethylene glycol, trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol, or aminoglycosides. In further embodiments, the lyoprotectant or cryoprotectant comprises up to 10% or up to 20% of the solution outside the liposome, inside the liposome, or both outside and inside the liposome.

[0219] In some embodiments, liposomes contain steric stabilizers that extend their lifespan in the circulation. One or more steric stabilizers, such as hydrophilic polymers (polyethylene glycol (PEG)), glycolipids (monosialoganglioside (GM1)), and others, occupy the space immediately adjacent to the liposome surface, excluding other macromolecules from this space. This prevents plasma opsonins from accessing and binding to the liposome surface, thereby inhibiting macrophage interaction with such liposomes or any other clearance mechanism, and extending the lifespan of liposomes in the circulation. In some embodiments, the steric stabilizer or stabilizers is PEG or a combination comprising PEG. In further embodiments, the steric stabilizer is PEG or a combination comprising PEG with a number-average molecular weight (Mn) of 200 to 5,000 daltons. These PEGs can be of any architecture, including linear, branched, star-shaped, or comb-shaped, and are commercially available.

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

[0221] The properties of liposomes are influenced by the nature of the lipids used to prepare the liposomes. A wide variety of lipids have been used to prepare liposomes. These include cationic, anionic, and neutral lipids. In some embodiments, liposomes containing γPTHF are anionic or neutral. In other embodiments, provided liposomes are cationic. The charge (e.g., anionic, neutral, or cationic) can be routinely determined by measuring the zeta potential of the liposomes. The zeta potential of the liposomes can be positive, zero, or negative. In some embodiments, the zeta potential of the liposomes is less than or equal to zero. In some embodiments, the zeta potential of the liposomes is in the range of 0 to -150 mV. In other embodiments, the zeta potential of the liposomes is in the range of -30 to -50 mV.

[0222] In some embodiments, cationic lipids are used to create cationic liposomes, which are often used as gene transfer agents. The positive charges on the cationic liposomes allow them to interact with the negative charges on the cell surface. After the cationic liposomes bind to cells, the liposomes are transported into the interior of the cells by endocytosis.

[0223] In some preferred embodiments, neutral to anionic liposomes are used. In preferred embodiments, anionic liposomes are used. For example, the use of a mixture of neutral lipids such as HSPC and anionic lipids such as PEG-DSPE forms anionic liposomes, which are less likely to nonspecifically bind to normal cells. Specific binding to tumor cells can be achieved using tumor-targeting antibodies, such as folate receptor antibodies, including folate receptor alpha antibodies, folate receptor beta antibodies, and / or folate receptor δ antibodies.

[0224] As an example, at least one (or some) lipid is an amphipathic lipid, defined as having a hydrophilic and a hydrophobic portion (usually a hydrophilic head and a hydrophobic tail). The hydrophobic portion typically faces the hydrophobic phase (e.g., within the bilayer), while the hydrophilic portion typically faces the aqueous phase (e.g., outside the bilayer). The hydrophilic portion can include polar or charged groups such as carbohydrates, phosphate, carboxylic acid, sulfato, amino, sulfhydryl, nitro, hydroxy, and other similar groups. The hydrophobic portion can include nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

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

[0226] The lipids comprising the liposomes provided herein can be anionic and neutral (including zwitterionic and polar) lipids, including anionic and neutral phospholipids. Neutral lipids exist in uncharged or neutral zwitterionic form at selected pHs. At physiological pH, such lipids include, for example, dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol. Examples of zwitterionic lipids include, but are not limited to, dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS). Anionic lipids are negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and anionic modifying groups attached to neutral lipids.

[0227] In summary, anionic and neutral lipids are referred to herein as non-cationic lipids.Such lipids may contain phosphorus, but they are not limited to such.Examples of non-cationic lipids include lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidyl ...DPPE), dioleoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DPPE), dioleoylphosphat 16-O-trans PE, 16-O-dimethyl PE, 18-1-trans PE, palmitoyloleoyl-phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), phosphatidylserine, phosphatidyl-inositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl-phosphate, and cholesterol.

[0228] Liposomes can be constructed using any liposome assembly method using liposomal components (also referred to as liposome components) known in the art. Liposome components include, for example, lipids such as DSPE, HSPC, cholesterol, and derivatives of these components. Other suitable lipids are commercially available, for example, from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). A partial list of available negatively or neutrally charged lipids suitable for making anionic liposomes can be, for example, at least one of the following: DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DOPE, DMPA·Na, DPPA·Na, DOPA·Na, DMPG·Na, DPPG·Na, DOPG·Na, DMPS·Na, DPPS·Na, DOPS·Na, DOPE-glutaryl·(Na)2, tetramyristoylcardiolipin·(Na)2, DSPE-mPEG-2000·Na, DSPE-mPEG-5000·Na, and DSPE-maleimidePEG-2000·Na.

[0229] In some embodiments, the γPTHF compositions provided herein are formulated in liposomes containing cationic l...

Claims

1. A liposome composition comprising a liposome encapsulating gamma polyglutamylated tetrahydrofolic acid, The gamma polyglutamylated tetrahydrofolate comprises two or more glutamyl groups linked by gamma carboxyl group bonds, and the gamma polyglutamylated tetrahydrofolate comprises (a) gamma polyglutamylated 5-formyl-THF (e.g., gamma polyglutamylated [6S]-5-formyl-THF); (b) gamma polyglutamylated 10-formyl-THF (e.g., gamma polyglutamylated [6R]-10-formyl-THF); (c) gamma polyglutamylated 5,10-methenyl-THF (e.g., gamma polyglutamylated [6R]-5,10-methenyl-THF); (d) gamma polyglutamylated 5-methyl-THF (e.g., gamma polyglutamylated [6S]-5-methyl-THF); (e) gamma polyglutamylated tetrahydrofolate (e.g., gamma polyglutamylated [6S]-tetrahydrofolate THF); (f) gamma polyglutamylated 5,10-methylene-THF (e.g., gamma polyglutamylated [6R]-5,10-methylene-THF); and (g) gamma polyglutamylated 5-formimino-THF (e.g., gamma polyglutamylated [6S]-5-formimino-THF); A liposomal composition, wherein the liposomes are pegylated and contain a targeting moiety that has specific affinity for a surface antigen on a target cell.

2. The liposome composition of claim 1 , wherein the gamma polyglutamylated tetrahydrofolate comprises a glutamyl group having a gamma carboxyl group bond.

3. The liposome composition of claim 1 , wherein the gamma polyglutamylated tetrahydrofolic acid is gamma tetraglutamylated tetrahydrofolic acid, gamma pentaglutamylated tetrahydrofolic acid, or gamma hexaglutamylated tetrahydrofolic acid.

4. The liposome composition of claim 1, wherein the gamma polyglutamylated tetrahydrofolate comprises 4-6 glutamyl groups with gamma carboxyl group linkages.

5. The liposome composition of claim 1 , wherein the targeting moiety is covalently attached to one or both of PEG and the exterior surface of the liposome.

6. 2. The liposome composition of claim 1, wherein the targeting moiety is one or more selected from the group consisting of a polypeptide or 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.

7. The liposome composition of claim 1, wherein the liposome comprises 30 to 200 targeting moieties.

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

9. The liposomes are formed from liposome components comprising at least one of anionic lipids and neutral lipids, and include at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide, and optionally the liposome components comprise 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 further comprises a steric stabilizer, and optionally polyethylene glycol (PEG); poly-L-lysine (PLL); monosodium EDTA (MSTA) or tert-butyl ether (TBE) or tert-butyl ether (TBE).

2. The liposome composition of claim 1, wherein the steric stabilizer is selected from the group consisting of aloganglioside (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; oligoglycerol, polyethylene glycol and polypropylene oxide containing copolymers, poloxamer 188, and polyvinyl alcohol, and optionally the steric stabilizer is PEG, and the PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons.

10. 2. The liposome composition of claim 1, wherein the liposomes are anionic, the liposomes are cationic, the liposomes are neutral, the liposomes have a zeta potential of less than zero, the liposomes have a zeta potential of 0 to -150 mV, or the liposomes have a zeta potential of -30 to -50 mV.

11. 2. The liposome composition of claim 1, wherein the liposome has an interior space comprising gamma polyglutaminated tetrahydrofolate and an aqueous pharma- ceutically acceptable carrier, and optionally comprises an isotonicity agent such as dextrose, mannitol, glycerin, 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, and optionally the liposome interior space has a pH of 5-8 or a pH of 6-7, or any range therebetween, and optionally the liposome comprises less than 500,000 or less than 200,000 of the gamma polyglutaminated tetrahydrofolate molecules, or comprises 10-100,000 gamma polyglutamated tetrahydrofolate molecules, or any range therebetween.

12. The liposome further comprises one or more of an immunostimulant, a detectable marker, and a maleimide, wherein the immunostimulant, detectable marker, or maleimide is attached to PEG or the outer surface of the liposome, and the immunostimulant is selected from the group consisting of fluorescein; fluorescein isothiocyanate (FITC); DNP; β-glucan; β-1,3-glucan; β-1,6-glucan; resolvin (e.g., DNP); ｎ－６ＤＰＡ Or D ｎ－３ＤＰＡ 2. The liposome composition of claim 1, further comprising at least one selected from the group consisting of: resolvin D, resolvin E, or T-series resolvins such as; and a Toll-like receptor (TLR) modulator such as oxidized low density lipoprotein (e.g., OXPAC, PGPC), and an erythrocyanate lipid (e.g., E5564), optionally further comprising at least one cryoprotectant selected from the group consisting of mannitol; trehalose; sorbitol; and sucrose, or further comprising fluorouracil or tetrahydrofolic acid.

13. 10. A pharmaceutical composition comprising the liposomal composition of claim 1 and, optionally, fluorouracil, such as 5FU.

14. The liposome composition according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 13 for use in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents, for the treatment of a disease, chemical induced toxicity, leukopenia, and / or to enhance the efficacy of or reduce toxic side effects associated with the therapeutic agent, optionally wherein the disease is cancer, a disorder of the immune system, or an infectious disease, and optionally an autoimmune disease, rheumatoid arthritis, or an inflammatory disease.

15. 13. A method of preparing a composition according to any one of claims 1 to 12, comprising forming a mixture in a solution comprising liposome components and gamma-polyglutamylated tetrahydrofolate; homogenizing the mixture in the solution to form liposomes; and processing the mixture to form liposomes containing gamma-polyglutamylated tetrahydrofolate, optionally 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.

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