Alpha polyglutamated antifolates and uses thereof

Abstract

To provide an alpha polyglutamated antifolate composition capable of overcoming pharmacological challenges associated with dose-limiting toxicity and treatment resistance related to antifolate therapy.SOLUTION: There is provided a liposomal composition comprising a liposome encapsulating an alpha polyglutamated antifolate, wherein the alpha polyglutamated antifolate comprises 2 to 15 glutamyl groups linked via alpha-carboxyl group bonds, wherein the liposome is pegylated, and comprises a targeting moiety having specific affinity for a surface antigen of a desired target cell.SELECTED DRAWING: None

Description

Background Art

[0001] The present disclosure generally relates to compositions of alpha-polyglutamated folic acid antimetabolites, including delivery carriers such as liposomes containing alpha-polyglutamated folic acid antimetabolite compositions, and methods for the manufacture and use of compositions for treating diseases including hyperproliferative diseases such as cancer, immune system disorders including inflammation and autoimmune diseases such as rheumatoid arthritis, and infectious diseases such as HIV, malaria, and schistosomiasis.

[0002] Folic acid is an essential cofactor that mediates the transfer of one-carbon units involved in nucleotide biosynthesis and DNA repair, remethylation of homocysteine (Hcy), and methylation of DNA, proteins, and lipids. The only circulating form of folic acid in the blood is monoglutamic acid, and folic acid monoglutamate is the only form of folic acid that is transported across the cell membrane. Similarly, monoglutamic acid-type polyglutamatable folic acid antimetabolites are also transported across the cell membrane. Once taken up into the cell, intracellular folic acid is converted to polyglutamic acid by the enzyme folylpoly-gamma-glutamate synthetase (FPGS).

[0003] Folic acid antagonists are transported into cells by the reduced folate carrier (RFC) system and folate receptors (FR) α and β, as well as by the proton-coupled folate transporter (PCFT), which is most active in a lower than normal pH environment. RFC is the major transporter of folic acid antagonists at physiological pH and is widely expressed in normal and diseased cells. Thus, folic acid antagonist therapy often suffers from dose-limiting toxicity, a major obstacle in cancer chemotherapy. Once inside the cell, folic acid antagonists are polyglutamylated by FPGS, which can add up to six glutamyl groups during the binding of the L-gamma carboxyl group to the folic acid antagonist. L-gamma polyglutamylation of folic acid antagonists by FPGS serves at least two major therapeutic purposes: (1) it greatly enhances the affinity and inhibitory activity of the folic acid antagonist for DHFR; and (2) it facilitates the accumulation of polyglutamylated folic acid antagonists, which, unlike the folic acid antagonist (monoglutamate), are not readily transported out of the cell by the cellular efflux pump.

[0004] Targeting folic acid metabolism and nucleotide biosynthesis is an established therapeutic strategy for cancer, but for folic acid antagonists, clinical efficacy is limited by a lack of tumor selectivity and the presence of new and acquired drug resistance. Folic acid antagonists often act during DNA and RNA synthesis and as a result have a major toxic effect on rapidly dividing cells such as malignant and myeloid cells. Myelosuppression is usually the dose-limiting toxicity of folic acid antagonist therapy and restricts the clinical application of folic acid antagonists.

[0005] Resistance to folic acid antagonist therapy is usually associated with one or more of the following: (a) increased cellular efflux pump activity, (b) decreased transport of the folic acid antagonist into the cell, (c) increased DHFR activity, (d) decreased activity of the holylpolyglutamate synthase (FPGS), and (e) increased activity of the gamma-glutamyl hydrolase (GGH), which cleaves the gamma-polyglutamate chain bound to folic acid and folic acid antagonists.

[0006] The problem with the long-term (>30 years) observation that higher levels of polyglutamate of various folic acid antagonists have far higher potency compared to lower levels of glutamate was that the scientific community has relied on the intracellular FPGS-mediated mechanism that converts lower levels of glutamate to their higher level forms. The present invention provides a means for directly delivering higher levels of polyglutamate forms of folic acid antagonists into cells without relying on the cell's machinery to achieve this goal.

[0007] The provided alpha-polyglutamylated folic acid antagonist composition provides a strategy to overcome the pharmacological issues related to dose-limiting toxicity and treatment resistance associated with folic acid antagonist therapy. In some embodiments, the provided method delivers an alpha-polyglutamylated form of a folic acid antagonist to cancer cells while simultaneously (1) minimizing / reducing exposure to normal tissue cells, (2) optimizing / improving the cytotoxic effect of the folic acid antagonist-based agent on cancer cells, and (3) minimizing / reducing the impact of efflux pumps and other resistance mechanisms that limit the therapeutic efficacy of folic acid antagonists. SUMMARY OF THE INVENTION

[0008] The present disclosure generally relates to novel alpha-polyglutamylated folic acid antagonist (αPANTIFOL) compositions, and methods of making and using the compositions for treating diseases including hyperproliferative diseases such as cancer, immune system disorders such as inflammation and rheumatoid arthritis, cardiovascular diseases such as coronary artery disease, and infectious diseases such as HIV, malaria, and schistosomiasis.

[0009] In some embodiments, the present disclosure provides the following. [1] A composition comprising an alpha-polyglutamylated folic acid antagonist, wherein at least one glutamyl group has an alpha-carboxyl group bond, the composition; [2] The composition of item [1], wherein the folic acid antagonist is selected from piritrexim, pralatrexate, AG2034, GW1843, and LY309887, or stereoisomers thereof; [3] The composition of item [1], wherein the folic acid antagonist is selected from PMX, MTX, RTX, and LMX, or stereoisomers thereof; [4] The composition according to item [1], wherein the folic acid antagonist is selected from the following: LV (etoposide), L-leucovorin (L-5-formyltetrahydrofolic acid); 5-CH3-THF, 5-methyltetrahydrofolic acid; FA, folic acid; PteGlu, pteroylglutamate (FA); MTX, methotrexate; 2-dMTX, 2-desamino-MTX; 2-CH3-MTX, 2-desamino-2-methyl-MTX; AMT, folic acid antagonist; 2-dAMT, 2-desamino-AMT; 2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM, 10-ethyl-10-deazaaminopterin; PT523, N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine; DDATHF (raltitrexed), 5,10-dideaza-5,6,7,8,-tetrahydrofolic acid; 5-d(i)H4PteGlu, 5-deaza-5,6,7,8-tetrahydroisofolic acid; N9-CH3-5-d(i)H4PteGlu, N9-methyl-5-deaza-5,6,7,8-tetrahydroisofolic acid; 5-dPteHCysA, N alpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, N alpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutyric acid; 5-dPteOrn, N alpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, N alpha-(5-deaza-5,6,7,8-tetrahydroapteroyl)-L-homocysteic acid; 5-dH4PteAPBA, N alpha-(5-deaza-5,6,7,8-tetrahydroapteroyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dH4PteOro, N alpha-(5-dideaza-5,6,7,8-tetrahydroapteroyl)-L-ornithine; CB3717, N10-propargyl-5,8-dideazafolic acid; ICI-198,583, 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid; 4-H-ICI-198,583, 4-deoxy-ICI-198,583:4-OCH3-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198;583;Glu-to-Sub-ICI-198,583, 2-aminosuberic acid-ICI-198,583; 7-CH3-ICI-198,583, 7-methyl-ICI-198,583; ZD1694, N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamic acid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89, (S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutamic acid; LY231514, N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidine-5-yl)ethyl)-benzoyl]-L-glutamic acid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ; 2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu, 5-dideazaisofolic acid; N9-CH3-5-d(i)PteGlu, N9-methyl-5-dideazaisofolic acid; N9-CHO-5-d(i)PteGlu, N9-formyl-5-dideazaisofolic acid; AG337, 3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)quinazoline; and 2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline; or stereoisomers thereof; [5] The composition according to item [1], wherein the folic acid antagonist is selected from methotrexate, raltitrexed, premetrexed, pemetrexed, lometrexol (LMX; 5,10-dideazatetrahydrofolic acid), cyclopenta[g]quinazoline having a dipeptide ligand, CB3717, CB300945, or stereoisomers thereof such as 6-R,S-BGC945 (ONX-0801), CB300638, and BW1843U89; [6] The composition according to any one of items [1] to [5], which is the following composition; (a) Each glutamyl group of the polyglutamylated folic acid antagonist other than the glutamyl group of the folic acid antagonist has an alpha carboxyl group bond; or (b) Two or more glutamyl groups of the polyglutamine oxidized folic acid antagonist have a gamma carboxyl group bond; [7] The composition according to any one of items [1] to [5], which is the following composition; (a) Each glutamyl group other than the C-terminal glutamyl group(s) and the glutamyl group of the folic acid antagonist has an alpha carboxyl group bond; or (b) Each glutamyl group other than the C-terminal glutamyl group(s) has an alpha carboxyl group bond; [8] The composition according to any one of items [1] to [7], wherein the alpha polyglutamine oxidized folic acid antagonist is the following: (a) Contains 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups; (b) Is an alpha pentaglutamine oxidized folic acid antagonist; or (c) Is an alpha hexaglutamine oxidized folic acid antagonist; [9] The composition according to any one of items [1] to [8], wherein the alpha polyglutamine oxidized folic acid antagonist contains 1 to 10 glutamyl groups having an alpha carboxyl group bond;

[10] The composition according to any one of items [1] to [9], which is the following composition: (a) At least two glutamyl groups of the alpha polyglutamine oxidized folic acid antagonist are of the L type; (b) Each glutamyl group of the alpha polyglutamine oxidized folic acid antagonist is of the L type; (c) At least one glutamyl group of the alpha polyglutamine oxidized folic acid antagonist is of the D type; (d) Each glutamyl group of the alpha polyglutamine oxidized folic acid antagonist other than the glutamyl group of the folic acid antagonist is of the D type; or (e) At least two glutamyl groups of the glutamyl group of the alpha polyglutamine oxidized folic acid antagonist are of the L type and at least one glutamyl group is of the D type;

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

[10] , wherein the polyglutamate is linear;

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

[10] , wherein the polyglutamate is branched;

[13] A liposome composition (Lp-αPANTIFOL) containing the alpha-polyglutamylated folic acid metabolism antagonist according to any one of items [1] to

[12] ;

[14] The Lp-αPANTIFOL composition of item

[13] , wherein the alpha-polyglutamylated folic acid metabolism antagonist is selected from the following: (a) AG2034, pyrimethamine, pterolymethamine, GW1843, folic acid metabolism antagonist, and LY309887; or (b) PMX, MTX, RTX, and LMX, or stereoisomers thereof;

[15] The Lp-αPANTIFOL composition of item

[13] , wherein the polyglutamated folic acid antagonist is selected from the following: LV (etoposide), L-leucovorin (L-5-formyltetrahydrofolic acid); 5-CH3-THF, 5-methyltetrahydrofolic acid; FA, folic acid; PteGlu, pteroylglutamate (FA); MTX, methotrexate; 2-dMTX, 2-desamino-MTX; 2-CH3-MTX, 2-desamino-2-methyl-MTX; AMT, aminopterin; 2-dAMT, 2-desamino-AMT; 2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM, 10-ethyl-10-deazaaminopterin; PT523, N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine; DDATHF (raltitrexed), 5,10-dideaza-5,6,7,8,-tetrahydrofolic acid; 5-d(i)H4PteGlu, 5-deaza-5,6,7,8-tetrahydroiso-folic acid; N9-CH3-5-d(i)H4PteGlu, N9-methyl-5-deaza-5,6,7,8-tetrahydroiso-folic acid; 5-dPteHCysA, N alpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, N alpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutyric acid; 5-dPteOrn, N alpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-homocysteic acid; 5-dH4PteAPBA, N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dH4PteOro, N alpha-(5-dideaza-5,6,7,8-tetrahydropteroyl)-L-ornithine; CB3717, N10-propargyl-5,8-dideazafolic acid; ICI-198,583, 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid; 4-H-ICI-198,583, 4-deoxy-ICI-198,583:4-OCH3-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198;583;Glu-to-Sub-ICI-198,583, 2-aminosuberic acid-ICI-198,583; 7-CH3-ICI-198,583, 7-methyl-ICI-198,583; ZD1694, N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamic acid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89, (S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutamic acid; LY231514, N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidine-5-yl)ethyl)-benzoyl]-L-glutamic acid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ; 2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu, 5-deazaisofolic acid; N9-CH3-5-d(i)PteGlu, N9-methyl-5-deazaisofolic acid; N9-CHO-5-d(i)PteGlu, N9-formyl-5-deazaisofolic acid; AG337, 3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)quinazoline; and AG377, 2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline; or their stereoisomers;

[16] The Lp-αPANTIFOL composition according to item

[13] , wherein the folic acid antagonist is selected from methotrexate, raltitrexed, premetrexed, pemetrexed, lometrexol (LMX; 5,10-dideazatetrahydrofolic acid), cyclopenta[g]quinazoline having a dipeptide ligand, CB3717, CB300945, or their stereoisomers such as 6-R,S-BGC945 (ONX-0801), CB300638, and BW1843U89;

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

[13] to

[16] , wherein the liposome contains an alphapolyglutamine oxidized folic acid antagonist containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups;

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

[13] to

[17] , wherein the liposome contains an alphatetraglutamine oxidized folic acid antagonist;

[19] The Lp-αPANTIFOL composition according to any one of items

[13] to

[17] , wherein the liposome contains an alphapentaglutamine oxidized folic acid antagonist;

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

[13] to

[17] , wherein the liposome contains an alphahexaglutamine oxidized folic acid antagonist;

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

[13] to

[20] , wherein the polyglutamate is linear or branched;

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

[13] to

[21] , wherein: (a) Each glutamyl group other than the glutamyl group of the folic acid antagonist has an alphacarboxyl group bond; or (b) Two or more glutamyl groups have a gammacarboxyl group bond;

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

[13] to

[21] , wherein: (a) The C-terminal glutamyl group(s) and each glutamyl group other than the glutamyl group of the folic acid antagonist have an alphacarboxyl group bond; or (b) Each glutamyl group other than the C-terminal glutamyl group(s) has an alphacarboxyl group bond;

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

[13] to

[23] , wherein: (a) At least two glutamyl groups of the alpha-polyglutamyl oxidized folic acid antimetabolite are of the L-type; (b) Each glutamyl group of the alpha-polyglutamyl oxidized folic acid antimetabolite is of the L-type; (c) At least one glutamyl group of the alpha-polyglutamyl oxidized folic acid antimetabolite is of the D-type; (d) Each glutamyl group of the alpha-polyglutamyl oxidized folic acid antimetabolite other than the glutamyl group of the folic acid antimetabolite is of the D-type; or (e) At least two glutamyl groups of the alpha-polyglutamyl oxidized folic acid antimetabolite are of the L-type and at least one glutamyl group is of the D-type;

[25] An Lp-αPANTIFOL composition according to any one of items

[13] to

[24] , wherein the liposome is pegylated (PαLp-αPANTIFOL), composition;

[26] An Lp-αPANTIFOL composition according to any one of items

[13] to

[24] , wherein the liposome is not pegylated, composition;

[27] An Lp-αPANTIFOL composition according to any one of items

[13] to

[26] , wherein the liposome has a diameter in the range of 20 nm to 200 nm, composition;

[28] An Lp-αPANTIFOL composition according to any one of items

[13] to

[27] , wherein the polyglutamate is linear or branched, composition;

[29] An Lp-αPANTIFOL composition according to any one of items

[13] to

[28] , wherein the liposome contains at least 1% by weight of the alpha-polyglutamyl oxidized folic acid antimetabolite, or during the process of preparing Lp-αPANTIFOL, at least 1% of the starting material of the alpha-polyglutamyl oxidized folic acid antimetabolite is encapsulated (enclosed) in Lp-αPANTIFOL, composition;

[30] An Lp-αPANTIFOL composition according to any one of items

[13] to

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

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

[13] to

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

[32] The Lp-αPANTIFOL composition according to any one of items

[13] to

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

[33] The Lp-αPANTIFOL composition according to item

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

[34] The Lp-αPANTIFOL composition according to item

[32] or

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

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

[32] to

[34] , wherein the liposome components include at least one selected from the following; DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC;

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

[32] to

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

[37] The Lp-αPANTIFOL composition according to item

[36] , wherein the steric stabilizer is at least one selected from polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymer; oligoglycerol, polyethylene glycol and polypropylene oxide-containing copolymer, poloxamer 188, and polyvinyl alcohol;

[38] The Lp-αPANTIFOL composition according to item

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

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

[13] to

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

[40] The Lp-αPANTIFOL composition according to any one of items

[13] to

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

[41] The Lp-αPANTIFOL composition according to any one of items

[13] to

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

[42] The Lp-αPANTIFOL composition according to any one of items

[13] to

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

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

[13] to

[38] , wherein the liposome is cationic;

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

[13] to

[43] , wherein the liposome has an internal space containing an alpha-polyglutamine oxidized folic acid antagonist and an aqueous pharmaceutically acceptable carrier;

[45] The Lp-αPANTIFOL composition of item

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

[46] The Lp-αPANTIFOL composition of item

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

[47] The Lp-αPANTIFOL composition of item

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

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

[44] to

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

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

[44] to

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

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

[44] to

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

[51] The Lp-αPANTIFOL composition according to any one of items

[44] to

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

[52] The Lp-αPANTIFOL composition according to any one of items

[13] to

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

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

[13] to

[52] , wherein the liposome contains less than 500,000 or less than 200,000 alpha-polyglutamylated folic acid antagonist molecules;

[54] The Lp-αPANTIFOL composition according to any one of items

[13] to

[53] , wherein the liposome contains 10 to 100,000 or any range therebetween of alpha-polyglutamylated folic acid antagonist molecules;

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

[13] to

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

[56] The Lp-αPANTIFOL composition according to item

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

[57] The Lp-αPANTIFOL composition according to item

[55] or

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

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

[55] to

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

[59] The Lp-αPANTIFOL composition according to any one of items

[55] to

[58] , wherein the targeting moiety binds to a surface antigen with an equilibrium dissociation constant (Kd) in the range of 0.5×10−10 to 10×10−6 as measured by BIACORE (registered trademark) analysis;

[60] The Lp-αPANTIFOL composition according to any one of items

[55] to

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

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

[55] to

[60] , wherein the targeting moiety comprises one or more selected from antibodies, humanized antibodies, antigen-binding fragments of antibodies, single-chain antibodies, single-domain antibodies, bispecific antibodies, synthetic antibodies, pegylated antibodies, and multimeric antibodies;

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

[55] to

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

[63] The Lp-αPANTIFOL composition according to any one of items

[44] to

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

[64] The Lp-αPANTIFOL composition of item

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

[65] The Lp-αPANTIFOL composition of item

[63] or

[64] , wherein the immunostimulant is at least one selected from toll-like receptor (TLR) regulators such as fluorescein, fluorescein isothiocyanate (FITC), DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan, resorcin (such as resorcin D like Dn-6DPA or Dn-3DPA, resorcin E, or T series resorcin), oxidized low density lipoprotein (such as OXPAC, PGPC), and erythran lipid (such as E5564);

[66] The Lp-αPANTIFOL composition according to any one of items

[63] to

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

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

[63] to

[66] , further comprising a hapten;

[68] The Lp-αPANTIFOL composition of item

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

[69] The Lp-αPANTIFOL composition according to any one of items

[13] to

[68] , further comprising at least one cryoprotective substance selected from mannitol, trehalose, sorbitol, and sucrose;

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

[69] ;

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

[54] and

[64] to

[69] ;

[72] An Lp-αPANTIFOL composition according to any one of items

[13] to

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

[73] A pharmaceutical composition comprising the liposomal alpha-polyglutamine oxidized folic acid antagonist composition according to any one of items

[13] to

[72] ;

[74] A pharmaceutical composition comprising the alpha-polyglutamine oxidized folic acid antagonist composition according to any one of items [1] to [8];

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

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

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

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

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

[75] to the subject;

[78] A method for treating or preventing a disease in a subject in need thereof, comprising the step of administering the liposomal alpha-polyglutamine oxidized folic acid antagonist composition according to any one of items

[13] to

[74] to the subject;

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

[74] ;

[80] A method for killing proliferating cells, comprising the step of contacting the proliferating cells with the liposomal alpha-polyglutamine oxidized folic acid antagonist composition according to any one of items

[13] to

[74] ;

[81] The method according to item

[79] or

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

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

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

[83] A method for treating cancer, comprising administering an effective amount of the liposomal alpha-polyglutamine oxidized folic acid antimetabolite composition according to any one of items

[13] to

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

[84] The method according to item

[82] or

[83] , wherein the cancer is a non-hematological tumor including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and a hematological tumor selected from, for example, leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias;

[85] The method according to item

[82] or

[83] , wherein the cancer is a member selected from breast cancer, advanced head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, villous adenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, central nervous system (CNS) lymphoma;

[86] The method according to item

[82] or

[83] , wherein the cancer is selected from colorectal cancer, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer;

[87] The method according to item

[82] or

[83] , wherein the cancer is a sarcoma such as osteosarcoma;

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

[55] to

[71] to a subject having or at risk of having cancer cells expressing a folate receptor bound by a targeting moiety on the surface;

[89] Maintenance therapy, comprising administering an effective amount of the composition according to any one of items [1] to

[74] to a subject who is receiving or has received cancer therapy;

[90] Maintenance therapy, comprising administering an effective amount of the liposomal alpha-polyglutamine oxidized folic acid antimetabolite composition according to any one of items

[13] to

[74] to a subject who is receiving or has received cancer therapy;

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

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

[92] A method for treating an immune system disorder, comprising administering an effective amount of the liposomal alpha-polyglutamine oxidized folic acid antagonist composition according to any one of items [9] to

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

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

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

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

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

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

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

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

[94] A method for treating an infectious disease, comprising administering to a subject having or at risk of having an infectious disease an effective amount of the liposomal alpha-polyglutamic acid oxidized folic acid metabolic antagonist composition according to any one of items

[13] to

[74] ;

[95] A method for delivering an alpha-polyglutamic acid oxidized folic acid metabolic antagonist to a tumor expressing a folate receptor on its surface, the method comprising administering to a subject having a tumor an Lp-αPANTIFOL composition according to any one of items [1] to

[74] in an amount sufficient to deliver a therapeutically effective amount of the alpha-polyglutamic acid oxidized folic acid metabolic antagonist to the tumor;

[96] A method for preparing an alpha-polyglutamic acid oxidized folic acid metabolic antagonist composition comprising the liposomal alpha-polyglutamic acid oxidized folic acid metabolic antagonist composition according to any one of items

[13] to

[74] , the method comprising forming a mixture comprising a liposomal component and an alpha-polyglutamic acid oxidized folic acid metabolic antagonist in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes containing the alpha-polyglutamic acid oxidized folic acid metabolic antagonist;

[97] A method for preparing an alpha-polyglutamic acid oxidized folic acid metabolic antagonist composition comprising the liposomal alpha-polyglutamic acid oxidized folic acid metabolic antagonist composition according to any one of items

[13] to

[74] , the method comprising forming a mixture comprising a liposomal component and an alpha-polyglutamic acid oxidized folic acid metabolic antagonist in solution; and treating the mixture to form liposomes containing the alpha-polyglutamic acid oxidized folic acid metabolic antagonist;

[98] The method of item

[97] , wherein the step of treating the mixture comprises the step of homogenizing the mixture in a solution to form liposomes;

[99] A method for producing the composition according to any one of items

[55] to

[74] , comprising the steps of: forming a mixture comprising liposome components and an alpha-polyglutamine oxidized folic acid antagonist in a solution; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes encapsulating and / or coating the alpha-polyglutamine oxidized folic acid antagonist; and imparting a targeting moiety on the surface of the liposomes, wherein the targeting moiety has specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ);

[0100] A method for producing the composition according to any one of items

[55] to

[74] , comprising the steps of: forming a mixture comprising liposome components and an alpha-polyglutamine oxidized folic acid antagonist in a solution; treating the mixture to form liposomes encapsulating and / or coating the alpha-polyglutamine oxidized folic acid antagonist; and imparting a targeting moiety on the surface of the liposomes, wherein the targeting moiety has specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ);

[0101] The method of item

[0100] , wherein the step of treating comprises the step of homogenizing the mixture in a solution to form liposomes;

[0102] The method according to any one of items

[99] to

[0101] , wherein the treatment step comprises one or more steps of thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw method, reverse phase evaporation method, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, freeze-dried double emulsion, 3D printing, membrane contactor method, and stirring;

[0103] The method according to any one of items

[99] to

[0102] , wherein the processing step includes one or more steps of changing the size of the liposome by one or more steps of extrusion, high-pressure microfluidization, and / or ultrasonic treatment; and / or

[0104] The method according to any one of items

[96] to

[0103] , wherein at least 1% of the starting material of the alpha-polyglutamine oxidized folic acid antimetabolite is encapsulated or enclosed in Lp-αPANTIFOL.

[0010] In some embodiments, the present disclosure provides an alphapolyglutamyl oxidized folic acid antagonist (αPANTIFOL) composition, wherein at least one glutamyl residue of the alphapolyglutamyl oxidized folic acid antagonist is bound by its alpha carboxyl group. In some embodiments, αPANTIFOL comprises 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl groups of the folic acid antagonist). In some embodiments, the alphapolyglutamyl oxidized folic acid antagonist is selected from (a) AG2034, pyrimethamine, pteropterin, GW1843, folic acid antagonist, and LY309887; or (b) PMX, MTX, RTX, and LMX, or their stereoisomers. In some embodiments, the alphapolyglutamyl oxidized folic acid antagonist is selected from the following: LV (etoposide), L-leucovorin (L-5-formyltetrahydrofolic acid); 5-CH3-THF, 5-methyltetrahydrofolic acid; FA, folic acid; PteGlu, pteroylglutamate (FA); MTX, methotrexate; 2-dMTX, 2-desamino-MTX; 2-CH3-MTX, 2-desamino-2-methyl-MTX; AMT, aminopterin; 2-dAMT, 2-desamino-AMT; 2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM, 10-ethyl-10-deazaaminopterin; PT523, N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine; DDATHF (raltitrexed), 5,10-dideaza-5,6,7,8,-tetrahydrofolic acid; 5-d(i)H4PteGlu, 5-deaza-5,6,7,8-tetrahydroiso folic acid; N9-CH3-5-d(i)H4PteGlu, N9-methyl-5-deaza-5,6,7,8-tetrahydroiso folic acid; 5-dPteHCysA, N alpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, N alpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutyric acid; 5-dPteOrn, N alpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-homocysteic acid;5-dH4PteAPBA, Nα-(5-Deaza-5,6,7,8-tetrahydropteroyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dH4PteOro, Nα-(5-Dideaza-5,6,7,8-tetrahydropteroyl)-L-ornithine; CB3717, N10-Propylgyl-5,8-dideazafolic acid; ICI-198,583, 2-Desamino-2-methyl-N10-propylgyl-5,8-dideazafolic acid; 4-H-ICI-198,583, 4-Deoxy-ICI-198,583: 4-OCH3-ICI-198,583, 4-Methoxy-ICI-198,583 Glu-to-Val-ICI-198,583; Val-ICI-198;583; Glu-to-Sub-ICI-198,583, 2-Aminosuberic acid-ICI-198,583; 7-CH3-ICI-198,583, 7-Methyl-ICI-198,583; ZD1694, N-[5(N-(3,4-Dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamic acid; 2-NH2-ZD1694, 2-Amino-ZD1694; BW1843U89, (S)-2[5-(((1,2-Dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutamic acid; LY231514, N-(4-(2-(2-Amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl)-benzoyl]-L-glutamic acid; IAHQ, 5,8-Dideazaisofolic acid; 2-dIAHQ, 2-Desamino-IAHQ; 2-CH3-dIAHQ, 2-Desamino-2-methyl-IAHQ; 5-d(i)PteGlu, 5-Deazaisofolic acid; N9-CH3-5-d(i)PteGlu, N9-Methyl-5-deazaisofolic acid; N9-CHO-5-d(i)PteGlu, N9-Formyl-5-deazaisofolic acid; AG337, 3,4-Dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)quanazoline; and AG377, 2,4-Diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline;Or their stereoisomers. In some embodiments, the alpha-polyglyglutamylated folate antimetabolite is selected from methotrexate, raltitrexed, premetrexed, pemetrexed, lometrexol (LMX; 5,10-dideazatetrahydrofolate), cyclopenta[g]quinazoline having a dipeptide ligand, CB3717, CB300945, or their stereoisomers such as 6-R,S-BGC945 (ONX-0801), CB300638, and BW1843U89. In some embodiments, αPANTIFOL contains two or more L-type glutamyl groups. In other embodiments, αPANTIFOL contains a D-type glutamyl group. In further embodiments, αPANTIFOL contains a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, αPANTIFOL contains two or more glutamyl groups having a gamma bond.;

[0011] In one embodiment, the αPANTIFOL composition comprises a chain of three glutamyl groups attached to the glutamyl group in the folic acid antagonist (i.e., tetraglutamine oxidized folic acid antagonist). In some embodiments, αPANTIFOL is the polyglutamate of the folic acid antagonist described in item [2] of the Summary section of the invention. In some embodiments, αPANTIFOL is the polyglutamate of the folic acid antagonist described in item [3] of the Summary section of the invention. In some embodiments, αPANTIFOL is the polyglutamate of the folic acid antagonist described in item [4] of the Summary section of the invention. In some embodiments, αPANTIFOL is the polyglutamate of the folic acid antagonist described in item [5] of the Summary section of the invention. In some embodiments, αPANTIFOL is the polyglutamine oxidized folic acid antagonist described in the Summary section of the invention. In some embodiments, the tetraglutamine oxidized folic acid antagonist comprises two or more L-type glutamyl groups. In other embodiments, the tetraglutamine oxidized folic acid antagonist comprises a D-type glutamyl group. In some embodiments, the tetraglutamine oxidized folic acid antagonist comprises two or more D-type glutamyl groups. In a further embodiment, the tetraglutamine oxidized folic acid antagonist comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the tetraglutamine oxidized folic acid antagonist comprises one, two, or three D-type glutamyl groups and three, two, or one L-type glutamyl groups, respectively. In a further embodiment, the alpha tetraglutamine oxidized folic acid antagonist comprises two or more glutamyl groups having gamma linkages.

[0012] In one embodiment, the αPANTIFOL composition comprises a chain of four glutamyl groups attached to the glutamyl group in the folic acid antagonist (i.e., pentaglutamylated folic acid antagonist). In some embodiments, αPANTIFOL is the polyglutamate of the folic acid antagonist described in item [2] of the Summary section of the invention. In some embodiments, αPANTIFOL is the polyglutamate of the folic acid antagonist described in item [3] of the Summary section of the invention. In some embodiments, αPANTIFOL is the polyglutamate of the folic acid antagonist described in item [4] of the Summary section of the invention. In some embodiments, αPANTIFOL is the polyglutamate of the folic acid antagonist described in item [5] of the Summary section of the invention. In some embodiments, αPANTIFOL is the pentaglutamylated folic acid antagonist described in the Summary section of the invention. In some embodiments, the pentaglutamylated folic acid antagonist comprises two or more L-type glutamyl groups. In other embodiments, the pentaglutamylated folic acid antagonist comprises a D-type glutamyl group. In some embodiments, the pentaglutamylated folic acid antagonist comprises two or more D-type glutamyl groups. In a further embodiment, the pentaglutamylated folic acid antagonist comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the pentaglutamylated folic acid antagonist comprises one, two, three, or four D-type glutamyl groups and four, three, two, or one L-type glutamyl group, respectively. In a further embodiment, the alpha pentaglutamylated folic acid antagonist comprises two or more glutamyl groups having a gamma linkage.

[0013] In one embodiment, the αPANTIFOL composition comprises a chain of 5 glutamyl groups attached to the glutamyl group in the folic acid antagonist (i.e., hexaglutaminyl methotrexate). In some embodiments, αPANTIFOL is the polyglutamate of the folic acid antagonist described in item [2] of the Summary of the Invention section. In some embodiments, αPANTIFOL is the polyglutamate of the folic acid antagonist described in item [3] of the Summary of the Invention section. In some embodiments, αPANTIFOL is the polyglutamate of the folic acid antagonist described in item [4] of the Summary of the Invention section. In some embodiments, αPANTIFOL is the polyglutamate of the folic acid antagonist described in item [5] of the Summary of the Invention section. In some embodiments, αPANTIFOL is the polyglutaminyl methotrexate described in the Summary of the Invention section. In some embodiments, the hexaglutaminyl methotrexate comprises 2 or more L-type glutamyl groups. In other embodiments, the hexaglutaminyl methotrexate comprises a D-type glutamyl group. In some embodiments, the hexaglutaminyl methotrexate comprises 2 or more D-type glutamyl groups. In a further embodiment, the hexaglutaminyl methotrexate comprises a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the pentaglutaminyl methotrexate each comprises 1, 2, 3, 4, or 5 D-type glutamyl groups and 5, 4, 3, 2, or 1 L-type glutamyl groups. In a further embodiment, the alpha hexaglutaminyl methotrexate comprises 2 or more glutamyl groups having a gamma bond.

[0014] In a further embodiment, the present disclosure provides a composition comprising a delivery carrier such as a liposome loaded (i.e., encapsulated) with and / or otherwise conjugated to an alpha-polyglutamylated folate antagonist, and a method of making an αPANTIFOL-loaded / conjugated delivery carrier composition (DV-αPANTIFOL) and using the same to deliver an alpha-polyglutamylated folate antagonist to diseased (e.g., cancerous) cells and / or target cells. These compositions have uses including, but not limited to, the treatment of diseases including hyperproliferative diseases such as cancer, immune system disorders such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV, malaria, and schistosomiasis. The αPANTIFOL-loaded / conjugated delivery carrier composition provides for the selective delivery of a higher cytotoxic payload (alpha-polyglutamylated folate antagonist) compared to the cytotoxicity of a folate antagonist administered in the monoglutamate state (ANTIFOL), resulting in improved efficacy and safety of delivery of the folate antagonist to cancer cells. In some embodiments, the alpha-polyglutamylated folate antagonist in DV-αPANTIFOL comprises 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5, or more than 20 glutamyl groups (including the glutamyl groups of the folate antagonist). In some embodiments, the delivery carrier comprises the polyglutamylated folate antagonist described in any one of items [1] to

[12] of the section of the Summary of the Invention. In some embodiments, the delivery carrier comprises the polyglutamylated folate antagonist described in the section of the Summary of the Invention.

[0015] In a further embodiment, the present disclosure provides a composition (Lp-αPANTIFOL) comprising liposomes encapsulating (filled with) an alpha-polyglytamyl oxidized folic acid antimetabolite. In some embodiments, the alpha-polyglytamyl oxidized folic acid antimetabolite in Lp-αPANTIFOL contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 20 glutamyl groups (including the glutamyl groups of the folic acid antimetabolite). In some embodiments, the alpha-polyglytamyl oxidized folic acid antimetabolite encapsulated by the liposome is selected from (a) AG2034, pyrithioxime, pralatrexate, GW1843, folic acid antimetabolites, and LY309887; or (b) PMX, MTX, RTX and LMX, or their stereoisomers. In some embodiments, the alpha-polyglytamyl oxidized folic acid antimetabolite encapsulated by the liposome is selected from the following: LV (etoposide), L-leucovorin (L-5-formyltetrahydrofolic acid); 5-CH3-THF, 5-methyltetrahydrofolic acid; FA, folic acid; PteGlu, pteroylglutamate (FA); MTX, methotrexate; 2-dMTX, 2-desamino-MTX; 2-CH3-MTX, 2-desamino-2-methyl-MTX; AMT, aminopterin; 2-dAMT, 2-desamino-AMT; 2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM, 10-ethyl-10-deazaaminopterin; PT523, N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine; DDATHF (raltitrexed), 5,10-dideaza-5,6,7,8,-tetrahydrofolic acid; 5-d(i)H4PteGlu, 5-deaza-5,6,7,8-tetrahydroiso-folic acid; N9-CH3-5-d(i)H4PteGlu, N9-methyl-5-deaza-5,6,7,8-tetrahydroiso-folic acid; 5-dPteHCysA, N alpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, N alpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutyric acid; 5-dPteOrn, N alpha-(5-deazapteroyl)-L-ornithine;5-dH4PteHCysA, N alpha-(5-deaza-5,6,7,8-tetrahydroptenoyl)-L-homocysteic acid; 5-dH4PteAPBA, N alpha-(5-deaza-5,6,7,8-tetrahydroptenoyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dH4PteOro, N alpha-(5-dideaza-5,6,7,8-tetrahydroptenoyl)-L-ornithine; CB3717, N10-prop-2-ynyl-5,8-dideazafolic acid; ICI-198,583, 2-desamino-2-methyl-N10-prop-2-ynyl-5,8-dideazafolic acid; 4-H-ICI-198,583, 4-deoxy-ICI-198,583:4-OCH3-ICI-198,583, 4-methoxy-ICI-198,583 Glu-to-Val-ICI-198,583; Val-ICI-198;583; Glu-to-Sub-ICI-198,583, 2-aminosuberic acid-ICI-198,583; 7-CH3-ICI-198,583, 7-methyl-ICI-198,583; ZD1694, N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamic acid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89, (S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutamic acid; LY231514, N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl)-benzoyl]-L-glutamic acid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ; 2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu, 5-dideazaisofolic acid; N9-CH3-5-d(i)PteGlu, N9-methyl-5-dideazaisofolic acid; N9-CHO-5-d(i)PteGlu, N9-formyl-5-dideazaisofolic acid; AG337, 3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)quinazoline;and AG377, 2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline; or a stereoisomer thereof. In some embodiments, the alpha-polyglutamyl oxidized folate antagonist encapsulated by the liposome is selected from methotrexate, raltitrexed, premetrexed, pemetrexed, lometrexol (LMX; 5,10-dideazatetrahydrofolic acid), cyclopenta[g]quinazoline having a dipeptide ligand, CB3717, CB300945, or a stereoisomer thereof such as 6-R,S-BGC945 (ONX-0801), CB300638, and BW1843U89. In some embodiments, the alpha-polyglutamyl oxidized folate antagonist in Lp-αPANTIFOL contains two or more L-type glutamyl groups. In other embodiments, the alpha-polyglutamyl oxidized folate antagonist in Lp-αPANTIFOL contains a D-type glutamyl group. In further embodiments, the alpha-polyglutamyl oxidized folate antagonist in Lp-αPANTIFOL contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the alpha-polyglutamyl oxidized folate antagonist in Lp-αPANTIFOL contains two or more glutamyl groups having a gamma bond. In further embodiments, the alpha-polyglutamyl oxidized folate antagonist in Lp-αPANTIFOL contains one or more glutamyl groups having both an alpha bond and a gamma bond. In some embodiments, the alpha-polyglutamyl oxidized folate antagonist in Lp-αPANTIFOL contains 2 to 10 or any range therebetween of glutamyl groups having both an alpha bond and a gamma bond. In some embodiments, the polyglutamate chain of the alpha-polyglutamyl oxidized folate antagonist is linear. In some embodiments, the polyglutamate chain of the alpha-polyglutamyl oxidized folate antagonist is branched.;

[0016] In one embodiment, the Lp-αPANTIFOL composition comprises an alpha-polyglutamylated folic acid antagonist comprising a chain of three glutamyl groups attached to the glutamyl group in the folic acid antagonist (i.e., a tetraglutamylated folic acid antagonist). In some embodiments, αPANTIFOL is a polyglutamate of the folic acid antagonist described in item [2] of the Summary of the Invention section. In some embodiments, αPANTIFOL is a polyglutamate of the folic acid antagonist described in item [3] of the Summary of the Invention section. In some embodiments, αPANTIFOL is a polyglutamate of the folic acid antagonist described in item [4] of the Summary of the Invention section. In some embodiments, αPANTIFOL is a polyglutamate of the folic acid antagonist described in item [5] of the Summary of the Invention section. In some embodiments, αPANTIFOL is the polyglutamylated folic acid antagonist described in the Summary of the Invention section. In some embodiments, the tetraglutamylated folic acid antagonist comprises two or more L-type glutamyl groups. In other embodiments, the tetraglutamylated folic acid antagonist comprises a D-type glutamyl group. In further embodiments, the tetraglutamylated folic acid antagonist comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the tetraglutamylated folic acid antagonist comprises two or more glutamyl groups having gamma linkages. In some embodiments, the polyglutamate chain of the alpha-polyglutamylated folic acid antagonist is linear. In some embodiments, the polyglutamate chain of the alpha-polyglutamylated folic acid antagonist is branched.

[0017] In one embodiment, the Lp-αPANTIFOL composition comprises an alphapolyglutamyl oxidized folate antagonist comprising a chain of four glutamyl groups attached to the glutamyl group in the folate antagonist (i.e., a pentaglutamyl oxidized folate antagonist). In some embodiments, αPANTIFOL is a polyglutamate of the folate antagonist described in item [2] of the Summary section of the invention. In some embodiments, αPANTIFOL is a polyglutamate of the folate antagonist described in item [3] of the Summary section of the invention. In some embodiments, αPANTIFOL is a polyglutamate of the folate antagonist described in item [4] of the Summary section of the invention. In some embodiments, αPANTIFOL is a polyglutamate of the folate antagonist described in item [5] of the Summary section of the invention. In some embodiments, αPANTIFOL is the polyglutamyl oxidized folate antagonist described in the Summary section of the invention. In some embodiments, the pentaglutamyl oxidized folate antagonist comprises two or more L-type glutamyl groups. In other embodiments, the pentaglutamyl oxidized folate antagonist comprises a D-type glutamyl group. In further embodiments, the pentaglutamyl oxidized folate antagonist comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the pentaglutamyl oxidized folate antagonist comprises two or more glutamyl groups having a gamma linkage. In some embodiments, the polyglutamate chain of the alphapolyglutamyl oxidized folate antagonist is linear. In some embodiments, the polyglutamate chain of the alphapolyglutamyl oxidized folate antagonist is branched.

[0018] In one embodiment, the Lp-αPANTIFOL composition comprises an alphapolyglutamylated folic acid antagonist comprising a chain of five glutamyl groups attached to the glutamyl group in the folic acid antagonist (i.e., a hexaglutamylated folic acid antagonist). In some embodiments, αPANTIFOL is a polyglutamate of the folic acid antagonist described in item [2] of the Summary section of the invention. In some embodiments, αPANTIFOL is a polyglutamate of the folic acid antagonist described in item [3] of the Summary section of the invention. In some embodiments, αPANTIFOL is a polyglutamate of the folic acid antagonist described in item [4] of the Summary section of the invention. In some embodiments, αPANTIFOL is a polyglutamate of the folic acid antagonist described in item [5] of the Summary section of the invention. In some embodiments, αPANTIFOL is a polyglutamylated folic acid antagonist described in the Summary section of the invention. In some embodiments, the hexaglutamylated folic acid antagonist comprises two or more L-type glutamyl groups. In other embodiments, the hexaglutamylated folic acid antagonist comprises a D-type glutamyl group. In some embodiments, the hexaglutamylated folic acid antagonist comprises two or more D-type glutamyl groups. In a further embodiment, the hexaglutamylated folic acid antagonist comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the pentaglutamylated folic acid antagonists each comprise one, two, three, four, or five D-type glutamyl groups and five, four, three, two, or one L-type glutamyl group. In a further embodiment, the hexaglutamylated folic acid antagonist comprises two or more glutamyl groups having gamma linkages. In some embodiments, the polyglutamate chain of the alphapolyglutamylated folic acid antagonist is linear. In some embodiments, the polyglutamate chain of the alphapolyglutamylated folic acid antagonist is branched.

[0019] In some embodiments, the Lp-αPANTIFOL composition is cationic. In some embodiments, the Lp-αPANTIFOL liposomes are cationic and have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In a further embodiment, the Lp-αPANTIFOL liposomes are cationic and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the cationic Lp-αPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of an alpha polyglutamyl oxidized folic acid antagonist. In some embodiments, during the production process of Lp-αPANTIFOL, 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 the alpha polyglutamyl oxidized folic acid antagonist is encapsulated (enclosed) in the cationic Lp-αPANTIFOL. In a further embodiment, the alpha polyglutamyl oxidized folic acid antagonist encapsulated by the liposome is present in the HEPES buffer within the liposome.

[0020] In other embodiments, the Lp-αPANTIFOL composition is anionic or neutral. In some embodiments, the Lp-αPANTIFOL composition is cationic. In some embodiments, the Lp-αPANTIFOL liposomes are anionic or neutral and have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-αPANTIFOL liposomes are anionic or neutral and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the Lp-αPANTIFOL liposomes are anionic and have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-αPANTIFOL liposomes are anionic and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the Lp-αPANTIFOL liposomes are neutral and have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-αPANTIFOL liposomes are neutral and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the anionic or neutral Lp-αPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of an alpha polyglutamyl oxidized folic acid antimetabolite. In some embodiments, during the preparation process of Lp-αPANTIFOL, 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 the alpha polyglutamyl oxidized folic acid antimetabolite is encapsulated (enclosed) in the anionic or neutral Lp-αPANTIFOL.In some embodiments, the anionic or neutral Lp-αPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of an alpha-tetraglutamyl oxidized folic acid antagonist. In some embodiments, the anionic or neutral Lp-αPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of an alpha-pentaglutamyl oxidized folic acid antagonist. In some embodiments, the anionic or neutral Lp-αPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of an alpha-hexaglutamyl oxidized folic acid antagonist. In a further embodiment, the alpha-polyglutamyl oxidized folic acid antagonist encapsulated by the liposome is present in the HEPES buffer within the liposome.

[0021] In a further embodiment, the liposomal alpha-polyglutamyl oxidized folic acid antagonist composition is pegylated (PLp-αPANTIFOL).

[0022] In some embodiments, the liposomal alpha-polyglutamyl oxidized folic acid antagonist composition is not targeted (NTLp-αPANTIFOL). That is, the NTLp-αPANTIFOL composition does not have a specific affinity for an epitope expressed on the surface of a target cell of interest (e.g., an epitope on a surface antigen). In some embodiments, the NTLp-αPANTIFOL composition does not contain a targeting moiety. In a further embodiment, the non-targeted liposomal alpha-polyglutamyl oxidized folic acid antagonist composition is pegylated (NTPLp-αPANTIFOL).

[0023] In other embodiments, the liposomal alpha polyglutamine antifolate metabolic antagonist composition is targeted (TLp-αPANTIFOL). That is, the TLp-αPANTIFOL composition includes a targeting moiety having specific affinity for an epitope (surface antigen) on the target cell of interest. In some embodiments, the targeting moiety of TLp-αPANTIFOL or TPLp-αPANTIFOL is not covalently bound to the liposome. In other embodiments, the targeting moiety of TLp-αPANTIFOL or TPLp-αPANTIFOL is bound to one or both of the PEG and the outer surface of the liposome. In some embodiments, the targeting moiety of TLp-αPANTIFOL or TPLp-αPANTIFOL is covalently bound to the liposome. The functions of the targeting moiety of the TLp-αPANTIFOL and / or TPLp-αPANTIFOL composition include, but are not limited to, targeting the liposome to the target cell of interest in vivo or in vitro; interacting with the surface antigen to which the targeting moiety has specific affinity; and delivering the liposome payload (αPANTIFOL) to the cell. Suitable targeting moieties are known in the art and include, but are not limited to, antibodies, antigen-binding antibody fragments, scaffold proteins, polypeptides, and peptides. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is a polypeptide comprising at least 3, 5, 10, 15, 20, 30, 40, 50, or 100 amino acid residues.

[0024] Targeted liposomal alpha-polyglutamylated folate antimetabolite compositions (TLp-αPANTIFOL and TPLp-αPANTIFOL) provide further improvements over the efficacy and safety profiles of folate antimetabolites by specifically delivering alpha-polyglutamylated (e.g., tetraglutamylated, pentaglutamylated, and hexaglutamylated) folate antimetabolites to target cells such as cancer cells. In further embodiments, the targeted liposomal alpha-polyglutamylated folate antimetabolite compositions are pegylated (TPLp-αPANTIFOL). The functions of the targeting moiety of the TLp-αPANTIFOL and / or TPLp-αPANTIFOL compositions include, but are not limited to, targeting liposomes to the target cells of interest in vivo or in vitro; interacting the targeting moiety with surface antigens having specific affinity; and delivering the liposomal payload (αPANTIFOL) to the cells.

[0025] 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. In some embodiments, the targeting moiety 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-αPANTIFOL or TPLp-αPANTIFOL has specific affinity for an epitope that is selectively expressed on target cells such as tumor cells as compared to normal or non-tumor cells. In some embodiments, the targeting moiety has specific affinity for an epitope on a tumor cell surface antigen that is present on tumor cells but not present on or difficult to access on non-tumor cells. In some embodiments, the targeting moiety binds to the target epitope with an equilibrium dissociation constant (Kd) in the range of 0.5x10 -10 ~10x10 -6 for the target epitope as measured by BIACORE® analysis.

[0026] In certain embodiments, the TLp-αPANTIFOL or TPLp-αPANTIFOL 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 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.

[0027] In a further embodiment, the αPANTIFOL composition comprises one or more of an immunostimulant, a detectable marker, and a maleimide, disposed on at least one of the PEG or outer surface of the liposome. In some embodiments, the liposomal αPANTIFOL composition (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL, or TPLp-αPANTIFOL) is cationic. In other embodiments, the liposomal αPANTIFOL composition (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL) is anionic or neutral. In a further embodiment, the liposomes of the liposomal αPANTIFOL composition (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL) have a diameter in the range of 20 nm to 200 nm, or any range therebetween. In a further embodiment, the liposomes of the liposomal αPANTIFOL composition have a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the liposomal αPANTIFOL composition is pegylated (e.g., PLp-αPANTIFOL, NTPLp-αPANTIFOL or TPLp-αPANTIFOL). In some embodiments, the liposomal αPANTIFOL composition comprises a targeting moiety (e.g., TLp-αPANTIFOL or TPLp-αPANTIFOL). In a further embodiment, the liposomal αPANTIFOL composition is pegylated and targeted (e.g., TPLp-αPANTIFOL). In some embodiments, the liposomal αPANTIFOL composition comprises an alpha polyglutamyl oxidized folic acid antagonist comprising 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomal αPANTIFOL composition comprises an alpha tetraglutamyl oxidized folic acid antagonist.In some embodiments, the liposomal αPANTIFOL composition comprises an alphapentaglutamyl oxidized folate antagonist. In other embodiments, the liposomal αPANTIFOL composition comprises an alphahexaglutamyl oxidized folate antagonist. In some embodiments, αPANTIFOL is a polyglutamate of the folate antagonist described in item [2] of the section of the Summary of the Invention. In some embodiments, αPANTIFOL is a polyglutamate of the folate antagonist described in item [3] of the section of the Summary of the Invention. In some embodiments, αPANTIFOL is a polyglutamate of the folate antagonist described in item [4] of the section of the Summary of the Invention. In some embodiments, αPANTIFOL is a polyglutamate of the folate antagonist described in item [5] of the section of the Summary of the Invention. In some embodiments, αPANTIFOL is a polyglutamyl oxidized folate antagonist described in the section of the Summary of the Invention.

[0028] In some embodiments, the liposome composition comprises an alpha-polyglutamine oxidized folic acid antagonist containing 4, 5, 6, 2 to 10, 4 to 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% w / w or more than 75% w / w of the alpha-polyglutamine oxidized folic acid antagonist. In some embodiments, the Lp-αPANTIFOL composition comprises an alpha-polyglutamine oxidized folic acid antagonist containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups and 1% to 98.5% w / w of the alpha-polyglutamine oxidized folic acid antagonist. In some embodiments, the liposome comprises an alpha-polyglutamine oxidized folic acid antagonist containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups, and during the preparation process of Lp-αPANTIFOL, starting materials of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of the alpha-polyglutamine oxidized folic acid antagonist are encapsulated (enclosed) in Lp-αPANTIFOL. In some embodiments, the liposome composition comprises the alpha-polyglutamine oxidized folic acid antagonist described in any one of items [1] to

[12] in the section of the summary of the invention. In some embodiments, the liposome composition comprises the liposome described in any one of items

[13] to

[72] in the section of the summary of the invention. In some embodiments, the composition comprises the alpha-polyglutamine oxidized folic acid antagonist described in the section of the summary of the invention.

[0029] In a further embodiment, the liposomal αPANTIFOL composition (i.e., Lp-αPANTIFOL such as PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL) comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% of liposome-encapsulated alpha polyglutamyl oxidized folic acid antagonist. In some embodiments, the liposomal αPANTIFOL composition comprises 1% to 98.5% of liposome-encapsulated alpha polyglutamyl oxidized folic acid antagonist. In a further embodiment, the liposomal αPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of liposome-encapsulated alpha polyglutamyl oxidized folic acid antagonist comprising 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomal αPANTIFOL composition comprises 1% to 98.5% of liposome-encapsulated alpha polyglutamyl oxidized folic acid antagonist comprising 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomal αPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of liposome-encapsulated alpha tetraglutamyl oxidized folic acid antagonist. In some embodiments, the liposomal composition comprises an alpha polyglutamyl oxidized folic acid antagonist as described in any one of items [1] to

[12] of the summary section of the invention. In some embodiments, the liposomal composition comprises a liposome as described in any one of items

[13] to

[72] of the summary section of the invention. In some embodiments, the liposomal composition comprises an alpha polyglutamyl oxidized folic acid antagonist as described in the summary section of the invention.

[0030] In some embodiments, the liposome composition consists of an alpha-tetraglutamyl oxidized folic acid antagonist and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of an alpha-tetraglutamyl oxidized folic acid antagonist. In some embodiments, the Lp-αPANTIFOL composition contains an alpha-tetraglutamyl oxidized folic acid antagonist and 1% to 98.5% w / w of an alpha-tetraglutamyl oxidized folic acid antagonist. In some embodiments, the liposome contains an alpha-tetraglutamyl oxidized folic acid antagonist, and during the preparation process of Lp-αPANTIFOL, starting materials of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of an alpha-tetraglutamyl oxidized folic acid antagonist are encapsulated (enclosed) in the Lp-αPANTIFOL. In some embodiments, the liposome composition contains an alpha-polyglutamyl oxidized folic acid antagonist described in any one of items [1] to

[12] in the summary section of the invention. In some embodiments, the liposome composition contains a liposome described in any one of items

[13] to

[72] in the summary section of the invention. In some embodiments, the liposome composition contains an alpha-polyglutamyl oxidized folic acid antagonist described in the summary section of the invention.

[0031] In some embodiments, the liposome composition consists of an alpha-pentaglutamyl oxidized folic acid antimetabolite and contains at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of the alpha-pentaglutamyl oxidized folic acid antimetabolite. In some embodiments, the Lp-αPANTIFOL composition contains an alpha-pentaglutamyl oxidized folic acid antimetabolite and 1% - 98.5% w / w of the alpha-pentaglutamyl oxidized folic acid antimetabolite. In some embodiments, the liposome contains an alpha-pentaglutamyl oxidized folic acid antimetabolite, and during the preparation process of Lp-αPANTIFOL, starting materials of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of the alpha-pentaglutamyl oxidized folic acid antimetabolite are encapsulated (enclosed) in Lp-αPANTIFOL. In some embodiments, the liposome composition consists of an alpha-hexaglutamyl oxidized folic acid antimetabolite and contains at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of the alpha-hexaglutamyl oxidized folic acid antimetabolite. In some embodiments, the Lp-αPANTIFOL composition contains an alpha-hexaglutamyl oxidized folic acid antimetabolite and 1% - 98.5% w / w of the alpha-hexaglutamyl oxidized folic acid antimetabolite. In some embodiments, the liposome contains an alpha-hexaglutamyl oxidized folic acid antimetabolite, and during the preparation process of Lp-αPANTIFOL, starting materials of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of the alpha-pentaglutamyl oxidized folic acid antimetabolite are encapsulated (enclosed) in Lp-αPANTIFOL. In some embodiments, the liposome αPANTIFOL composition contains 1% - 98.5% of the liposome-encapsulated alpha-pentaglutamyl oxidized folic acid antimetabolite.In some embodiments, the liposome composition comprises an alpha polyglutamine oxidized folic acid antagonist as described in any one of items [1] to

[12] of the section of the Summary of the Invention. In some embodiments, the liposome composition comprises a liposome as described in any one of items

[13] to

[72] of the section of the Summary of the Invention. In some embodiments, the liposome composition comprises an alpha polyglutamine oxidized folic acid antagonist as described in the section of the Summary of the Invention.

[0032] In some embodiments, the liposome αPANTIFOL composition comprises 1% to 98.5% of a liposome-encapsulated alpha tetraglutamine oxidized folic acid antagonist. In some embodiments, the liposome αPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of a liposome-encapsulated alpha pentaglutamine oxidized folic acid antagonist. In some embodiments, the liposome αPANTIFOL composition comprises 1% to 98.5% of a liposome-encapsulated alpha pentaglutamine oxidized folic acid antagonist. In some embodiments, the liposome αPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of a liposome-encapsulated alpha hexaglutamine oxidized folic acid antagonist. In some embodiments, the liposome composition comprises an alpha polyglutamine oxidized folic acid antagonist as described in any one of items [1] to

[12] of the section of the Summary of the Invention. In some embodiments, the liposome composition comprises a liposome as described in any one of items

[13] to

[72] of the section of the Summary of the Invention. In some embodiments, the liposome composition comprises an alpha polyglutamine oxidized folic acid antagonist as described in the section of the Summary of the Invention.

[0033] Liposomal compositions comprising αPANTIFOL-encapsulated liposomes are also provided. In some embodiments, the liposomal composition comprises a pegylated αPANTIFOL composition. In some embodiments, the liposomal composition comprises an αPANTIFOL composition linked or otherwise bound to a targeting moiety. In further embodiments, the liposomal composition comprises a pegylated αPANTIFOL composition linked or otherwise bound to a targeting moiety. In some embodiments, the liposomal composition comprises αPANTIFOL comprising 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomal composition comprises an alpha-tetraglutamyl methotrexate. In some embodiments, the liposomal composition comprises an alpha-pentaglutamyl methotrexate. In other embodiments, the liposomal composition comprises an alpha-hexaglutamyl methotrexate. In some embodiments, the liposomal composition comprises an alpha-polyglutamyl methotrexate antagonist as described in any one of items [1] to

[12] of the summary section of the invention. In some embodiments, the liposomal composition comprises a liposome as described in any one of items

[13] to

[72] of the summary section of the invention. In some embodiments, the liposomal composition comprises an alpha-polyglutamyl methotrexate antagonist as described in the summary section of the invention.

[0034] In some embodiments, the liposome composition comprises liposomal αPANTIFOL (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL, and TPLp-αPANTIFOL). In some embodiments, the liposomal αPANTIFOL is pegylated (e.g., NTPLp-αPANTIFOL, and TPLp-αPANTIFOL). In some embodiments, the pharmaceutical composition comprises αPANTIFOL containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition comprises an alpha-tetraglutamyl oxidized folate antagonist. In some embodiments, the pharmaceutical composition comprises an alpha-pentaglutamyl oxidized folate antagonist. In other embodiments, the pharmaceutical composition comprises an alpha-hexaglutamyl oxidized folate antagonist. In some embodiments, the liposome composition comprises an alpha-polyg lutamyl oxidized folate antagonist described in any one of items [1] to

[12] of the section of the summary of the invention. In some embodiments, the liposome composition comprises a liposome described in any one of items

[13] to

[72] of the section of the summary of the invention. In some embodiments, the liposome composition comprises an alpha-polyg lutamyl oxidized folate antagonist described in the section of the summary of the invention. In some embodiments, the liposomal αPANTIFOL comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TLp-αPANTIFOL or TPLp-αPANTIFOL). In a further embodiment, the liposome composition comprises pegylated liposomal αPANTIFOL and further comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TPLp-αPANTIFOL). In some embodiments, the liposome composition comprises liposomal αPANTIFOL that is cationic. In other embodiments, the liposome composition comprises liposomal αPANTIFOL that is anionic or neutral.In further embodiments, the liposome composition comprises liposomal αPANTIFOL having a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, or any range therebetween. In further embodiments, liposomal αPANTIFOL has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

[0035] There is also provided a pharmaceutical composition comprising an alphapolyglutamyl oxidized folic acid antagonist (αPANTIFOL) including a delivery carrier such as liposomal αPANTIFOL. In some embodiments, the pharmaceutical composition comprises a pegylated αPANTIFOL composition. In some embodiments, the pharmaceutical composition comprises an αPANTIFOL composition linked or otherwise bound to a targeting moiety. In further embodiments, the pharmaceutical composition comprises a pegylated αPANTIFOL composition linked or otherwise bound to a targeting moiety. In some embodiments, the pharmaceutical composition comprises αPANTIFOL comprising 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition comprises an alphatetraglutamyl oxidized folic acid antagonist. In some embodiments, the pharmaceutical composition comprises an alphapentaglutamyl oxidized folic acid antagonist. In other embodiments, the pharmaceutical composition comprises an alphahexaglutamyl oxidized folic acid antagonist. In other embodiments, the pharmaceutical composition comprises an alphahexaglutamyl oxidized folic acid antagonist. In some embodiments, the composition comprises an alphapolyglutamyl oxidized folic acid antagonist according to any one of items [1] to

[12] in the summary section of the invention. In some embodiments, the pharmaceutical composition comprises a liposome composition according to any one of items

[13] to

[74] in the summary section of the invention. In some embodiments, the pharmaceutical composition comprises an alphapolyglutamyl oxidized folic acid antagonist described in the summary section of the invention. In some embodiments, the pharmaceutical composition comprises a polyglutamyl oxidized folic acid antagonist described in the summary section of the invention.

[0036] In some embodiments, the pharmaceutical composition comprises liposomal αPANTIFOL (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL, and TPLp-αPANTIFOL). In some embodiments, the liposomal αPANTIFOL composition is pegylated (e.g., NTPLp-αPANTIFOL, and TPLp-αPANTIFOL). In some embodiments, liposomal αPANTIFOL comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TLp-αPANTIFOL or TPLp-αPANTIFOL). In further embodiments, the pharmaceutical composition comprises a pegylated liposomal αPANTIFOL composition and further comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TPLp-αPANTIFOL). In some embodiments, the pharmaceutical composition comprises liposomal αPANTIFOL that is cationic. In other embodiments, the pharmaceutical composition comprises liposomal αPANTIFOL that is anionic or neutral. In further embodiments, the pharmaceutical composition comprises liposomal αPANTIFOL having a diameter in the range of 20 nm to 500 nm or 20 nm to 500 nm, or any range therebetween. In further embodiments, the liposomal αPANTIFOL composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the pharmaceutical composition comprises αPANTIFOL comprising 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition comprises an alpha-tetraglutamyl methotrexate. In some embodiments, the pharmaceutical composition comprises an alpha-pentaglutamyl methotrexate. In some embodiments, the pharmaceutical composition comprises an alpha-polyglutamyl methotrexate as described in any one of items [1] to

[12] of the section of the Summary of the Invention. In some embodiments, the pharmaceutical composition comprises a liposomal composition as described in any one of items

[13] to

[74] of the section of the Summary of the Invention.In some embodiments, the pharmaceutical composition comprises the alpha-polyglutamyl oxidized folic acid antimetabolite described in the Summary of the Invention section.

[0037] In further embodiments, the present disclosure provides a method of modulating cell activation, chemokine production, or metabolic activity, the method comprising contacting a cell with a composition comprising an alpha-polyglutamyl oxidized folic acid antimetabolite (αPANTIFOL) composition. In some embodiments, the cell to be contacted is a mammalian cell. In further embodiments, the cell to be contacted is a human cell. In some embodiments, the cell to be contacted is a hyperproliferative cell. In further embodiments, the cell is an immune cell. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, αPANTIFOL comprises 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the αPANTIFOL composition comprises an alpha-tetraglutamyl oxidized folic acid antimetabolite. In some embodiments, the αPANTIFOL composition comprises an alpha-pentaglutamyl oxidized folic acid antimetabolite. In other embodiments, the αPANTIFOL composition comprises an alpha-hexaglutamyl oxidized folic acid antimetabolite. In some embodiments, the αPANTIFOL composition comprises the alpha-polyglutamyl oxidized folic acid antimetabolite described in any one of items [1] to

[12] in the Summary of the Invention section. In some embodiments, the composition comprises the liposomal composition described in any one of items

[13] to

[72] in the Summary of the Invention section. In some embodiments, the composition comprises the alpha-polyglutamyl oxidized folic acid antimetabolite described in the Summary of the Invention section.

[0038] In further embodiments, the present disclosure provides a method of modulating cell activation, chemokine production, or metabolic activity, the method comprising contacting the cells with liposomes comprising an alpha-polyglutamylated folate antagonist (αPANTIFOL) composition. In some embodiments, the cells to be contacted are mammalian cells. In further embodiments, the cells to be contacted are human cells. In some embodiments, the cells to be contacted are hyperproliferative cells. In further embodiments, the cells are immune cells. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, αPANTIFOL comprises 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the αPANTIFOL composition comprises an alpha-tetraglutamylated folate antagonist. In some embodiments, the αPANTIFOL composition comprises an alpha-pentaglutamylated folate antagonist. In other embodiments, the αPANTIFOL composition comprises an alpha-hexaglutamylated folate antagonist. In some embodiments, the liposomes comprise an alpha-polyglutamylated folate antagonist as described in any one of items [1] to

[12] of the section of the Summary of the Invention. In some embodiments, the liposomes are the liposomes as described in any one of items

[13] to

[72] of the section of the Summary of the Invention. In some embodiments, the liposomes comprise an alpha-polyglutamylated folate antagonist as described in the section of the Summary of the Invention.

[0039] In further embodiments, the present disclosure provides a method for killing cells, the method comprising contacting the cells with a composition comprising an alpha polyglutamylated folic acid antagonist (αPANTIFOL) composition. In some embodiments, the cells to be contacted are mammalian cells. In further embodiments, the cells to be contacted are human cells. In some embodiments, the cells to be contacted are hyperproliferative cells. In further embodiments, the hyperproliferative cells are cancer cells. In further embodiments, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained from cancers selected from, for example, non-blood tumors including lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and blood tumors such as, for example, leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dysplasias or malignancies. In some embodiments, the cancer cells are primary cells or cells derived from cell lines obtained from cancers selected from breast cancer, advanced head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, villous adenoma, aleukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, αPANTIFOL contains 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the αPANTIFOL composition comprises an alpha tetraglutamylated folic acid antagonist. In some embodiments, the αPANTIFOL composition comprises an alpha pentaglutamylated folic acid antagonist. In other embodiments, the αPANTIFOL composition comprises an alpha hexaglutamylated folic acid antagonist. In some embodiments, αPANTIFOL is the polyglutamylated folic acid antagonist described in any one of items [1] to

[12] of the summary section of the invention.In some embodiments, αPANTIFOL is a polyglutamylated folic acid antagonist as described in the Summary of the Invention section. In some embodiments, the αPANTIFOL composition comprises a liposome as described in any one of items

[13] to

[72] in the Summary of the Invention section.

[0040] In further embodiments, the present disclosure provides a method of killing cells, the method comprising contacting the cells with liposomes comprising an alpha-polyglutamylated folate antagonist (e.g., Lp-αPANTIFOL such as PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL). In some embodiments, the cells to be contacted are mammalian cells. In further embodiments, the cells to be contacted are human cells. In some embodiments, the cells to be contacted are hyperproliferative cells. In further embodiments, the hyperproliferative cells to be contacted are cancer cells. In further embodiments, the cancer cells are primary cells or cells derived from cell lines obtained from cancers selected from, for example, non-hematological tumors including lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors such as, for example, leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias or malignancies. In some embodiments, the cells are primary cells or cells derived from cell lines obtained from cancers selected from breast cancer, advanced head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, villous adenoma, aleukemic meningeal gliomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, central nervous system (CNS) lymphoma. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the liposomes comprise αPANTIFOL comprising 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes comprise an alpha-tetra-glutamylated folate antagonist. In some embodiments, the liposomes comprise an alpha-penta-glutamylated folate antagonist. In other embodiments, the liposomes comprise an alpha-hexa-glutamylated folate antagonist.In some embodiments, the liposome comprises a polyglutamylated folic acid antagonist described in any one of items [1] to

[12] of the section of the Summary of the Invention. In some embodiments, the liposome comprises a polyglutamylated folic acid antagonist described in the section of the Summary of the Invention. In some embodiments, the liposome composition comprises a liposome described in any one of items

[13] to

[72] of the section of the Summary of the Invention.

[0041] In further embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a delivery carrier (e.g., an antibody immune complex or a liposome) comprising an alpha-polyglutamyl oxidized folate antagonist. In some embodiments, the delivery carrier is an antibody-containing immune complex (e.g., comprising a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery carrier is a liposome (e.g., an Lp-αPANTIFOL such as PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments, the administered delivery carrier is pegylated. In some embodiments, the administered delivery carrier is not pegylated. In further embodiments, the administered delivery carrier comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of cancer cells.In a further embodiment, the delivery carrier comprises a targeting moiety that specifically binds to a cell surface antigen selected from the following: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, EGFR, IGFR-1, EGFRvIII, CD2, CD3, CD4, CD5, CD6, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD105, CD133, CD138, cripto, CD38, EphA receptor, EphB receptor, EphA2, integrin (e.g., integrin α. v β3, α v β5, or α vβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CD98, CD56, CanAg, and CALLA. In some embodiments, the delivery carrier comprises a targeting moiety that specifically binds to a cell surface antigen derived from or determined to be expressed on a particular subject's cancer (tumor), such as a neoantigen. In some embodiments, the targeting moiety specifically binds to a cell surface antigen derived from or determined to be expressed on a particular tumor of the subject, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the administered delivery carrier comprises αPANTIFOL comprising 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the administered delivery carrier comprises an alpha-tetraglutamyl oxidized folic acid antagonist. In some embodiments, the administered delivery carrier comprises an alpha-pentaglutamyl oxidized folic acid antagonist. In other embodiments, the administered delivery carrier comprises an alpha-hexaglutamyl oxidized folic acid antagonist. In some embodiments, the administered delivery carrier comprises an L-alpha-polyglutamyl oxidized folic acid antagonist. In some embodiments, the administered delivery carrier comprises 2, 3, 4, 5, or more than 5 L-alpha-glutamyl groups. In some embodiments, the administered delivery carrier comprises a D-alpha-polyglutamyl oxidized folic acid antagonist. In some embodiments, the administered delivery carrier comprises 2, 3, 4, 5, or more than 5 D-alpha-glutamyl groups. In some embodiments, the administered delivery carrier comprises L and D alpha-polyglutamyl oxidized folic acid antagonists. In some embodiments, the administered delivery carrier comprises 2, 3, 4, 5, or more than 5 L-alpha-glutamyl groups and 2, 3, 4, 5, or more than 5 D-alpha-glutamyl groups.In some embodiments, the cancer is a non-hematological tumor including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and a hematological tumor selected from, for example, leukemia, lymphoma and other B cell malignancies, multiple myeloma and other plasma cell dyscrasias or cachexia. In some embodiments, the cancer cells are cells derived from a cell line obtained from / derived from a primary cell or a cancer selected from breast cancer, advanced head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T cell lymphoma) choriocarcinoma, villous adenoma, aleukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, central nervous system (CNS) cancer.

[0042] In some embodiments, the cancer cells to be contacted are cells from a primary cell or a cell line obtained from / derived from lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer cells to be contacted are cells from a primary cell or a cell line obtained from / derived from breast cancer (e.g., HER2++ or triple-negative breast cancer). In some embodiments, the cancer cells to be contacted are cells from a primary cell or a cell line obtained from / derived from colorectal cancer. In some embodiments, the cancer cells to be contacted are cells from a primary cell or a cell line obtained from / derived from ovarian cancer. In some embodiments, the cancer cells to be contacted are cells from a primary cell or a cell line obtained from / derived from endometrial cancer. In some embodiments, the cancer cells to be contacted are cells from a primary cell or a cell line obtained from / derived from pancreatic cancer. In some embodiments, the cancer cells to be contacted are cells from a primary cell or a cell line obtained from / derived from liver cancer. In some embodiments, the cancer cells to be contacted are cells from a primary cell or a cell line obtained from / derived from head and neck cancer. In some embodiments, the cancer cells to be contacted are cells from a primary cell or a cell line obtained from / derived from osteosarcoma. In some embodiments, the delivery carrier to be administered comprises a polyglutamylated folic acid antimetabolite as described in any one of items [1] to

[12] of the section of the Summary of the Invention. In some embodiments, the delivery carrier comprises a polyglutamylated folic acid antimetabolite as described in the section of the Summary of the Invention. In some embodiments, the liposome composition comprises a liposome as described in any one of items

[13] to

[72] of the section of the Summary of the Invention.

[0043] In a further embodiment, the present disclosure provides a method of treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a liposome comprising an alpha-polyglutamyl oxidized folic acid antagonist (e.g., Lp-αPANTIFOL such as PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL). In some embodiments, the liposome is pegylated. In some embodiments, the liposome is not pegylated. In a further embodiment, the liposome comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of cancer cells. In a further embodiment, the liposome comprises a targeting moiety that specifically binds to a cell surface antigen selected from the following: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, EGFR, IGFR-1, EGFRvIII, CD2, CD3, CD4, CD5, CD6, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD30, CD33, CD34, CD37, CD38, CD40, CD40L, CD44, CD56, CD70, CD74, CD79, CD79b, CD105, CD133, CD138, cripto, CD38, EphA receptor, EphB receptor, EphA2, integrin (e.g., integrin α v β3, α v β5, or α vβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CD98, CD56, CanAg, and CALLA. This also includes the use of cancer stem cell targeting moieties such as those targeting CD34, CD133 and CD44, CD138, and CD15. In some embodiments, the liposome comprises a targeting moiety that specifically binds to a cell surface antigen derived from or determined to be expressed on a particular subject's tumor, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the liposome comprises αPANTIFOL containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposome comprises an alpha-tetraglutamine oxidized folate antagonist. In some embodiments, the liposome comprises an alpha-pentaglutamine oxidized folate antagonist. In other embodiments, the liposome comprises an alpha-hexaglutamine oxidized folate antagonist. In some embodiments, the liposome comprises a polyglutamine oxidized folate antagonist described in any one of items [1] to

[12] of the section of the summary of the invention. In some embodiments, αPANTIFOL is a polyglutamine oxidized folate antagonist described in the section of the summary of the invention. In some embodiments, the liposome composition is a liposome described in any one of items

[13] to

[72] of the section of the summary of the invention. In some embodiments, the liposome comprises 2, 3, 4, 5, or more than 5 L-alpha-glutamyl groups. In some embodiments, the liposome comprises a D-alpha-polyglutamine oxidized folate antagonist. In some embodiments, the liposome comprises 2, 3, 4, 5, or more than 5 D-alpha-glutamyl groups. In some embodiments, the liposome comprises L and D alpha-polyglutamine oxidized folate antagonists. In some embodiments, the liposome comprises 2, 3, 4, 5, or more than 5 L-alpha-glutamyl groups and 2, 3, 4, 5, or more than 5 D-alpha-glutamyl groups.In some embodiments, the cancer is selected from lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, and hematological tumors (e.g., leukemia or lymphoma). In some embodiments, the cancer is selected from breast cancer, advanced head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, villous adenoma, aleukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) cancer. In some embodiments, the cancer is lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer is breast cancer (e.g., HER2++ or triple-negative breast cancer). In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is osteosarcoma.

[0044] In a further embodiment, the present disclosure provides a method of treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a liposomal composition comprising a liposome comprising an alpha-polyglutamyl oxidized folic acid antagonist and a targeting moiety having specific affinity for an epitope of an antigen on the cancer surface. In some embodiments, the liposome comprises a targeting moiety that specifically binds to a cell surface antigen selected from the following: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, EGFR, IGFR-1, EGFRvIII, CD2, CD3, CD4, CD5, CD6, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD30, CD33, CD34, CD37, CD38, CD40, CD40L, CD44, CD56, CD70, CD74, CD79, CD79b, CD105, CD133, CD138, cripto, CD38, EphA receptor, EphB receptor, EphA2, integrin (e.g., integrin α v β3, α v β5, or α vβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CD98, CD56, CanAg, and CALLA. In some embodiments, the liposomes to be administered include a targeting moiety that specifically binds to a cell surface antigen derived from or determined to be expressed on a particular subject's tumor, such as a neoantigen. In some embodiments, the liposome composition to be administered includes pegylated liposomes (e.g., TPLp-αPANTIFOL). In some embodiments, the liposome composition to be administered includes non-pegylated liposomes. In some embodiments, the liposomes of the liposome composition to be administered include αPANTIFOL containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the liposome composition to be administered include an alpha-tetraglutamine oxidized folic acid antagonist. In some embodiments, the liposomes of the liposome composition to be administered include an alpha-pentaglutamine oxidized folic acid antagonist. In other embodiments, the liposomes of the liposome composition to be administered include an alpha-hexaglutamine oxidized folic acid antagonist. In some embodiments, the liposomes include a polyglutamine oxidized folic acid antagonist described in any one of items [1] to

[12] of the summary section of the invention. In some embodiments, αPANTIFOL is a polyglutamine oxidized folic acid antagonist described in the summary section of the invention. In some embodiments, the liposome composition includes a liposome described in any one of items

[13] to

[72] of the summary section of the invention. In some embodiments, the liposome composition is administered for treating cancer selected from lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, multiple myeloma and other plasma cell dysplasia or cachexia, as well as leukemia, lymphoma and other B cell malignancies.In some embodiments, the liposome composition is administered to treat cancer selected from breast cancer, advanced head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, villous adenoma, aleukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) cancer. In some embodiments, the liposome composition is administered to treat lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the liposome composition is administered to treat breast cancer (e.g., HER2++ or triple negative breast cancer). In some embodiments, the liposome composition is administered to treat colorectal cancer. In some embodiments, the liposome composition is administered to treat ovarian cancer. In some embodiments, the liposome composition is administered to treat endometrial cancer. In some embodiments, the liposome composition is administered to treat pancreatic cancer. In some embodiments, the liposome composition is administered to treat liver cancer. In some embodiments, the liposome composition is administered to treat head and neck cancer. In some embodiments, the liposome composition is administered to treat osteosarcoma.

[0045] In a further embodiment, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer that expresses a folate receptor on its cell surface an effective amount of a liposomal composition, the liposomal composition comprising liposomes that contain (a) an alphapolyglutamylated folate antagonist (αPANTIFOL) and (b) a targeting moiety having specific binding affinity for the folate receptor. In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α) and folate receptor beta (FR-β). In some embodiments, the liposomal composition to be administered comprises pegylated liposomes (e.g., TPLp-αPANTIFOL). In some embodiments, the liposomal composition to be administered comprises non-pegylated liposomes. In some embodiments, the liposomes of the liposomal composition to be administered contain αPANTIFOL that contains 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered contain an alphatetraglutamylated folate antagonist. In some embodiments, the liposomes of the liposomal composition to be administered contain an alphapentaglutamylated folate antagonist. In other embodiments, the liposomes of the liposomal composition to be administered contain an alphahexaglutamylated folate antagonist. In some embodiments, the liposomes contain a polyglutamylated folate antagonist as described in any one of items [1] to

[12] of the section of the summary of the invention. In some embodiments, αPANTIFOL is the polyglutamylated folate antagonist described in the section of the summary of the invention. In some embodiments, the liposomal composition comprises liposomes as described in any one of items

[13] to

[72] of the section of the summary of the invention.In some embodiments, the liposome composition is administered to treat cancer selected from, for example, non - hematological tumors including lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors such as leukemia, lymphoma and other B - cell malignancies, multiple myeloma and other plasma cell dyscrasias or malignancies. In some embodiments, the liposome composition is administered to treat cancer selected from breast cancer, advanced head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non - Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T - cell lymphoma), choriocarcinoma, villous adenoma, aleukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) cancer. In some embodiments, the liposome composition is administered to treat lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the liposome composition is administered to treat breast cancer (e.g., HER2 ++ or triple - negative breast cancer). In some embodiments, the liposome composition is administered to treat colorectal cancer. In some embodiments, the liposome composition is administered to treat ovarian cancer. In some embodiments, the liposome composition is administered to treat endometrial cancer. In some embodiments, the liposome composition is administered to treat pancreatic cancer. In some embodiments, the liposome composition is administered to treat liver cancer. In some embodiments, the liposome composition is administered to treat head and neck cancer. In some embodiments, the liposome composition is administered to treat osteosarcoma.

[0046] In further embodiments, the present disclosure provides a method for maintenance therapy of cancer, the method comprising administering to a subject who is receiving or has received cancer therapy an effective amount of a liposomal composition comprising a liposome (Lp-αPANTIFOL) comprising an alpha-polyglutamylated folate antagonist. In some embodiments, the liposomal composition to be administered is PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL. In some embodiments, the liposomes of the liposomal composition to be administered comprise pegylated liposomes (e.g., PLp-αPANTIFOL, NTPLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments, the liposomal composition to be administered comprises targeted liposomes (e.g., TLp-αPANTIFOL or TPLp-αPANTIFOL). In some embodiments, the liposomal composition to be administered comprises liposomes that are pegylated and comprise a targeting moiety (e.g., TPLp-αPANTIFOL). In some embodiments, the liposomes of the liposomal composition to be administered comprise an alpha-polyglutamylated folate antagonist comprising 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise an alpha-tetraglutamylated folate antagonist. In some embodiments, the liposomes of the liposomal composition to be administered comprise an alpha-pentaglutamylated folate antagonist. In other embodiments, the liposomes of the liposomal composition to be administered comprise an alpha-hexaglutamylated folate antagonist. In some embodiments, the liposomal composition comprises a polyglutamylated folate antagonist described in any one of items [1]-

[12] of the section of the Summary of the Invention. In some embodiments, αPANTIFOL is a polyglutamylated folate antagonist described in the section of the Summary of the Invention. In some embodiments, the liposomal composition comprises a liposome described in any one of items

[13] -

[72] of the section of the Summary of the Invention.

[0047] In further embodiments, the present disclosure provides a method for treating a disorder of the immune system, the method comprising administering to a subject having or at risk of having a disorder of the immune system an effective amount of a liposomal composition comprising an alpha-polyglutamylated folic acid antimetabolite (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL). In some embodiments, the liposomal composition is administered to treat an autoimmune disease. In further embodiments, the liposomal composition is administered to treat rheumatoid arthritis. In another embodiment, the liposomal composition is administered to treat inflammation. In some embodiments, the disorder of the immune system is selected from inflammation (e.g., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, systemic lupus erythematosus, Takayasu disease, and psoriasis. In some embodiments, the liposomal composition administered comprises a pegylated liposome (e.g., PLp-αPANTIFOL, NTPLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments, the liposomal composition administered comprises a targeted liposome (e.g., TLp-αPANTIFOL or TPLp-αPANTIFOL) comprising a targeting moiety having specific affinity for a surface antigen on a target cell (e.g., an immune cell) of interest. In further embodiments, the liposomal composition administered comprises a liposome that is pegylated and comprises a targeting moiety (e.g., TPLp-αPANTIFOL). In some embodiments, the liposomes of the liposomal composition administered comprise an alpha-polyglutamylated folic acid antimetabolite comprising 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the liposomal composition administered comprise an alpha-tetraglutamylated folic acid antimetabolite. In some embodiments, the liposomes of the liposomal composition administered comprise an alpha-pentaglutamylated folic acid antimetabolite. In other embodiments, the liposomes of the liposomal composition administered comprise an alpha-hexaglutamylated folic acid antimetabolite.In some embodiments, the administered liposomal composition comprises an alpha-polyglutamyl oxidized folic acid antagonist as described in any one of items [1] to

[12] of the section of the Summary of the Invention. In some embodiments, αPANTIFOL is a polyglutamyl oxidized folic acid antagonist as described in the section of the Summary of the Invention. In some embodiments, the liposomal composition comprises a liposome as described in any one of items

[13] to

[72] of the section of the Summary of the Invention.

[0048] In a further embodiment, the present disclosure provides a method for treating an autoimmune disease, the method comprising administering to a subject having or at risk of having an autoimmune disease an effective amount of a liposomal composition comprising an alpha-polyglutamylated folic acid antagonist (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL) encapsulated within liposomes. In some embodiments, the autoimmune disease is rheumatoid arthritis. In some embodiments, the autoimmune disease is selected from inflammatory bowel disease (IBD), Crohn's disease, systemic lupus erythematosus, and psoriasis. In some embodiments, the autoimmune disease is a disease or disorder selected from the following: Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, diabetes (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis. In some embodiments, the liposomal composition administered comprises pegylated liposomes (e.g., PLp-αPANTIFOL, NTPLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments, the liposomal composition administered comprises targeted liposomes (e.g., TLp-αPANTIFOL or TPLp-αPANTIFOL) comprising a targeting moiety having specific affinity for a surface antigen on a target cell of interest (e.g., an immune cell). In a further embodiment, the liposomal composition administered comprises liposomes that are pegylated and comprise a targeting moiety (e.g., TPLp-αPANTIFOL). In some embodiments, the liposomes of the liposomal composition administered comprise an alpha-polyglutamylated folic acid antagonist comprising 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the liposomal composition administered comprise an alpha-tetraglutamylated folic acid antagonist.In some embodiments, the liposomes of the administered liposomal composition comprise an alpha pentaglutamine oxidized folic acid antagonist. In other embodiments, the liposomes of the administered liposomal composition comprise an alpha hexaglutamine oxidized folic acid antagonist. In some embodiments, the liposomes comprise a polyglutamine oxidized folic acid antagonist described in any one of items [1] to

[12] of the section of the summary of the invention. In some embodiments, αPANTIFOL is a polyglutamine oxidized folic acid antagonist described in the section of the summary of the invention. In some embodiments, the liposomal composition comprises a liposome described in any one of items

[13] to

[72] of the section of the summary of the invention.

[0049] In a further embodiment, the present disclosure provides a method for treating an inflammatory disease, the method comprising administering to a subject having or at risk of having an inflammatory disease an effective amount of a liposomal composition comprising an alpha-polyglutamyl oxidized folic acid antimetabolite (e.g., Lp-αPANTIFOL, PLp-αPANTIFOL, NTLp-αPANTIFOL, NTPLp-αPANTIFOL, TLp-αPANTIFOL or TPLp-αPANTIFOL). In some embodiments, the inflammatory disease is a disease selected from acute inflammation, chronic inflammation, systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, and systemic lupus erythematosus. In some embodiments, the inflammatory disease is a rheumatic-like disease or other arthritis disease (e.g., acute arthritis, acute gouty arthritis, bacterial arthritis, chronic inflammatory arthritis, osteoarthritis (osteoarthritis), infectious arthritis, juvenile arthritis, fungal arthritis, neuropathic arthritis, polyarthritis, proliferative arthritis, psoriatic arthritis, venereal arthritis, viral arthritis), conjunctivitis, pelvic inflammatory disease, acne, psoriasis, actinomycosis, dysentery, biliary cirrhosis, Lyme disease, aspergillosis, Stevens-Johnson syndrome, mumps, pemphigus vulgaris, and blastomycosis. In some embodiments, the inflammatory disease is inflammatory bowel disease. Inflammatory bowel disease is a chronic inflammatory disease of the gastrointestinal tract including, but not limited to, Crohn's disease, ulcerative colitis, and unclassified colitis. In some embodiments, the administered liposomal composition comprises a pegylated liposome (e.g., PLp-αPANTIFOL, NTPLp-αPANTIFOL, or TPLp-αPANTIFOL). In some embodiments, the administered liposomal composition comprises a targeted liposome (e.g., TLp-αPANTIFOL or TPLp-αPANTIFOL) comprising a targeting moiety having specific affinity for a surface antigen on a target cell of interest (e.g., an immune cell). In a further embodiment, the administered liposomal composition comprises a liposome that is pegylated and comprises a targeting moiety (e.g., TPLp-αPANTIFOL).In some embodiments, the liposomes of the administered liposomal composition comprise an alpha-pentaglutamine oxidized folic acid antagonist containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise an alpha-tetraglutamine oxidized folic acid antagonist. In some embodiments, the liposomes of the administered liposomal composition comprise an alpha-pentaglutamine oxidized folic acid antagonist. In other embodiments, the liposomes of the administered liposomal composition comprise an alpha-hexaglutamine oxidized folic acid antagonist. In some embodiments, the liposomes comprise a polyglutamine oxidized folic acid antagonist described in any one of items [1] to

[12] of the section of the Summary of the Invention. In some embodiments, αPANTIFOL is a polyglutamine oxidized folic acid antagonist described in the section of the Summary of the Invention. In some embodiments, the liposomal composition comprises a liposome described in any one of items

[13] to

[72] of the section of the Summary of the Invention.

[0050] The present disclosure also provides a method for delivering an alpha-polyglutamylated folic acid antimetabolite to an inflamed site of a subject, the method comprising administering to a subject having inflammation a composition comprising an alpha-polyglutamylated folic acid antimetabolite (L-αPANTIFOL) and a targeting moiety having specific binding affinity for an epitope on the surface antigen of a cell that is at the site of inflammation or that otherwise affects inflammation (e.g., affects through production of pro-inflammatory cytokines). In some embodiments, the targeting moiety being administered is bound to a delivery carrier. In some embodiments, the delivery carrier is an antibody or an antigen-binding fragment of an antibody. In further embodiments, the delivery carrier is a liposome. In further embodiments, the antibody, antigen-binding antibody fragment, or liposome is a pegylated liposome (e.g., TPLp-αPANTIFOL). In some embodiments, the composition being administered comprises an alpha-polyglutamylated folic acid antimetabolite comprising 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the composition being administered comprises an alpha-tetraglutamylated folic acid antimetabolite. In some embodiments, the composition being administered comprises an alpha-pentaglutamylated folic acid antimetabolite. In other embodiments, the composition being administered comprises an alpha-hexaglutamylated folic acid antimetabolite. In some embodiments, αPANTIFOL is a polyglutamylated folic acid antimetabolite described in any one of items [1] to

[12] of the section of the summary of the invention. In some embodiments, αPANTIFOL is a polyglutamylated folic acid antimetabolite described in the section of the summary of the invention. In some embodiments, the delivery carrier is a liposome described in any one of items

[13] to

[72] of the section of the summary of the invention.

[0051] The present disclosure also provides a method for delivering an alpha-polyglutamylated folic acid antimetabolite to tumor or cancer cells, the method comprising administering to a subject having a tumor a composition comprising an alpha-polyglutamylated folic acid antimetabolite (L-αPANTIFOL) and a targeting moiety having specific binding affinity for an epitope on the surface antigen of a tumor cell or cancer cell. In some embodiments, the targeting moiety to be administered is bound to a delivery carrier. In some embodiments, the delivery carrier is an antibody or an antigen-binding fragment of an antibody. In further embodiments, the delivery carrier is a liposome. In further embodiments, the antibody, antigen-binding antibody fragment, or liposome is a pegylated liposome (e.g., TPLp-αPANTIFOL). In some embodiments, the composition to be administered comprises an alpha-polyglutamylated folic acid antimetabolite containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the composition to be administered comprises an alpha-tetra-glutamylated folic acid antimetabolite. In some embodiments, the composition to be administered comprises an alpha-penta-glutamylated folic acid antimetabolite. In other embodiments, the composition to be administered comprises an alpha-hexa-glutamylated folic acid antimetabolite. In some embodiments, αPANTIFOL is the polyglutamylated folic acid antimetabolite described in any one of items [1] to

[12] of the section of the summary of the invention. In some embodiments, αPANTIFOL is the polyglutamylated folic acid antimetabolite described in the section of the summary of the invention. In some embodiments, the delivery carrier is the liposome described in any one of items

[13] to

[72] of the section of the summary of the invention.

[0052] In a further embodiment, the present disclosure provides a method for preparing a liposomal composition comprising a liposomal alpha polyglutamate antifolate (αPANTIFOL) composition, the method comprising: forming a mixture comprising liposomal components and an alpha polyglutamate antifolate in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes comprising an alpha polyglutamate antifolate. In some embodiments, the alpha polyglutamate antifolate comprises 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the polyglutamate antifolate composition comprises an alpha tetraglutamate antifolate. In some embodiments, the polyglutamate antifolate composition comprises an alpha pentaglutamate antifolate. In other embodiments, the polyglutamate antifolate composition comprises an alpha hexaglutamate antifolate. In some embodiments, αPANTIFOL is the polyglutamate antifolate described in any one of items [1] to

[12] of the section of the summary of the invention. In some embodiments, αPANTIFOL is the polyglutamate antifolate described in the section of the summary of the invention. In some embodiments, the liposomal composition comprises liposomes described in any one of items

[13] to

[72] of the section of the summary of the invention.

[0053] In one embodiment, the present disclosure provides a kit comprising an αPANTIFOL delivery carrier such as a liposome comprising a folate antagonist alpha polyglutamate composition and / or αPANTIFOL and an αPANTIFOL immune complex (e.g., an ADC described herein). BRIEF DESCRIPTION OF THE DRAWINGS

[0054]

Figure 1-1

Figure 1-2

Figure 1-3

Figure 1-4

Figure 1-5

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

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Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Mode for Carrying Out the Invention

[0055] Generally, the present disclosure relates to novel alpha-polyglutamylated folate antimetabolite compositions. The compositions provide an advance over prior treatments for hyperproliferative diseases such as cancer. Methods of making, delivering, and using the alpha-polyglutamylated folate antimetabolite compositions are also provided. The alpha-polyglutamylated compositions have uses including, but not limited to, the treatment or prevention of hyperproliferative diseases such as cancer, disorders of the immune system including inflammation and autoimmune diseases such as rheumatoid arthritis, and infectious diseases such as HIV, malaria, and schistosomiasis.

[0056] I. Definitions Unless otherwise specified, 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.

[0057] When embodiments are described herein with the term "comprising", other similar embodiments described with the terms "containing", "consisting of", and / or "consisting essentially of" are provided as well. However, when used as a transitional phrase in the claims, each should be construed separately and in the appropriate legal and factual context (e.g., in the claims, the transitional phrase "comprising" is considered a more open-ended phrase, "consisting of" is considered more exclusive, and "consisting essentially of" is considered intermediate between these).

[0058] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise or unless it is otherwise clear from the context that only a single referent is intended.

[0059] As used herein, the term "and / or" in expressions such as "A and / or B" is intended to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" in expressions such as "A, B, and / or C" encompasses the following embodiments respectively: 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.

[0060] Headings and subheadings are used for convenience and / or only for compliance with official rules, and do not limit the subject technology nor are they referred to in relation to the interpretation of the description of the subject technology. Features described under one heading or one subheading of the subject disclosure may be combined with features described under other headings or subheadings in various embodiments. Furthermore, not all features under a single heading or a single subheading are necessarily used together in some embodiments.

[0061] Unless otherwise indicated, the terms "antifol" and "ANTIFOL" are used interchangeably and include salts, acids and / or free base forms of antifols (e.g., antifol disodium). Compositions containing ANTIFOL salts may further contain any of various cations, such as Na+, Mg2+, K+, NH4+, and / or Ca2+. In certain embodiments, typically, the salts are pharmaceutically acceptable salts. In further particular embodiments, the antifol salt contains Na+. Antifol typically contains one L-gamma-glutamyl group and is thus considered monoglutamylated for the purposes of the present disclosure.

[0062] The compounds of the present invention can exist as mixtures of stereoisomers, but they are preferably resolved into one optically active isomeric form. Such requirements complicate the synthesis of the compounds and thus they preferably contain as few asymmetric carbon atoms as possible that are compatible with the achievement of the desired activity.

[0063] However, as previously shown, the cyclopenta[g]quinazolines of the present invention contain at least three asymmetric carbon atoms. Among these, the asymmetric carbon atoms at six positions in the ring system preferably have a 6S orientation rather than a 6R orientation. The preferred compound (I) herein is thus preferably one having such an arrangement at the positions of the asymmetric carbon atoms, and those of somewhat lesser preference are mixtures in which one or both of these asymmetric carbon atoms are undivided.

[0064] The folic acid metabolic antagonist can be any known one or a folic acid metabolic antagonist that will be derived in the future or is polyglutaminated and oxidized folic acid. In some embodiments, the folic acid metabolic antagonist is LV (etoposide), L-leucovorin (L-5-formyltetrahydrofolic acid); 5-CH3-THF, 5-methyltetrahydrofolic acid; FA, folic acid; PteGlu, pteroylglutamate (FA); MTX, methotrexate; 2-dMTX, 2-desamino-MTX; 2-CH3-MTX, 2-desamino-2-methyl-MTX; AMT, aminopterin; 2-dAMT, 2-desamino-AMT; 2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM, 10-ethyl-10-deazaaminopterin; PT523, N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine; DDATHF (raltitrexed), 5,10-dideaza-5,6,7,8,-tetrahydrofolic acid; 5-d(i)H4PteGlu, 5-deaza-5,6,7,8-tetrahydroiso-folic acid; N9-CH3-5-d(i)H4PteGlu, N9-methyl-5-deaza-5,6,7,8-tetrahydroiso-folic acid; 5-dPteHCysA, N alpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, N alpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutyric acid; 5-dPteOrn, N alpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, N alpha-(5-deaza-5,6,7,8-tetrahydroapteroyl)-L-homocysteic acid; 5-dH4PteAPBA, N alpha-(5-deaza-5,6,7,8-tetrahydroapteroyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dH4PteOro, N alpha-(5-dideaza-5,6,7,8-tetrahydroapteroyl)-L-ornithine; CB3717, N10-propynyl-5,8-dideazafolic acid; ICI-198,583, 2-desamino-2-methyl-N10-propynyl-5,8-dideazafolic acid; 4-H-ICI-198,583, 4-deoxy-ICI-198,583:4-OCH3-ICI-198,583 Glu-to-Val-ICI-198,583;Valine-ICI-198;583; Glu-to-Sub-ICI-198,583, 2-Aminosuccinic acid-ICI-198,583; 7-CH3-ICI-198,583, 7-Methyl-ICI-198,583; ZD1694, N-[5(N-(3,4-Dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamic acid; 2-NH2-ZD1694, 2-Amino-ZD1694; BW1843U89, (S)-2[5-(((1,2-Dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutamic acid; LY231514, N-(4-(2-(2-Amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidin-5-yl)ethyl)-benzoyl]-L-glutamic acid; IAHQ, 5,8-Dideazaisofolic acid; 2-dIAHQ, 2-Desamino-IAHQ; 2-CH3-dIAHQ, 2-Desamino-2-methyl-IAHQ; 5-d(i)PteGlu, 5-Deazaisofolic acid; N9-CH3-5-d(i)PteGlu, N9-Methyl-5-deazaisofolic acid; N9-CHO-5-d(i)PteGlu, N9-Formyl-5-deazaisofolic acid; AG337, 3,4-Dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)quinazoline; and AG377, 2,4-Diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline; or a stereoisomer thereof.;

[0065] In some embodiments, the folic acid antagonist is a member selected from the following: aminopterin, methotrexate, raltitrexed (also called TOMUDEX, ZD1694 (RTX)), premetrexed (BGC9331; also called ZD9331), pemetrexed (also called ALIMTA, LY231514), lometrexol (LMX) (5,10-dideazatetrahydrofolic acid), cyclopenta[g]quinazoline having a dipeptide ligand, CB3717, CB300945 (also called BGC945), or 6-R,S-BGC945 (ONX-0801), CB300638 (also called BGC638), and their stereoisomers such as BW1843U89.

[0066] The terms "polyglutamate", "polyglutamine oxidation", or variations thereof, mean a composition containing at least one chain of two or more linked glutamyl groups. The polyglutamate chain can be linear or branched. A linear polyglutamate chain can include, for example, a glutamyl group containing an alpha carboxyl group or a gamma carboxyl group bond. A branched polyglutamate chain can include, for example, one or more glutamyl groups containing both an alpha carboxyl group and a gamma carboxyl group bond to another glutamyl group, thereby providing a branch point for the polyglutamate. Representative branched polyglutamates are shown in FIGS. 1O-1R. The polyglutamate chain includes an N-terminal glutamyl group and one or more C-terminal glutamyl groups. The N-terminal glutamyl group of the polyglutamate chain does not bind to another glutamyl group via its amino group, but binds to one or more glutamyl groups via its carboxylic acid group. In some embodiments, the N-terminal glutamyl group of the polyglutamine oxidized folic acid antagonist is the glutamyl group of the folic acid antagonist. The C-terminal glutamyl group(s) of the polyglutamate chain bind to another glutamyl group via their amino groups, but do not bind to another glutamyl group via their carboxylic acid groups.

[0067] The terms "polyglutamylated folate antagonist", "polyglutamylated ANTIFOL", "ANTIFOL-PG", and "PANTIFOL" are used interchangeably herein and refer to a folate antagonist composition that contains at least one glutamyl group in addition to the glutamyl group in the folate antagonist (i.e., ANTIFOL-PGn, n≧1). References herein to the number of glutamyl groups in αPANTIFOL (ANTIFOL-PG) include the glutamyl groups in the folate antagonist. For example, an ANTIFOL-PG composition that contains five glutamyl residues in addition to the glutamyl group of ANTIFOL is referred to herein as a hexaglutamylated folate antagonist or folate antagonist hexaglutamate.

[0068] The terms "alpha glutamyl group", "alpha glutamate", and "alpha bond", when referring to the bond of a glutamyl group, refer to a glutamyl group that includes an alpha carboxyl group bond. In some embodiments, the alpha bond is an amide bond between the alpha carboxyl group of one glutamyl group and a second glutamyl group. The alpha bond can be a bond between a glutamyl group and a glutamyl group in a folate antagonist, or between a glutamyl group and a second glutamyl group such as a glutamyl group in a polyglutamate chain that is not present in the folate antagonist but is attached to the folate antagonist.

[0069] The terms "gamma-glutamyl group", "gamma-glutamate", and "gamma linkage", when referring to the linkage of glutamyl groups, mean a glutamyl group including a gamma-carboxyl group linkage. As discussed herein, when a folate antagonist enters a cell, it is polyglutamylated by the enzyme folylpolyglutamate synthetase (FPGS), which sequentially adds L-glutamyl groups to the glutamyl groups within the folate antagonist. Thus, an alpha-polyglutamylated folate antagonist composition is not formed intracellularly during folate antagonist therapy. In some embodiments, a gamma linkage is an amide bond between the gamma-carboxyl group of one glutamyl group and a second glutamyl group. A gamma linkage can be between a glutamyl group and a glutamyl group in a folate antagonist, or between a glutamyl group and a second glutamyl group such as a glutamyl group within a polyglutamate chain that is not present in the folate antagonist but is attached to the folate antagonist. In some embodiments, a gamma linkage means an amide bond of a glutamyl group of a folate antagonist. References to gamma linkages include gamma linkages of glutamyl groups of folate antagonists, unless specifically stated otherwise or clearly apparent from the context that it is not intended.

[0070] Unless otherwise indicated, the terms "alpha-polyglutamylated folate antagonists", "αPANTIFOL", "alpha ANTIFOL-PG", and iterations thereof are used interchangeably herein and mean a polyglutamylated folate antagonist composition that includes at least one glutamyl group that includes an alpha bond. For example, a pentaglutamylated ANTIFOL composition in which the third glutamyl group has an alpha bond but each of the other glutamyl groups has a gamma bond is considered an alpha ANTIFOL-PG in the present disclosure. In some embodiments, each glutamyl group of ANTIFOL-PG other than the glutamyl groups of ANTIFOL has an alpha bond (e.g., ANTIFOL-PGn where n = 5 and each of G1, G2, G3, G4, and G5 has an alpha bond). In some embodiments, each glutamyl group of ANTIFOL-PG other than the C-terminal glutamyl group(s) and the glutamyl groups of the folate antagonist has an alpha bond (e.g., ANTIFOL-PG n ) where n = 5 and each of G1, G2, G3, and G4 has an alpha bond). In some embodiments, each glutamyl group of PMX-PG other than the C-terminal glutamyl group(s) has an alpha bond (e.g., ANTIFOL-PG where n = 5 and the glutamyl groups of the folate antagonist and each of G1, G2, G3, and G4 has an alpha bond n ).

[0071] As used herein, the term "isolated" means a composition in a form not found in nature. Isolated alpha-polyglutamine oxidized compositions include those that are purified to the extent that they are no longer in the form found in nature. In some embodiments, the isolated alpha-polyglutamine oxidized folate antagonists are substantially pure. An isolated composition is free or substantially free of other cellular components such as proteins and nucleic acids that may potentially be found in nature or in the environment in which they are produced (e.g., cell culture). Alpha-polyglutamine oxidized compositions can be formulated with diluents or adjuvants and further isolated for practical purposes - for example, when used in a diagnostic or therapeutic, an alpha-polyglutamine oxidized composition is typically mixed with a pharmaceutically acceptable carrier or diluent. In some embodiments, an isolated alpha-polyglutamine oxidized composition (e.g., a delivery vehicle such as an alpha-polyglutamate and liposomes containing alpha-polyglutamate) contains less than 1% or less than 0.1% of undesired DNA or protein contaminants. In some embodiments, an alpha-polyglutamate composition (e.g., a delivery vehicle such as an alpha-polyglutamate and liposomes containing alpha-polyglutamate) is "isolated."

[0072] As used herein, the term "targeting moiety" means a molecule that confers enhanced affinity for a selected target, such as a cell, cell type, tissue, organ, region of the body, or compartment, e.g., a compartment of a cell, tissue or organ. A targeting moiety can include a wide variety of entities. Targeting moieties include natural molecules, or recombinant or synthetic molecules. In some embodiments, the targeting moiety is an antibody 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.

[0073] The terms "specific affinity" or "specifically binds" mean that a targeting moiety, such as an antibody or an antigen-binding antibody fragment, reacts with or binds to an epitope, protein, or target molecule with a higher frequency, more rapidly, for a longer period of time, with a greater affinity, or in some combination of these, than it does to another substance, including a protein unrelated to the target epitope. Due to sequence identity between homologous proteins in different species, in some embodiments, a specific affinity involves a binding substance that recognizes a protein or target in two or more species. Similarly, due to homology within a specific region of the polypeptide sequences of different proteins, the terms "specific affinity" or "specific binding" can include a binding substance that recognizes two or more proteins or targets. In certain embodiments, it is understood that a targeting moiety that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific affinity" does not necessarily (although it can) require exclusive binding, e.g., binding to only one target. Thus, in certain embodiments, a targeting moiety can specifically bind to two or more targets. In certain embodiments, multiple targets can be bound by the same targeting moiety.

[0074] The term "epitope" means a portion of an antigen that is recognized and can be specifically bound by a targeting moiety (i.e., a binding portion), such as an antibody. When the antigen is a polypeptide, the epitope can be formed from both contiguous and non-contiguous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed from contiguous amino acids are usually retained upon protein denaturation, whereas epitopes formed by tertiary folding are usually lost upon protein denaturation. An epitope typically contains at least 3 amino acids, more generally at least 5 or 8 - 10 amino acids, in a unique spatial higher-order structure.

[0075] Expressions such as "binding affinity for a target" and "binding to a target", which are known in the art, and similar expressions, refer to properties of a targeting moiety that can be directly measured by determining an affinity constant, e.g., the amount of targeting moiety that binds and dissociates at a given antigen concentration. Intermolecular interactions can be characterized using other methods, including, but not limited to, competitive analysis, equilibrium analysis, and microcalorimetry, and real-time interaction analysis based on surface plasmon resonance interactions (e.g., using a BIACORE® instrument). These methods are well known to those skilled in the art and are described, for example, in Neri et al., Tibtech 14:465-470 (1996), and Jansson et al., J. Biol. Chem. 272:8189-8197 (1997).

[0076] The term "delivery vehicle" generally means any composition that acts to assist, facilitate, or ease the entry of an alpha-polygulamine oxidized folic acid antagonist into a cell. 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), antigen-binding antibody fragments, and derivatives thereof), cell components, cyclic oligosaccharides (e.g., cyclodextrins), micelles, microparticles (e.g., microspheres), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), hydrogels, lipoprotein particles, viral sequences, viral materials, or lipid or liposome formulations, and combinations thereof. A delivery vehicle can be directly or indirectly bound to a targeting moiety. In some embodiments, the targeting moiety is selected from a polymer, protein, peptide, monoclonal antibody, or fatty acid lipid.

[0077] "Subject" means a vertebrate mammal, including but not limited to humans, dogs, cats, horses, goats, and primates such as monkeys. Thus, the present invention can also be used to treat diseases or conditions in non-human subjects. For example, cancer is one of the leading causes of death in companion animals (i.e., cats and dogs). In some embodiments of the present invention, the subject is a human. In the present disclosure, the terms "subject" and "patient" are used interchangeably and have the same meaning. Generally, it is preferred that the maximum dose, i.e., the maximum safe dose, is used according to sound medical judgment.

[0078] As used herein, "effective amount" means an amount of a drug sufficient to produce a medically desired result. The effective amount can vary depending on the desired outcome, the particular condition 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 therapies (if any), the specific route of administration, and similar factors within the knowledge and professional opinion of the health care provider. The "effective amount" can be determined experimentally or routinely in relation to the indicated purpose. In the case of cancer, an effective amount of a drug can reduce the number of cancer cells; reduce the size of the tumor; inhibit the invasion of cancer cells into surrounding organs (i.e., slow down and preferably stop to some extent); inhibit the metastasis of the tumor (i.e., slow down and preferably stop to some extent); inhibit the growth of the tumor to some extent; and / or alleviate one or more of the symptoms associated with the disorder to some extent. Depending on the extent to which the drug can prevent and / or kill the growth of existing cancer cells, the drug can be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured by, for example, evaluating survival time, progression-free survival (PFS) period, response rate (RR), response duration, and / or quality of life.

[0079] The terms "hyperproliferative disorder", "proliferative disease", and "proliferative disorder" are used interchangeably herein and relate to unwanted or uncontrolled cell proliferation of undesirable or abnormal cells, such as neoplastic or hyperplastic proliferation, regardless of in vitro or in vivo. In some embodiments, the proliferative disease is a cancer or tumor disease (including benign or cancerous) and / or any tumor metastasis, regardless of the location of the cancer, tumor and / or tumor metastasis. In some embodiments, the proliferative disease is a benign or malignant tumor. In some embodiments, the proliferative disease is a non-cancerous disease. In some embodiments, the proliferative disease is a hyperproliferative condition such as hyperplasia, fibrosis (particularly pulmonary, but also other types such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis, and smooth muscle proliferation in blood vessels such as stenosis or restenosis after angioplasty.

[0080] "Cancer", "tumor", or "malignant tumor" are used as synonymous terms and mean any of a number of diseases characterized by uncontrolled, abnormal cell growth, local or spread (metastasis) of infected cells to other parts of the body via the bloodstream and lymphatic system, as well as a number of characteristic structures and / or molecular features. As used herein, "tumor" means all neoplastic cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. "Cancerous tumor", or "malignant cells" are understood to be cells that have specific structural characteristics, lack differentiation, and are capable of invasion and metastasis. Cancers that can be treated with the αPANTIFOL compositions provided herein include, but are not limited to, for example, non-blood system tumors such as lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and, for example, blood system tumors such as leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias or cachexia. In some embodiments, the cancer is breast cancer, advanced head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, villous adenoma, aleukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) cancer. In some embodiments, the cancer is lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer is breast cancer (e.g., HER2++ or triple negative breast cancer). In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is osteosarcoma. Other types of cancers and tumors that can be treated with the αPANTIFOL compositions are described herein or are known in the art.The term "metastasis" means the spread or seeding of a tumor, cancer, or other site, location, region, organ, or tissue system within the target of the tumor, and the site, location, region, organ, or tissue system in the target is different from the primary tumor, cancer, or neoplasm. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder", and "tumor" are not mutually exclusive when referred to in this specification.

[0081] The terms "treating", or "treatment", or "treat" mean both (a) therapeutic means to cure, slow down, reduce the symptoms of, and / or halt the progression of a diagnosed medical condition or disorder and (b) prophylactic or preventive means to prevent and / or delay the occurrence of a targeted disease or condition. Thus, subjects in need of treatment include subjects already having the cancer, disorder or disease, subjects at risk of becoming a cancer or condition, and subjects in whom an infection or condition is to be prevented. A subject is identified as "at risk of having" a cancer, infectious disease, immune system disorder, hyperproliferative disease, or another disease or disorder mentioned herein using well-known medical and diagnostic techniques. In certain embodiments, where a subject exhibits an overall, partial, or temporary remission or removal of symptoms associated with a disease or condition (e.g., cancer, rheumatoid arthritis), the subject is "being treated successfully" by the methods provided herein. In certain embodiments, the term "treating" or "treatment" or "treat" means an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which may not be distinguishable by the patient. In other embodiments, the term "treating" or "treatment" or "treat" means inhibiting the progression of a proliferative disorder, e.g., physically by stabilization of distinguishable symptoms or physiologically by stabilization of physical parameters, or both. In other embodiments, the term "treating" or "treatment" or "treat" means a decrease or stabilization in size, tumor cell proliferation or survival, or cancer cell number. The treatment can use the α-PANTIFOL composition alone or in combination with additional therapeutic agents.

[0082] "Subject", "patient", and "animal" are used synonymously and mean mammalian subjects such as human patients and non-human primates, as well as laboratory animals such as rabbits, rats, mice, and other animals. Animals include all vertebrates, for example, mammals and non-mammals such as chickens, amphibians, and reptiles. As used herein, "mammal" includes, but is not limited to, human and non-human primate animals such as chimpanzees and other apes and monkey species; livestock animals such as cows, sheep, pigs, goats, and horses; pet mammals such as dogs and cats; laboratory animals such as rodents such as mice, rats, guinea pigs, and other members of the mammalian class known in the art. In certain embodiments, the patient is human.

[0083] As used herein, "treatment of a proliferative disorder" includes maintaining or reducing the tumor size of a subject with a proliferative disorder, inducing tumor shrinkage (partial or complete), suppressing tumor growth, and / or prolonging lifespan. In one embodiment, the proliferative disorder is a solid tumor. Such tumors include, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma. In one embodiment, the proliferative disorder is a hematological tumor. Such hematological tumors include, for example, leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias or malignancies. In some embodiments, the cancer is selected from breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, villous adenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) cancer. In some embodiments, the cancer is lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer is breast cancer (e.g., HER2++ or triple-negative breast cancer). In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is osteosarcoma.

[0084] As used herein, the term "autoimmune disease" is defined as a disorder resulting from an autoimmune reaction. Autoimmune diseases are the result of an inappropriate and excessive reaction against self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis.

[0085] The terms "inflammation" and "inflammatory disease" are used interchangeably and mean a disease or disorder characterized by or caused by inflammation. "Inflammation" means a local reaction to cellular injury characterized by capillary dilation, leukocyte infiltration, redness, heat, and pain, which serves as a mechanism to initiate the removal of harmful agents and damaged tissues. Sites of inflammation include the lung, pleura, tendon, lymph node or gland, uvula, vagina, brain, spinal cord, nasal and pharyngeal mucosa, muscle, skin, bone or bone tissue, joint, bladder, retina, cervix, canthus, intestinal tract, vertebra, rectum, anus, bursa, hair follicle, etc. Such inflammatory diseases include, but are not limited to, inflammatory bowel disease, rheumatic-like diseases (e.g., rheumatoid arthritis), other arthritis diseases (e.g., acute arthritis, acute gouty arthritis, bacterial arthritis, chronic inflammatory arthritis, osteoarthritis (degenerative joint disease), infectious arthritis, juvenile arthritis, fungal arthritis, neuropathic arthritis, polyarthritis, proliferative arthritis, psoriatic arthritis, gonococcal arthritis, viral arthritis), conjunctivitis, pelvic inflammatory disease, acne, psoriasis, actinomycosis, dysentery, biliary cirrhosis, Lyme disease, aspergillosis, Stevens-Johnson syndrome, mumps, pemphigus vulgaris, and blastomycosis. Inflammatory bowel disease is a chronic inflammatory disease of the gastrointestinal tract including, but not limited to, Crohn's disease, ulcerative colitis, and unclassified colitis. Rheumatoid arthritis is a chronic inflammatory disease of the joints, usually polyarticular, characterized by inflammatory changes in the synovium and joint structures as well as muscle spasm and osteoporosis of the bone.

[0086] As used herein, the term "therapeutic agent" means an agent, or derivative thereof, or prodrug 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 agents, platinum-based agents (e.g., cisplatin, carboplatin, oxaliplatin), taxanes (e.g., Taxol®), etoposide, alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., antifolates (ANTIFOL)), 5-fluorouracil, gemcitabine, or derivatives thereof), antitumor antibiotics (e.g., mitomycin, doxorubicin), plant-derived antitumor agents (e.g., vincristine, vindesine, Taxol®). Such agents further include, but are not limited to, the anticancer agents trimethoprim, temozolomide, raltitrexed, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), bis-chloronitrosourea (BCNU) and camptothecin (trademark), or any therapeutic derivatives thereof. Further examples of therapeutic agents that may be suitable for use by the methods of the present disclosure include, but are not limited to, anti-restenosis agents, proliferative or anti-proliferative agents, anti-inflammatory agents, anti-neoplastic agents, anti-mitotic agents, antiplatelet agents, anticoagulants, antifibrin agents, antithrombin agents, cytostatic agents, antibiotics and other anti-infective agents, anti-enzymatic agents, antimetabolites, angiogenesis agents, cytoprotective agents, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists and / or cardioprotective agents. "Therapeutic agent" also means salts, acids, and free base forms of the above agents.

[0087] As used herein, the term "chemotherapeutic agent", when used in the context of cancer therapy, means any agent that causes the death of cancer cells or inhibits the growth or spread of cancer cells. Examples of such chemotherapeutic agents include alkylating agents, antibiotics, antimetabolites, plant-derived agents, and hormones. 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.

[0088] As used herein, the term "antimetabolite" means a therapeutic agent that inhibits the utilization of a metabolite or its prodrug. Examples of antimetabolites include folic acid antimetabolites, pemetrexed, 5-fluorouracil, 5-fluorouracil prodrugs such as capecitabine, 5-fluorodeoxyuridine monophosphate, cytarabine, cytarabine prodrugs such as nelarabine, 5-azacitidine, gemcitabine, mercaptopurine, thioguanine, azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, and cladribine. Antimetabolites useful for practicing the disclosed methods include nucleoside analogs that include purine or pyrimidine analogs. In some embodiments, the alpha-polyglutamyl oxidized folic acid antimetabolite composition is used in combination with an antimetabolite selected from fluoropyrimidines, 5-fluorouracil, 5-fluoro-2'-deoxycytidine, cytarabine, gemcitabine, troxacitabine, decitabine, azacitidine, pseudoisocytidine, zebularine, ancitabine, fazarabine, 6-azacitidine, capecitabine, N4-octadecylcytarabine, elaidic acid cytarabine, fludarabine, cladribine, clofarabine, nelarabine, folotyn, and pentostatin, or derivatives thereof. In one example, the nucleoside analog is a substrate of a nucleoside deaminase that is adenosine deaminase or cytidine deaminase. In some examples, the nucleoside analog is selected from fludarabine, cytarabine, gemcitabine, decitabine, and azacitidine or derivatives thereof. In certain embodiments, the antimetabolite is 5-fluorouracil.

[0089] As used herein, "taxane" is an anti-cancer agent that interferes with or disrupts microtubule stability, formation, and / or function. Taxane agents include paclitaxel and docetaxel and their derivatives, which function in the same mode of action with respect to microtubules as the taxanes from which they are derived. In certain embodiments, the taxane is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the taxane is paclitaxel (Taxol®), docetaxel (Taxotere®), albumin-bound paclitaxel (nab-paclitaxel; Abraxane®), DHA-paclitaxel, or PG-paclitaxel.

[0090] The terms "pharmaceutically-acceptable carrier" and "pharmaceutically acceptable carrier" mean components that are non-toxic to a subject other than the active ingredient in a pharmaceutical formulation. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. Pharmaceutically acceptable carriers include, for example, one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other subjects.

[0091] The present disclosure generally relates to novel alpha-polyglutamylated antifolate (ANTIFOL) compositions, and methods of making and using the compositions for treating diseases including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV, malaria, and schistosomiasis.

[0092] In some embodiments, the present disclosure provides the following. [1] A composition comprising an alpha-polyglutamylated antifolate, wherein at least one glutamyl group has an alpha-carboxyl group linkage. [2] The composition of item [1], wherein the folic acid antagonist is selected from piritrexim, pralatrexate, AG2034, GW1843, and LY309887, or stereoisomers thereof; [3] The composition of item [1], wherein the folic acid antagonist is selected from PMX, MTX, RTX, and LMX, or stereoisomers thereof; [4] The composition according to item [1], wherein the folic acid antagonist is selected from the following: LV (etoposide), L-leucovorin (L-5-formyltetrahydrofolic acid); 5-CH3-THF, 5-methyltetrahydrofolic acid; FA, folic acid; PteGlu, pteroylglutamate (FA); MTX, methotrexate; 2-dMTX, 2-desamino-MTX; 2-CH3-MTX, 2-desamino-2-methyl-MTX; AMT, folic acid antagonist; 2-dAMT, 2-desamino-AMT; 2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM, 10-ethyl-10-deazaaminopterin; PT523, N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine; DDATHF (raltitrexed), 5,10-dideaza-5,6,7,8,-tetrahydrofolic acid; 5-d(i)H4PteGlu, 5-deaza-5,6,7,8-tetrahydroiso-folic acid; N9-CH3-5-d(i)H4PteGlu, N9-methyl-5-deaza-5,6,7,8-tetrahydroiso-folic acid; 5-dPteHCysA, N alpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, N alpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutyric acid; 5-dPteOrn, N alpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-homocysteic acid; 5-dH4PteAPBA, N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dH4PteOro, N alpha-(5-dideaza-5,6,7,8-tetrahydropteroyl)-L-ornithine; CB3717, N10-propargyl-5,8-dideazafolic acid; ICI-198,583, 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid; 4-H-ICI-198,583, 4-deoxy-ICI-198,583:4-OCH3-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198;583;Glu-to-Sub-ICI-198,583, 2-aminosuberic acid-ICI-198,583; 7-CH3-ICI-198,583, 7-methyl-ICI-198,583; ZD1694, N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamic acid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89, (S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutamic acid; LY231514, N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidine-5-yl)ethyl)-benzoyl]-L-glutamic acid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ; 2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu, 5-deazaisofolic acid; N9-CH3-5-d(i)PteGlu, N9-methyl-5-deazaisofolic acid; N9-CHO-5-d(i)PteGlu, N9-formyl-5-deazaisofolic acid; AG337, 3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)quinazoline; and 2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline; or their stereoisomers; [5] The composition according to item [1], wherein the folic acid metabolism antagonist is selected from methotrexate, raltitrexed, premetrexed, pemetrexed, lometrexol (LMX; 5,10-dideazatetrahydrofolic acid), cyclopenta[g]quinazoline having a dipeptide ligand, CB3717, CB300945, or their stereoisomers such as 6-R,S-BGC945 (ONX-0801), CB300638, and BW1843U89; [6] The composition according to any one of items [1] to [5], which is the following composition; (a) Each glutamyl group of the polyglutamylated folic acid metabolism antagonist other than the glutamyl group of the folic acid metabolism antagonist has an alpha carboxyl group bond; or (b) Two or more glutamyl groups of the polyglutamine oxidized folic acid antagonist have a gamma carboxyl group bond; [7] The composition according to any one of items [1] to [5], which is the following composition; (a) Each glutamyl group other than the C-terminal glutamyl group(s) and the glutamyl group of the folic acid antagonist has an alpha carboxyl group bond; or (b) Each glutamyl group other than the C-terminal glutamyl group(s) has an alpha carboxyl group bond; [8] The composition according to any one of items [1] to [7], which is the following composition: (a) Containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups; (b) An alpha pentaglutamine oxidized folic acid antagonist; or (c) An alpha hexaglutamine oxidized folic acid antagonist; [9] The composition according to any one of items [1] to [8], which is a composition in which the alpha polyglutamine oxidized folic acid antagonist contains 1 to 10 glutamyl groups having an alpha carboxyl group bond;

[10] The composition according to any one of items [1] to [9], which is the following composition: (a) At least two glutamyl groups of the alpha polyglutamine oxidized folic acid antagonist are of the L type; (b) Each glutamyl group of the alpha polyglutamine oxidized folic acid antagonist is of the L type; (c) At least one glutamyl group of the alpha polyglutamine oxidized folic acid antagonist is of the D type; (d) Each glutamyl group of the alpha polyglutamine oxidized folic acid antagonist other than the glutamyl group of the folic acid antagonist is of the D type; or (e) At least two glutamyl groups of the alpha polyglutamine oxidized folic acid antagonist are of the L type and at least one glutamyl group is of the D type;

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

[10] , wherein the polyglutamate is linear;

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

[10] , wherein the polyglutamate is branched;

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

[12] ;

[14] The Lp-αPANTIFOL composition of item

[13] , wherein the alpha-polyglutamylated folic acid antimetabolite is selected from the following: (a) AG2034, pyrithioxime, pralatrexate, GW1843, a folic acid antimetabolite, and LY309887; or (b) PMX, MTX, RTX and LMX, or their stereoisomers;

[15] The Lp-αPANTIFOL composition of item

[13] , wherein the polyglutaminated folic acid antimetabolite is a composition selected from the following: LV (etoposide), L-leucovorin (L-5-formyltetrahydrofolic acid); 5-CH3-THF, 5-methyltetrahydrofolic acid; FA, folic acid; PteGlu, pteroylglutamate (FA); MTX, methotrexate; 2-dMTX, 2-desamino-MTX; 2-CH3-MTX, 2-desamino-2-methyl-MTX; AMT, aminopterin; 2-dAMT, 2-desamino-AMT; 2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM, 10-ethyl-10-deazaaminopterin; PT523, N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine; DDATHF (raltitrexed), 5,10-dideaza-5,6,7,8,-tetrahydrofolic acid; 5-d(i)H4PteGlu, 5-deaza-5,6,7,8-tetrahydroiso-folic acid; N9-CH3-5-d(i)H4PteGlu, N9-methyl-5-deaza-5,6,7,8-tetrahydroiso-folic acid; 5-dPteHCysA, N alpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, N alpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutyric acid; 5-dPteOrn, N alpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, N alpha-(5-deaza-5,6,7,8-tetrahydroapteroyl)-L-homocysteic acid; 5-dH4PteAPBA, N alpha-(5-deaza-5,6,7,8-tetrahydroapteroyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dH4PteOro, N alpha-(5-dideaza-5,6,7,8-tetrahydroapteroyl)-L-ornithine; CB3717, N10-propargyl-5,8-dideazafolic acid; ICI-198,583, 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid; 4-H-ICI-198,583, 4-deoxy-ICI-198,583:4-OCH3-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198;583;Glu-to-Sub-ICI-198,583, 2-aminosuberic acid-ICI-198,583; 7-CH3-ICI-198,583, 7-methyl-ICI-198,583; ZD1694, N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamic acid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89, (S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutamic acid; LY231514, N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidin-5-yl)ethyl)-benzoyl]-L-glutamic acid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ; 2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu, 5-deazaisofolic acid; N9-CH3-5-d(i)PteGlu, N9-methyl-5-deazaisofolic acid; N9-CHO-5-d(i)PteGlu, N9-formyl-5-deazaisofolic acid; AG337, 3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)quinazoline; and AG377, 2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline; or stereoisomers thereof;

[16] The Lp-αPANTIFOL composition according to item

[13] , wherein the folic acid antagonist is selected from methotrexate, raltitrexed, premetrexed, pemetrexed, lometrexol (LMX; 5,10-dideazatetrahydrofolic acid), cyclopenta[g]quinazoline having a dipeptide ligand, CB3717, CB300945, or stereoisomers thereof such as 6-R,S-BGC945 (ONX-0801), CB300638, and BW1843U89;

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

[13] to

[16] , wherein the liposome contains an alphapolyglutamine oxidized folic acid antagonist containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups;

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

[13] to

[17] , wherein the liposome contains an alphatetraglutamine oxidized folic acid antagonist;

[19] The Lp-αPANTIFOL composition according to any one of items

[13] to

[17] , wherein the liposome contains an alphapentaglutamine oxidized folic acid antagonist;

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

[13] to

[17] , wherein the liposome contains an alphahexaglutamine oxidized folic acid antagonist;

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

[13] to

[20] , wherein the polyglutamate is linear or branched;

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

[13] to

[21] , wherein: (a) Each glutamyl group other than the glutamyl group of the folic acid antagonist has an alphacarboxyl group bond; or (b) Two or more glutamyl groups have a gammacarboxyl group bond;

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

[13] to

[21] , wherein: (a) The C-terminal glutamyl group(s) and each glutamyl group other than the glutamyl group of the folic acid antagonist have an alphacarboxyl group bond; or (b) Each glutamyl group other than the C-terminal glutamyl group(s) has an alphacarboxyl group bond;

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

[13] to

[23] , wherein: (a) At least two glutamyl groups of the alpha-polyglutamyl oxidized folic acid antimetabolite are of the L-type; (b) Each glutamyl group of the alpha-polyglutamyl oxidized folic acid antimetabolite is of the L-type; (c) At least one glutamyl group of the alpha-polyglutamyl oxidized folic acid antimetabolite is of the D-type; (d) Each glutamyl group of the alpha-polyglutamyl oxidized folic acid antimetabolite other than the glutamyl group of the folic acid antimetabolite is of the D-type; or (e) At least two glutamyl groups of the alpha-polyglutamyl oxidized folic acid antimetabolite are of the L-type and at least one glutamyl group is of the D-type;

[25] An Lp-αPANTIFOL composition according to any one of items

[13] to

[24] , wherein the liposome is pegylated (PαLp-αPANTIFOL), the composition;

[26] An Lp-αPANTIFOL composition according to any one of items

[13] to

[24] , wherein the liposome is not pegylated, the composition;

[27] An Lp-αPANTIFOL composition according to any one of items

[13] to

[26] , wherein the liposome has a diameter in the range of 20 nm to 200 nm, the composition;

[28] An Lp-αPANTIFOL composition according to any one of items

[13] to

[27] , wherein the polyglutamate is linear or branched, the composition;

[29] An Lp-αPANTIFOL composition according to any one of items

[13] to

[28] , wherein the liposome contains at least 1% by weight of the alpha-polyglutamyl oxidized folic acid antimetabolite, or during the process of preparing Lp-αPANTIFOL, at least 1% of the starting material of the alpha-polyglutamyl oxidized folic acid antimetabolite is encapsulated (enclosed) in Lp-αPANTIFOL, the composition;

[30] An Lp-αPANTIFOL composition according to any one of items

[13] to

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

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

[13] to

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

[32] The Lp-αPANTIFOL composition according to any one of items

[13] to

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

[33] The Lp-αPANTIFOL composition according to item

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

[34] The Lp-αPANTIFOL composition according to item

[32] or

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

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

[32] to

[34] , wherein the liposome components include at least one selected from the following: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC;

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

[32] to

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

[37] The Lp-αPANTIFOL composition according to item

[36] , wherein the steric stabilizer is at least one selected from polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymer; oligoglycerol, polyethylene glycol and polypropylene oxide-containing copolymer, poloxamer 188, and polyvinyl alcohol;

[38] The Lp-αPANTIFOL composition according to item

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

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

[13] to

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

[40] The Lp-αPANTIFOL composition according to any one of items

[13] to

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

[41] The Lp-αPANTIFOL composition according to any one of items

[13] to

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

[42] The Lp-αPANTIFOL composition according to any one of items

[13] to

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

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

[13] to

[38] , wherein the liposome is cationic;

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

[13] to

[43] , wherein the liposome has an internal space containing an alpha-polyglutamine oxidized folic acid antagonist and an aqueous pharmaceutically acceptable carrier;

[45] The Lp-αPANTIFOL composition according to item

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

[46] The Lp-αPANTIFOL composition according to item

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

[47] The Lp-αPANTIFOL composition according to item

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

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

[44] to

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

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

[44] to

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

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

[44] to

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

[51] The Lp-αPANTIFOL composition according to any one of items

[44] to

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

[52] The Lp-αPANTIFOL composition according to any one of items

[13] to

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

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

[13] to

[52] , wherein the liposome contains less than 500,000 or less than 200,000 alpha-polyglutamylated folic acid antagonist molecules;

[54] The Lp-αPANTIFOL composition according to any one of items

[13] to

[53] , wherein the liposome contains 10 to 100,000 or any range therebetween of alpha-polyglutamylated folic acid antagonist molecules;

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

[13] to

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

[56] The Lp-αPANTIFOL composition according to item

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

[57] The Lp-αPANTIFOL composition according to item

[55] or

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

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

[55] to

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

[59] The Lp-αPANTIFOL composition according to any one of items

[55] to

[58] , wherein the targeting moiety binds to a surface antigen with an equilibrium dissociation constant (Kd) in the range of 0.5x10-10 to 10x10-6 as measured by BIACORE (registered trademark) analysis;

[60] The Lp-αPANTIFOL composition according to any one of items

[55] to

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

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

[55] to

[60] , wherein the targeting moiety comprises one or more selected from antibodies, humanized antibodies, antigen-binding fragments of antibodies, single-chain antibodies, single-domain antibodies, bispecific antibodies, synthetic antibodies, pegylated antibodies, and multimeric antibodies;

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

[55] to

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

[63] The Lp-αPANTIFOL composition according to any one of items

[44] to

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

[64] The Lp-αPANTIFOL composition of item

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

[65] The Lp-αPANTIFOL composition of item

[63] or

[64] , wherein the immunostimulant is at least one selected from toll-like receptor (TLR) modulators such as fluorescein, fluorescein isothiocyanate (FITC), DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan, resorcin (such as resorcin D like Dn-6DPA or Dn-3DPA, resorcin E, or T series resorcin), oxidized low-density lipoprotein (such as OXPAC, PGPC), and erythran lipid (such as E5564);

[66] The Lp-αPANTIFOL composition according to any one of items

[63] to

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

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

[63] to

[66] , further comprising a hapten;

[68] The Lp-αPANTIFOL composition of item

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

[69] The Lp-αPANTIFOL composition according to any one of items

[13] to

[68] , further comprising at least one cryoprotective substance selected from mannitol, trehalose, sorbitol, and sucrose;

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

[69] ;

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

[54] and

[64] to

[69] ;

[72] An Lp-αPANTIFOL composition according to any one of items

[13] to

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

[73] A pharmaceutical composition comprising the liposomal alpha-polyglutamine oxidized folic acid antagonist composition according to any one of items

[13] to

[72] ;

[74] A pharmaceutical composition comprising the alpha-polyglutamine oxidized folic acid antagonist composition according to any one of items [1] to [8];

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

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

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

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

[77] A method for treating or preventing a disease in a subject in need of treatment or prevention, comprising the step of administering the composition according to any one of items [1] to

[75] to the subject;

[78] A method for treating or preventing a disease in a subject in need of treatment or prevention, comprising the step of administering the liposomal alpha-polyglutamine oxidized folic acid antagonist composition according to any one of items

[13] to

[74] to the subject;

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

[74] ;

[80] A method for killing hyperproliferative cells, comprising the step of contacting the hyperproliferative cells with the liposomal alpha-polyglutamine oxidized folic acid antagonist composition according to any one of items

[13] to

[74] ;

[81] The method according to item

[79] or

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

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

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

[83] A method for treating cancer, comprising the step of administering an effective amount of the liposomal alpha-polyglutamyl oxidized folic acid antimetabolite composition according to any one of items

[13] to

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

[84] The method according to item

[82] or

[83] , wherein the cancer is a non-hematological tumor including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and a hematological tumor selected from, for example, leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias;

[85] The method according to item

[82] or

[83] , wherein the cancer is a member selected from breast cancer, advanced head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, villous adenoma, aleukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, central nervous system (CNS) lymphoma;

[86] The method according to item

[82] or

[83] , wherein the cancer is selected from colorectal cancer, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer;

[87] The method according to item

[82] or

[83] , wherein the cancer is a sarcoma such as osteosarcoma;

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

[55] to

[71] to a subject having or at risk of having cancer cells expressing a folate receptor bound by a targeting moiety on the surface;

[89] Maintenance therapy comprising the step of administering an effective amount of the composition according to any one of items [1] to

[74] to a subject who is receiving or has received cancer therapy;

[90] Maintenance therapy comprising the step of administering an effective amount of the liposomal alpha-polyglutamyl oxidized folic acid antimetabolite composition according to any one of items

[13] to

[74] to a subject who is receiving or has received cancer therapy;

[91] A method for treating an immune disorder, comprising the step of administering to a subject having or at risk of having an immune disorder an effective amount of the composition according to any one of items [1] to

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

[92] A method for treating an immune disorder, comprising the step of administering to a subject having or at risk of having an immune disorder an effective amount of the liposomal alpha-polyglutamine oxidized folic acid metabolic antagonist composition according to any one of items [9] to

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

[93] The following treatment methods: (a) A method for treating an infectious disease, comprising the step of administering to a subject having or at risk of having an infectious disease an effective amount of the composition according to any one of items [1] to

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

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

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

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

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

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

[94] A method for treating an infectious disease, comprising administering to a subject having or at risk of having an infectious disease an effective amount of the liposomal alpha-polyglutamic acid oxidized folic acid metabolic antagonist composition according to any one of items

[13] to

[74] ;

[95] A method for delivering an alpha-polyglutamic acid oxidized folic acid metabolic antagonist to a tumor expressing a folate receptor on its surface, the method comprising administering to a subject having a tumor an Lp-αPANTIFOL composition according to any one of items [1] to

[74] in an amount sufficient to deliver a therapeutically effective amount of the alpha-polyglutamic acid oxidized folic acid metabolic antagonist to the tumor;

[96] A method for preparing an alpha-polyglutamic acid oxidized folic acid metabolic antagonist composition comprising the liposomal alpha-polyglutamic acid oxidized folic acid metabolic antagonist composition according to any one of items

[13] to

[74] , the method comprising forming a mixture comprising a liposomal component and an alpha-polyglutamic acid oxidized folic acid metabolic antagonist in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes comprising the alpha-polyglutamic acid oxidized folic acid metabolic antagonist;

[97] A method for preparing an alpha-polyglutamic acid oxidized folic acid metabolic antagonist composition comprising the liposomal alpha-polyglutamic acid oxidized folic acid metabolic antagonist composition according to any one of items

[13] to

[74] , the method comprising forming a mixture comprising a liposomal component and an alpha-polyglutamic acid oxidized folic acid metabolic antagonist in solution; and treating the mixture to form liposomes comprising the alpha-polyglutamic acid oxidized folic acid metabolic antagonist;

[98] The method according to item

[97] , wherein the step of treating the mixture comprises the step of homogenizing the mixture in a solution to form liposomes;

[99] A method for producing the composition according to any one of items

[55] to

[74] , comprising the steps of: forming a mixture containing liposome components and an alpha-polyglutamine oxidized folic acid antagonist in a solution; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes encapsulating and / or coating the alpha-polyglutamine oxidized folic acid antagonist; and imparting a targeting moiety on the surface of the liposomes, wherein the targeting moiety has specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β) and folate receptor delta (FR-δ);

[0100] A method for producing the composition according to any one of items

[55] to

[74] , comprising the steps of: forming a mixture containing liposome components and an alpha-polyglutamine oxidized folic acid antagonist in a solution; treating the mixture to form liposomes encapsulating and / or coating the alpha-polyglutamine oxidized folic acid antagonist; and imparting a targeting moiety on the surface of the liposomes, wherein the targeting moiety has specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β) and folate receptor delta (FR-δ);

[0101] The method according to item

[0100] , wherein the treating step comprises the step of homogenizing the mixture in a solution to form liposomes;

[0102] The method according to any one of items

[99] to

[0101] , wherein the treating step comprises one or more steps of thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw method, reverse phase evaporation method, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, freeze-dried double emulsion, 3D printing, membrane contactor method, and stirring; and The method according to any one of items

[99] to

[0102] , wherein the processing step includes one or more steps of changing the size of the liposome by one or more steps of extrusion, high-pressure microfluidization, and / or ultrasonic treatment; The method according to any one of items

[96] to

[0103] , wherein at least 1% of the starting material of the alpha-polyglutamine oxidized folic acid antimetabolite is encapsulated or enclosed in Lp-αPANTIFOL.

[0093] II. Alpha-polyglutamine oxidized folic acid antimetabolite (αPANTIFOL) Generally, the present disclosure relates to alpha-polyglutamine oxidized folic acid antimetabolite (αPANTIFOL) compositions. The αPANTIFOL compositions contain at least one glutamyl group having an alpha bond. These compositions are structurally different from the L-gamma-polyglutamine oxidized form of the folic acid antimetabolite (LαPANTIFOL) produced by the enzyme holylpoly-gamma-glutamate synthase (FPGS) in cells during folic acid antimetabolite therapy.

[0094] In some embodiments, the αPANTIFOL composition comprises 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl group of the folic acid antagonist). In some embodiments, each glutamyl group in αPANTIFOL other than the glutamyl group of the folic acid antagonist has an alpha bond. In some embodiments, each glutamyl group in αPANTIFOL other than the C-terminal glutamyl group(s) and the glutamyl group of the folic acid antagonist has an alpha bond. In some embodiments, each glutamyl group in αPANTIFOL other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, two or more glutamyl groups in αPANTIFOL have a gamma bond. In some embodiments, at least one glutamyl group of the alphapolyg lutamine oxidized folic acid antagonist has an alphacarboxyl group bond and a gammacarboxyl group bond. In some embodiments, each glutamyl group in αPANTIFOL is of the L-type. In some embodiments, each glutamyl group in αPANTIFOL other than the glutamyl group of the folic acid antagonist is of the D-type. In some embodiments, αPANTIFOL comprises two or more L-type glutamyl groups and one or more D-type glutamyl groups. In some embodiments, the polyglutamate chain of αPANTIFOL is linear (not branched). In some embodiments, the polyglutamate chain of αPANTIFOL is branched.

[0095] In some embodiments, the folic acid antagonist is selected from PMX, MTX, RTX, and LMX, or stereoisomers thereof.

[0096] In some embodiments, the folic acid antagonist is selected from the following: LV (etoposide), L-leucovorin (L-5-formyltetrahydrofolic acid); 5-CH3-THF, 5-methyltetrahydrofolic acid; FA, folic acid; PteGlu, pteroylglutamate (FA); MTX, methotrexate; 2-dMTX, 2-desamino-MTX; 2-CH3-MTX, 2-desamino-2-methyl-MTX; AMT, aminopterin; 2-dAMT, 2-desamino-AMT; 2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM, 10-ethyl-10-deazaaminopterin; PT523, N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine; DDATHF (raltitrexed), 5,10-dideaza-5,6,7,8,-tetrahydrofolic acid; 5-d(i)H4PteGlu, 5-deaza-5,6,7,8-tetrahydroiso-folic acid; N9-CH3-5-d(i)H4PteGlu, N9-methyl-5-deaza-5,6,7,8-tetrahydroiso-folic acid; 5-dPteHCysA, N alpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, N alpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutyric acid; 5-dPteOrn, N alpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-homocysteic acid; 5-dH4PteAPBA, N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dH4PteOro, N alpha-(5-dideaza-5,6,7,8-tetrahydropteroyl)-L-ornithine; CB3717, N10-prop-2-ynyl-5,8-dideazafolic acid; ICI-198,583, 2-desamino-2-methyl-N10-prop-2-ynyl-5,8-dideazafolic acid; 4-H-ICI-198,583, 4-deoxy-ICI-198,583:4-OCH3-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198;583;Glu-to-Sub-ICI-198,583, 2-aminosuberic acid-ICI-198,583; 7-CH3-ICI-198,583, 7-methyl-ICI-198,583; ZD1694, N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamic acid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89, (S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutamic acid; LY231514, N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidine-5-yl)ethyl)-benzoyl]-L-glutamic acid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ; 2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu, 5-deazaisofolic acid; N9-CH3-5-d(i)PteGlu, N9-methyl-5-deazaisofolic acid; N9-CHO-5-d(i)PteGlu, N9-formyl-5-deazaisofolic acid; AG337, 3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)quinazoline; and AG377, 2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline; or stereoisomers thereof.;

[0097] In some embodiments, the folic acid antagonist is selected from methotrexate, raltitrexed, premetrexed, pemetrexed, lometrexol (LMX; 5,10-dideazatetrahydrofolic acid), cyclopenta[g]quinazoline having a dipeptide ligand, CB3717, CB300945, or stereoisomers thereof such as 6-R,S-BGC945 (ONX-0801), CB300638, and BW1843U89.;

[0098] In some embodiments, the folic acid antagonist is a 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl antifolate. In some embodiments, the folic acid antagonist is a 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl antifolate having a carbon bridge with a length of 1 to 6 carbons (for example, a compound having the structure of formula (I) below, n1 = 1 to 6). In some embodiments, the folic acid antagonist is a 6-substituted thieno[2,3-d]pyrimidine benzoyl antifolate having a bridge with a length of 2 to 8 carbons (for example, a compound having the structure of formula (II), n2 = 7 to 13). In some embodiments, the folic acid antagonist is a 6-substituted pyrrolo[2,3-d]pyrimidine antifolate having a bridge with a length of 2 to 8 carbons and with the benzoyl moiety replaced by thienoyl (for example, a compound having the structure of formula (III), n1 = 1 to 6). In some embodiments, the folic acid antagonist has a structure according to any of formulas (I)-(III), and x = 4, 5, 6, 2 to 10, 4 to 6, or greater than 5.

Chemical formula

[0099] In some embodiments, the folic acid antagonist is selected from the following: indoline ring and modified ornithine-containing methotrexate derivatives, indoline ring and modified glutamate-containing methotrexate derivatives, alkyl-substituted benzene ring C-containing methotrexate derivatives, benzoxazine moiety-containing methotrexate derivatives, benzothiazine moiety-containing methotrexate derivatives, 10-deazaaminopterin analogs, 5-deazaaminopterin methotrexate analogs, 5,10-dideazaaminopterin methotrexate analogs, indoline moiety-containing methotrexate derivatives, lipophilic amide methotrexate derivatives, L-threo-(2S,4S)-4-fluoroglutamic acid-containing methotrexate analogs, DL-3,3-difluoroglutamic acid-containing methotrexate analogs, methotrexate tetrahydroquinazoline analogs, N-(ac-aminoacyl)methotrexate derivatives, biotin methotrexate derivatives, D-glutamic acid methotrexate analogs, D-erythro,threo-4-fluoroglutamic acid methotrexate analogs, β,γ-methano methotrexate analogs, 10-deazaaminopterin (10-EDAM) analogs, γ-tetrazole methotrexate analogs, N-(L-α-aminoacyl)methotrexate derivatives, meta-isomers of aminopterin, ortho-isomers of aminopterin, hydroxymethylmethotrexate, γ-fluoromethotrexate, polyglutamyl methotrexate derivatives, gem-diphosphonate methotrexate analogs (see, e.g., International Application No. 1988 / 06158, the content of which is hereby incorporated by reference in its entirety), α-substituted methotrexate analogs, γ-substituted methotrexate analogs, 5-methyl-5-deazamethotrexate analogs (e.g., U.S. Patent No. 4,725,Reference 687 (the entire contents of which are incorporated herein by reference), Nδ-acyl-Nα-(4-amino-4-deoxypteroyl)-L-ornithine derivatives, 8-deazamethotrexate analogs, asibicine methotrexate analogs, polymer platinumol methotrexate derivatives, methotrexate-γ-dimyristoyl phosphatidylethanolamine, methotrexate polyglutamate analogs, poly-γ-glutamyl methotrexate derivatives, deoxyuridylate methotrexate derivatives, iodoacetyl lysine methotrexate analogs, 2,ω-diaminoalkanoic acid-containing methotrexate analogs, polyglutamate derivatives, 5-methyl-5-deaza analogs, quinazoline methotrexate analogs, pyrazine methotrexate analogs, cysteine or homocysteic acid methotrexate analogs (see, e.g., U.S. Patent No. 4,490,529 and EPA0142220, the entire contents of which are incorporated herein by reference), γ-tert-butyl methotrexate ester, fluorinated methotrexate analogs, folic acid methotrexate analogs, phosphonoglutamic acid analogs, poly(L-lysine) methotrexate complexes, dilysine or trilysine methotrexate derivatives, 7-hydroxymethotrexate, poly-γ-glutamyl methotrexate analogs, 3‘,5’-dichloromethotrexate, diazoketone or chloromethyl ketone methotrexate analogs, 10-propylgylaminopterin, alkyl methotrexate homologs, lectin derivatives of methotrexate, polyglutamate methotrexate derivatives, halogenated methotrexate derivatives, 8-alkyl-7,8-dihydro analogs, 7-methylmethotrexate derivatives, dichloromethotrexate, lipophilic methotrexate derivatives, 3’,5’-dichloromethotrexate, deazaamethopterin analogs, and MX068; or stereoisomers thereof.,

[0100] In some embodiments, the folic acid antagonist is of formula (IV):

Chemical formula

[0101] In some embodiments, the folic acid antagonist has the formula (IV), wherein X = CH2; R1 = Me or Et; R2 = H, Cl, F, OH, or R2 = R3; and R3 = H, Cl, F, OH, Me, or Br. In some embodiments, X = CH2; R1 = Me; R2 = H, Cl, F, OH; and R3 = H, Cl, Me, or Br. In some embodiments, X = O(CH2)3O; R1 = Me; and R2 = R3 = H.

[0102] In some embodiments, the folic acid antagonist has the formula (V):

Chem.

[0103] In some embodiments, the folic acid antagonist has the formula (V), wherein X = C2H4; and R1 = R2 = H or CL. In some embodiments, X = C2H4; R1 = Cl; and R2 = H. In some embodiments, X = C4H8; and R1 = R2 = H. In some embodiments, X = C6H 12 ; and R1 = R2 = H.

[0104] In some embodiments, the folic acid antagonist has the formula (VI):

Chem.

[0105] In some embodiments, the folic acid antagonist has the formula (VI), where X = CH2; Y = 2,5-thiophene; and R = H2F, Cn, Et, or CH2OH. In some embodiments, X = C2H4; Y = 2,5-thiophene; and R = Me.

[0106] In some embodiments, the folic acid antagonist has the formula (VII):

Chemical formula

[0107] In some embodiments, the folic acid antagonist has the formula (VII), wherein (a) X = N; Y = NH2; and R = H; (b) X = N; Y = NH2; and R = CH3; (c) X = N; Y = NH2; and R = CHO; (d) X = CH, Y = NH2, R = H; (e) X = CH, Y = H, R = H; or (f) X = CH, Y = CH3, and R = H.

[0108] In some embodiments, the folic acid antagonist has the formula (VIII):

Chemical formula

[0109] In some embodiments, the folic acid antagonist has the formula (IX):

Chemical formula

[0110] In a further embodiment, the folic acid antagonist is a cyclopenta[g]quinazoline derivative. In some embodiments, the cyclopenta[g]quinazoline derivative is N-{N-{4-[N-(2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamic acid; or N-{N-{4-[N-(2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]-quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamic acid; or a pharmaceutically acceptable salt or ester thereof.

[0111] In some embodiments, the folic acid antagonist has the formula (X):

Chemical formula

[0112] In some embodiments, the folic acid antagonist has formula (X), wherein R1 is C1-4 alkyl or C1-4 hydroxyalkyl (e.g., methyl or hydroxymethyl); R2 is (a) methyl, ethyl, propyl, prop-2-enyl, prop-2-ynyl, 2-hydroxyethyl, 2-fluoroethyl, 2-bromoethyl or 2-cyanoethyl, (b) methyl or (c) prop-2-ynyl; Ar is 1,4-phenylene or 1,4-phenylene having one or two substituents selected from chloro and fluoro (e.g., 2-fluoro substituent, e.g., 2-fluoro-1,4-phenylene or 2,6-difluoro-1,4-phenylene), thiophene-2,5-diyl, thiazole-2,5-diyl or pyridine-2,5-diyl.

[0113] In some embodiments, the folic acid antagonist has formula (X), wherein R1 is methyl or hydroxymethyl; R2 is methyl or prop-2-ynyl; Ar is 1,4-phenylene or 1,4-phenylene having a 2-fluoro substituent such as 2,6-difluoro-1,4-phenylene or in particular 2-fluoro-1,4-phenylene, or pyridine 2,5-diyl. In some embodiments, Ar is 1,4 phenylene or 2-fluoro-1,4 phenylene.

[0114] In other embodiments, the alpha-polyglutamyl oxidized folic acid antagonist is a cyclopenta[g]quinazoline disclosed in International Publication No. WO 2009 / 115776, International Publication No. WO 2003 / 020300, International Publication No. WO 2003 / 020706, International Publication No. WO 2003 / 020748, Gibbs et al., Cancer Research 65(15):11721-11728(2005), and Bavetsias et al., Tetrahedron 63(7):1537-1543(2007). The entire contents of each of these are incorporated herein by reference.

[0115] In some embodiments, the alpha-polyglutamylated folate antagonist is diglutamylated. That is, the alpha-polyglutamylated folate antagonist contains, in addition to the glutamyl group in the folate antagonist, one additional glutamyl group (αANTIFOL-PG1), and the additional glutamyl group is attached to the glutamyl group in the folate antagonist via an alpha bond. In some embodiments, each glutamyl group of the alpha-diglutamylated folate antagonist is of the L-type. In other embodiments, the alpha-diglutamylated folate antagonist contains a D-type glutamyl group.

[0116] In some embodiments, the alpha-polyglutamylated folate antagonist is triglutamylated. That is, the alpha-polyglutamylated folate antagonist contains two additional glutamyl groups in addition to the glutamyl group in the folate antagonist (αANTIFOL-PG2). In some embodiments, each of the two glutamyl groups has an alpha bond. In other embodiments, one of the two additional glutamyl groups has an alpha bond and the other glutamyl group has a gamma bond. In some embodiments, one of the two additional glutamyl groups has an alpha bond. In some embodiments, one of the two additional glutamyl groups has a gamma bond. In some embodiments, two of the three glutamyl groups have an alpha bond. In other embodiments, one of the three glutamyl groups has an alpha bond and another glutamyl group has a gamma bond. In some embodiments, one glutamyl group has both an alpha bond and a gamma bond. In some embodiments, each glutamyl group of the alpha-triglutamylated folate antagonist is of the L-type. In other embodiments, the alpha-triglutamylated folate antagonist contains a D-type glutamyl group. In further embodiments, each glutamyl group of the alpha-triglutamylated folate antagonist other than the glutamyl group of the folate antagonist is of the D-type. In further embodiments, the triglutamylated folate antagonist contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0117] In some embodiments, the alpha-polyglutamylated folate antagonist is tetraglutamylated and thus contains, in addition to the glutamyl groups in the folate antagonist, three additional glutamyl groups (αANTIFOL-PG3). In some embodiments, each of the three glutamyl groups has an alpha bond. In other embodiments, one or two of the three additional glutamyl groups have an alpha bond and the remaining two or one glutamyl groups each have a gamma bond. In some embodiments, two of the three additional glutamyl groups have an alpha bond. In other embodiments, one of the three glutamyl groups has an alpha bond and another additional glutamyl group has a gamma bond. In other embodiments, one of the three additional glutamyl groups has both an alpha bond and a gamma bond. In other embodiments, three of the four glutamyl groups have an alpha bond. In some embodiments, at least one glutamyl group has both an alpha bond and a gamma bond. In some embodiments, the alpha-tetraglutamylated folate antagonist contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha-tetraglutamylated folate antagonist is of the L-type. In other embodiments, the alpha-tetraglutamylated folate antagonist contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha-tetraglutamylated folate antagonist other than the glutamyl groups of the folate antagonist is of the D-type. In a further embodiment, the tetraglutamylated folate antagonist contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0118] In some embodiments, the alpha-pentaglutamylated antifolate contains a chain of four additional glutamyl groups attached to the glutamyl group in the antifolate, where the antifolate is pentaglutamylated (αANTIFOL-PG4). In some embodiments, each of the four additional glutamyl groups in the chain has an alpha bond. In some embodiments, each of the four additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In other embodiments, one, two, or three of the four additional glutamyl groups have an alpha bond, and the remaining three, two, or one glutamyl groups are each attached to the glutamyl group of the molecule via a gamma bond. In other embodiments, one or two of the four additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the five glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the five glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, the alpha-pentaglutamylated antifolate contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha-pentaglutamylated antifolate is of the L-type. In other embodiments, the alpha-pentaglutamylated antifolate contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha-pentaglutamylated antifolate other than the glutamyl group of the antifolate is of the D-type. In a further embodiment, the pentaglutamylated antifolate contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0119] In some embodiments, the alpha polyglutamylated folate antagonist is hexaglutamylated (αANTIFOL-PG5) and contains a chain of five additional glutamyl groups attached to the glutamyl group in the folate antagonist. In some embodiments, each of the five additional glutamyl groups in the chain has an alpha bond. In some embodiments, each of the five additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, four of the five additional glutamyl groups in the chain have an alpha bond. In other embodiments, one, two, three, or four of the five additional glutamyl groups are attached to the glutamyl group of the molecule via an alpha bond, and the remaining four, three, two, or one glutamyl groups are each attached to the glutamyl group of the molecule via a gamma bond. In other embodiments, one, two, three, or four of the five additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the six glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the six glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, five of the six glutamyl groups have an alpha bond. In some embodiments, the alpha hexaglutamylated folate antagonist contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha hexaglutamylated folate antagonist is of the L-type. In other embodiments, the alpha hexaglutamylated folate antagonist contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha hexaglutamylated folate antagonist other than the glutamyl group of the folate antagonist is of the D-type. In a further embodiment, the hexaglutamylated folate antagonist contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0120] In some embodiments, the alpha polyglutamylated folate antagonist is heptaglutamylated (αANTIFOL-PG6) and thus contains a chain of six additional glutamyl groups attached to the glutamyl group in the folate antagonist. In some embodiments, each of the six additional glutamyl groups has an alpha bond. In some embodiments, each of the six additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, five of the six additional glutamyl groups in the chain have an alpha bond. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have an alpha bond and the remaining five, four, three, two, or one glutamyl groups each have a gamma bond. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have an alpha bond and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the seven glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the seven glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, six of the seven glutamyl groups have an alpha bond. In some embodiments, the alpha heptaglutamylated folate antagonist contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha heptaglutamylated folate antagonist is of the L-type. In other embodiments, the alpha heptaglutamylated folate antagonist contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha heptaglutamylated folate antagonist other than the glutamyl group of the folate antagonist is of the D-type. In a further embodiment, the heptaglutamylated folate antagonist contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0121] In some embodiments, the alpha-polyglutamylated antifolate is octaglutamylated (αANTIFOL-PG7) and thus contains a chain of seven additional glutamyl groups attached to the glutamyl group in the antifolate. In some embodiments, each of the seven additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, six of the seven additional glutamyl groups in the chain have an alpha bond. In some embodiments, each of the seven additional glutamyl groups has an alpha bond. In other embodiments, one, two, three, four, five, or six of the seven additional glutamyl groups have an alpha bond and the remaining six, five, four, three, two, or one glutamyl group has a gamma bond, respectively. In other embodiments, one, two, three, four, five, or six of the seven additional glutamyl groups have an alpha bond and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the eight glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the eight glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, seven of the eight glutamyl groups have an alpha bond. In some embodiments, the alpha-octaglutamylated antifolate contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha-octaglutamylated antifolate is of the L-type. In other embodiments, the alpha-octaglutamylated antifolate contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha-octaglutamylated antifolate other than the glutamyl group of the antifolate is of the D-type. In a further embodiment, the octaglutamylated antifolate contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0122] In some embodiments, the alpha polyglutamylated folate antagonist is nona-glutamylated (αANTIFOL-PG8) and contains a chain of 8 additional glutamyl groups attached to the glutamyl group in the folate antagonist. In some embodiments, each of the 8 additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha bond. In some embodiments, 7 of the 8 additional glutamyl groups in the chain have an alpha bond. In some embodiments, each of the 8 additional glutamyl groups has an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, or 7 of the 8 additional glutamyl groups have an alpha bond, and the remaining 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, or 7 of the 8 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 9 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 9 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 8 of the 9 glutamyl groups have an alpha bond. In some embodiments, the alpha nona-glutamylated folate antagonist contains 2 or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha nona-glutamylated folate antagonist is of the L-type. In other embodiments, the alpha nona-glutamylated folate antagonist contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha nona-glutamylated folate antagonist other than the glutamyl group of the folate antagonist is of the D-type. In a further embodiment, the nona-glutamylated folate antagonist contains a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0123] In some embodiments, the alpha polyglutamylated antifolate is decaglutamylated (αANTIFOL-PG9) (i.e., it contains a chain of 9 additional glutamyl groups attached to the glutamyl group in the antifolate). In some embodiments, each of the 9 additional glutamyl groups has an alpha bond. In some embodiments, each of the 9 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 8 of the 9 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 of the 9 additional glutamyl groups have an alpha bond, and the remaining 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 of the 9 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 10 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 10 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 9 of the 10 glutamyl groups have an alpha bond. In some embodiments, the alpha decaglutamylated antifolate contains 2 or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha decaglutamylated antifolate is of the L-type. In other embodiments, the alpha decaglutamylated antifolate contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha decaglutamylated antifolate other than the glutamyl group of the antifolate is of the D-type. In a further embodiment, the decaglutamylated antifolate contains a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0124] In some embodiments, the alpha polyglutamylated antifolate is undecaglutamylated (αANTIFOL-PG10). In some embodiments, each of the 10 additional glutamyl groups has an alpha bond. In some embodiments, each of the 10 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 9 of the 10 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an alpha bond and the remaining 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an alpha bond and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 11 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 11 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 10 of the 11 glutamyl groups have an alpha bond. In some embodiments, the alpha undecaglutamylated antifolate contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha undecaglutamylated antifolate is of the L-type. In other embodiments, the alpha undecaglutamylated antifolate contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha undecaglutamylated antifolate other than the glutamyl groups of the antifolate is of the D-type. In a further embodiment, the undecaglutamylated antifolate contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0125] In some embodiments, the alpha polyglutamylated antifolate is dodecaglutamylated (αANTIFOL-PG11). In some embodiments, each of the 11 additional glutamyl groups has an alpha bond. In some embodiments, each of the 11 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 10 of the 11 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alpha bond, and the remaining 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group(s) each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 12 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 12 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 11 of the 12 glutamyl groups have an alpha bond. In some embodiments, the alpha dodecaglutamylated antifolate contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha dodecaglutamylated antifolate is of the L-type. In other embodiments, the alpha dodecaglutamylated antifolate contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha dodecaglutamylated antifolate other than the glutamyl group of the antifolate is of the D-type. In a further embodiment, the dodecaglutamylated antifolate contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0126] In some embodiments, the alpha-polyglutamylated antifolate is tridecaglutamylated (αANTIFOL-PG12). In some embodiments, each of the 12 additional glutamyl groups has an alpha bond. In some embodiments, each of the 12 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 11 of the 12 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alpha bond, and the remaining 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group has a gamma bond, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 13 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 13 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 12 of the 13 glutamyl groups have an alpha bond. In some embodiments, the alpha-tridecaglutamylated antifolate contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha-tridecaglutamylated antifolate is of the L-type. In other embodiments, the alpha-tridecaglutamylated antifolate contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha-tridecaglutamylated antifolate other than the glutamyl groups of the antifolate contains a D-type glutamyl group. In a further embodiment, the tridecaglutamylated antifolate contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0127] In some embodiments, the alpha polyglutamylated antifolate is tetradecaglutamylated (αANTIFOL-PG13). In some embodiments, each of the 13 additional glutamyl groups has an alpha bond. In some embodiments, each of the 13 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 12 of the 13 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have an alpha bond, and the remaining 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 14 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 14 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 13 of the 14 glutamyl groups have an alpha bond. In some embodiments, the alpha tetradecaglutamylated antifolate contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha tetradecaglutamylated antifolate is of the L-type. In other embodiments, the alpha tetradecaglutamylated antifolate contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha tetradecaglutamylated antifolate other than the glutamyl group of the antifolate contains a D-type. In a further embodiment, the tetradecaglutamylated antifolate contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear.In other embodiments, the polyglutamate chain is a branched chain.

[0128] In some embodiments, the alpha polyglutamylated antifolate is pentadeca-glutamylated (αANTIFOL-PG14). In some embodiments, each of the 14 additional glutamyl groups has an alpha bond. In some embodiments, each of the 14 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 13 of the 14 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14 additional glutamyl groups have an alpha bond, and the remaining 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group has a gamma bond, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 15 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 15 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 14 of the 15 glutamyl groups have an alpha bond. In some embodiments, the alpha pentadeca-glutamylated antifolate contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha pentadeca-glutamylated antifolate is of the L-type. In other embodiments, the alpha pentadeca-glutamylated antifolate contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha pentadeca-glutamylated antifolate other than the glutamyl group of the antifolate contains a D-type glutamyl group. In a further embodiment, the pentadeca-glutamylated antifolate contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear.In other embodiments, the polyglutamate chain is a branched chain.

[0129] In some embodiments, the alpha-polyglytamylated antifolate is hexadeca-glytamylated (αANTIFOL-PG15). In some embodiments, each of the 15 additional glutamyl groups has an alpha bond. In some embodiments, each of the 15 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 14 of the 15 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15 additional glutamyl groups have an alpha bond, and the remaining 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 16 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 16 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 15 of the 16 glutamyl groups have an alpha bond. In some embodiments, the alpha-hexadeca-glytamylated antifolate contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha-hexadeca-glytamylated antifolate is of the L-type. In other embodiments, the alpha-hexadeca-glytamylated antifolate contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha-hexadeca-glytamylated antifolate other than the glutamyl groups of the antifolate is of the D-type. In a further embodiment, the hexadeca-glytamylated antifolate contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear.In other embodiments, the polyglutamate chain is a branched chain.

[0130] In other embodiments, the alpha polyglutamylated folate antagonist is heptadeca-glutamylated (αANTIFOL-PG16). In some embodiments, each of the 16 additional glutamyl groups has an alpha bond. In some embodiments, each of the 16 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 15 of the 16 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the 16 additional glutamyl groups have an alpha bond, and the remaining 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each has a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the 16 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 17 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 17 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 16 of the 17 glutamyl groups have an alpha bond. In some embodiments, the alpha heptadeca-glutamylated folate antagonist comprises two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha heptadeca-glutamylated folate antagonist is of the L-type. In other embodiments, the alpha heptadeca-glutamylated folate antagonist comprises a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha heptadeca-glutamylated folate antagonist other than the glutamyl group of the folate antagonist is of the D-type. In a further embodiment, the heptadeca-glutamylated folate antagonist comprises a D-type glutamyl group and two or more L-type glutamyl groups.In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0131] In some embodiments, the alpha polyglutamylated antifolate is octadeca-glutamylated (αANTIFOL-PG17). In some embodiments, each of the 17 additional glutamyl groups has an alpha bond. In some embodiments, each of the 17 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 16 of the 17 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the 17 additional glutamyl groups have an alpha bond, and the remaining 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the 17 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 18 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 18 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 17 of the 18 glutamyl groups have an alpha bond. In some embodiments, the alpha octadeca-glutamylated antifolate contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha octadeca-glutamylated antifolate is of the L-type. In other embodiments, the alpha octadeca-glutamylated antifolate contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha octadeca-glutamylated antifolate other than the glutamyl group of the antifolate contains a D-type. In a further embodiment, the octadeca-glutamylated antifolate contains a D-type glutamyl group and two or more L-type glutamyl groups.In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0132] In some embodiments, the alpha polyglutamylated antifolate is nonadeca-glutamylated (αANTIFOL-PG18). In some embodiments, each of the 18 additional glutamyl groups has an alpha bond. In some embodiments, each of the 18 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 17 of the 18 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have an alpha bond, and the remaining 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group(s) each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 19 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 19 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 18 of the 19 glutamyl groups have an alpha bond. In some embodiments, the alpha nonadeca-glutamylated antifolate contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of the alpha nonadeca-glutamylated antifolate is of the L-type. In other embodiments, the alpha nonadeca-glutamylated antifolate contains a D-type glutamyl group. In further embodiments, each glutamyl group of the alpha nonadeca-glutamylated antifolate other than the glutamyl group of the antifolate is of the D-type. In further embodiments, the nonadeca-glutamylated antifolate contains a D-type glutamyl group and two or more L-type glutamyl groups.In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0133] In some embodiments, the alpha polyglutamyl oxidized folic acid antagonist is eicosaglutamin oxidized (αANTIFOL-PG19). In some embodiments, each of the 19 additional glutamyl groups has an alpha bond. In some embodiments, each of the 19 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 18 of the 19 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups have an alpha bond, and the remaining 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each has a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 20 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 20 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 19 of the 20 glutamyl groups have an alpha bond. In some embodiments, the alpha eicosaglutamin oxidized folic acid antagonist contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the alpha eicosaglutamin oxidized folic acid antagonist is of the L-type. In other embodiments, the alpha eicosaglutamin oxidized folic acid antagonist contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the alpha eicosaglutamin oxidized folic acid antagonist other than the glutamyl groups of the folic acid antagonist is of the D-type. In a further embodiment, the eicosaglutamin oxidized folic acid antagonist contains a D-type glutamyl group and two or more L-type glutamyl groups.In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0134] In some embodiments, the alpha polyglutamylated antifolate is henicosaglutamylated (αANTIFOL-PG20). In some embodiments, each of the 20 additional glutamyl groups has an alpha bond. In some embodiments, each of the 20 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 19 of the 20 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have an alpha bond, and the remaining 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each has a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 21 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 21 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 20 of the 21 glutamyl groups have an alpha bond. In some embodiments, the alpha henicosaglutamylated antifolate contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of the alpha henicosaglutamylated antifolate is of the L-type. In other embodiments, the alpha henicosaglutamylated antifolate contains a D-type glutamyl group. In further embodiments, each glutamyl group of the alpha henicosaglutamylated antifolate other than the glutamyl group of the antifolate is of the D-type.In a further embodiment, the henicosaglutamine oxidized folic acid antagonist contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0135] In some embodiments, the alpha polyglutamine oxidized folic acid antagonist contains 4 to 7 glutamyl groups attached to the folic acid antagonist (i.e., αANTIFOL-PGn, n = 4 to 7), and each of the 4 to 7 attached glutamyl groups has an alpha bond. In some embodiments, the alpha polyglutamine oxidized folic acid antagonist contains 4 to 7 glutamyl groups attached to the folic acid antagonist (i.e., αANTIFOL-PGn, n = 4 to 7), and each of the 4 to 7 attached glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, each of the 4 to 7 attached glutamyl groups is of the L-type. In other embodiments, each of the 4 to 7 attached glutamyl groups is of the D-type. In other embodiments, the 4 to 7 attached glutamyl groups are of the L-type and D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0136] In some embodiments, the alpha-polyglutamyl methotrexate (αPANTIFOL) contains a total of 1 to 15, 1 to 10, 2 to 15, 2 to 10, 3 to 15, 3 to 10, 3 to 6, 3 to 5, 4 to 10, 4 to 7, or 4 to 6 glutamyl groups, including the glutamyl group of the methotrexate. In some embodiments, each glutamyl group in αPANTIFOL other than the glutamyl group of the methotrexate has an alpha bond. In some embodiments, the C-terminal glutamyl group(s) and each glutamyl group in αPANTIFOL other than the glutamyl group of the methotrexate have an alpha bond. In some embodiments, each glutamyl group in αPANTIFOL other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPANTIFOL have an alpha bond. In some embodiments, αPANTIFOL contains L-type and D-type glutamyl groups. In a further embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPANTIFOL have an alpha bond, and 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 of the glutamyl groups each have a gamma bond. In some embodiments, each glutamyl group in the polyglutamate structure of the polyglutamyl methotrexate is of the L-type. In some embodiments, each glutamyl group in αPANTIFOL other than the glutamyl group of the methotrexate is of the D-type. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the glutamyl groups in αPANTIFOL are of the L-type. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPANTIFOL are of the D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0137] In some embodiments, the alpha-polyglytamyl antifolate (αPANTIFOL) contains a total of 2 to 20, 2 to 15, 2 to 10, 2 to 5, or any range in between glutamyl groups, including the glutamyl group of the antifolate. In some embodiments, each glutamyl group in αPANTIFOL other than the glutamyl group of the antifolate has an alpha bond. In some embodiments, the C-terminal glutamyl group(s) and each glutamyl group in αPANTIFOL other than the glutamyl group of the antifolate have an alpha bond. In some embodiments, each glutamyl group in αPANTIFOL other than the C-terminal glutamyl group(s) has an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups have an alpha bond. In some embodiments, αPANTIFOL contains two or more glutamyl groups having a gamma bond. In a further embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups in αPANTIFOL other than the glutamyl group of the antifolate. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups have an alpha bond. In a further embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups in αPANTIFOL other than the glutamyl group of the antifolate have an alpha bond, and 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 glutamyl groups each have a gamma bond. In some embodiments, each glutamyl group in αPANTIFOL is of the L-type. In some embodiments, each glutamyl group in αPANTIFOL other than the glutamyl group of the antifolate is of the D-type.In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the glutamyl groups in αPANTIFOL are of the L-type. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the glutamyl groups in αPANTIFOL are of the D-type.

[0138] In some embodiments, the alpha polyglutamylated folic acid antagonist includes a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in addition to the glutamyl groups of the folic acid antagonist. In further embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additional glutamyl groups have an alpha bond. In further embodiments, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the glutamyl groups in the alpha polyglutamylated folic acid antagonist have a gamma bond. In some embodiments, at least one glutamyl group has both an alpha bond and a gamma bond. In some embodiments, the glutamyl groups in the folic acid antagonist have an alpha bond. In some embodiments, the glutamyl groups in the folic acid antagonist have both an alpha bond and a gamma bond.

[0139] In some embodiments, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in an alpha-polyglutamylated folic acid antagonist are of the L-form, D-form, or a combination of L-form and D-form. In some embodiments, each glutamyl group of an alpha-polyglutamylated folic acid antagonist is of the L-form. In other embodiments, each glutamyl group of an alpha-polyglutamylated folic acid antagonist other than the glutamyl group of the folic acid antagonist is of the D-form. In alternative embodiments, at least two of the glutamyl groups of an alpha-polyglutamylated folic acid antagonist are of the L-form and at least one of the glutamyl groups in the alpha-polyglutamylated folic acid antagonist is of the D-form. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 glutamyl groups in an alpha-polyglutamylated folic acid antagonist are of the L-form. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 glutamyl groups in an alpha-polyglutamylated folic acid antagonist are of the D-form.

[0140] In further embodiments, an alpha-polyglutamylated folic acid antagonist contains 20 to 100, 20 to 75, 20 to 50, 20 to 40, 20 to 30, 20 to 25, or more than 100 glutamyl groups, or any range therebetween. In some embodiments, each glutamyl group of an alpha-polyglutamylated folic acid antagonist is of the L-form. In other embodiments, each glutamyl group of an alpha-polyglutamylated folic acid antagonist other than the glutamyl group of the folic acid antagonist is of the D-form. In alternative embodiments, at least two of the glutamyl groups of an alpha-polyglutamylated folic acid antagonist are of the L-form and at least one of the glutamyl groups in the alpha-polyglutamylated folic acid antagonist is of the D-form.

[0141] In a further embodiment, the provided composition comprises an alpha-polyglutamyl oxidized folic acid antagonist comprising a glutamyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 alpha linkages. In some embodiments, the alpha-polyglutamyl oxidized folic acid antagonist comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 L-type glutamyl groups. In some embodiments, the alpha-polyglutamyl oxidized folic acid antagonist comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 D-type glutamyl groups. In some embodiments, the alpha-polyglutamyl oxidized folic acid antagonist comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 L-type glutamyl groups and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 D-type glutamyl groups.

[0142] In other embodiments, the alpha-polyglutamyl oxidized folic acid antagonist comprises at least one glutamyl group having both alpha and gamma linkages. In some embodiments, the alpha-polyglutamyl oxidized folic acid antagonist comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or more than 10 glutamyl groups having both alpha and gamma linkages.

[0143] In some embodiments, the alpha-polyglytamylated folate antimetabolite comprises at least 1 glutamyl group having an alpha bond and comprises 2, 3, 4, 5, 6, 7, 8, 9, 1-10, 1-20, or more glutamyl groups having a gamma bond. For example, in some embodiments, the alpha-polyglytamylated folate antimetabolite comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10 L-alpha-glutamyl group bonds and further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 L-gamma-glutamyl group bonds. In some further embodiments, the alpha-polyglytamylated folate antimetabolite comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 L-alpha-glutamyl group bonds and further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma-glutamyl group bonds. In additional further embodiments, the alpha-polyglytamylated folate antimetabolite comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-alpha-glutamyl group bonds and further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma-glutamyl group bonds. In additional further embodiments, the alpha-polyglytamylated folate antimetabolite comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-alpha-glutamyl group bonds and further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma-glutamyl group bonds. In other further embodiments, the alpha-polyglytamylated folate antimetabolite comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma-glutamyl group bonds and further comprises 1, 2, 3, 4, 5, 6, or 1-10 L-gamma-glutamyl group bonds. In other embodiments, the alpha-polyglytamylated folate antimetabolite comprises at least 1 glutamyl group having both an alpha bond and a gamma bond. In some embodiments, the alpha-polyglytamylated folate antimetabolite comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or more than 10 glutamyl groups having both an alpha bond and a gamma bond.

[0144] In some embodiments, the alpha-polyglutamylated folate antimetabolite compositions provided herein can accept one or more additional glutamyl groups, i.e., the compositions can serve as substrates for FPGS (folylpolyglutamate synthetase). Reagents and assays for measuring the ability of an alpha-polyglutamylated folate antimetabolite composition to act as a substrate for FPGS (e.g., human FPGS, or rat liver FPGS) are readily available and can be performed as routine business.

[0145] In some embodiments, the rate of uptake of the naked alphaPANTIFOL compositions disclosed herein (e.g., alphaPANTIFOL not conjugated to a delivery carrier) by liver cells is significantly reduced compared to the rate of uptake of folate antimetabolites under physiological conditions. In some embodiments, the rate of liver cell uptake of the naked alphaPANTIFOL composition is less than 30%, 20%, 15%, or 10% compared to the rate of folate antimetabolites. In further embodiments, the rate of efflux (transport) of the alphaPANTIFOL compositions disclosed herein from liver cells occurs at a significantly slower rate (less than 30%, 20%, 15%, or 10%) compared to folate antimetabolites.

[0146] In some embodiments, the alpha-polyglutamylated folate antimetabolite compositions provided herein have higher cytotoxicity against proliferating cells than folate antimetabolites. In some embodiments, the proliferating cells are cancer cells. In some embodiments, the proliferating cells are colorectal cancer cells, colon cancer cells, breast cancer cells, or ovarian cancer cells. In some embodiments, the cancer cells are mesothelioma cells or non-small cell lung cancer cells. In some embodiments, the cytotoxicity is measured in an in vitro assay. In some embodiments, the alpha-polyglutamylated folate antimetabolite is a hexaglutamylated folate antimetabolite.

[0147] In some embodiments, the alpha-polyglutamylated folate antimetabolite compositions provided herein have lower toxic side effects than folate antimetabolites. In some embodiments, the alpha-polyglutamylated folate antimetabolite compositions provided herein are less toxic to non-proliferating cells than folate antimetabolites. In some embodiments, the alpha-polyglutamylated folate antimetabolite compositions provided herein are less toxic to neutrophils, liver cells, or colon epithelial cells than folate antimetabolites. In some embodiments, the neutrophils are human neutrophils, differentiated human neutrophils, or neutrophils differentiated from CD34+ cells. In some embodiments, the liver cells are AML12 liver cells. In some embodiments, the colon epithelial cells are CCD841 colon epithelial cells. In some embodiments, the toxicity is measured in an in vitro assay. In some embodiments, the alpha-polyglutamylated folate antimetabolite is a hexaglutamylated folate antimetabolite.

[0148] In some embodiments, the alpha-polyglutamylated folate antimetabolite compositions provided herein have lower toxic side effects than folate antimetabolites. In some embodiments, the alpha-polyglutamylated folate antimetabolite compositions provided herein result in fewer or less severe toxic side effects than folate antimetabolites in in vivo assays. In some embodiments, the in vivo assay is performed in an in vivo mouse model. In some embodiments, the alpha-polyglutamylated folate antimetabolite compositions provided herein result in fewer or less severe hematological or liver toxic side effects than folate antimetabolites. In some embodiments, the hematological side effects are evaluated by mean neutrophils, mean white blood cells, or mean platelet counts. In some embodiments, the liver toxic side effects are evaluated by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the in vivo assay comprises administering the alpha-polyglutamylated folate antimetabolite composition at 40 mg / kg or 80 mg / kg once a week for 4 weeks. In some embodiments, the alpha-polyglutamylated folate antimetabolite is a hexaglutamylated folate antimetabolite.

[0149] In some embodiments, treatment with the alpha-polyglutamylated folate antagonist compositions provided herein does not induce significant hematological or liver toxic side effects in an in vivo mouse model. In some embodiments, hematological side effects are evaluated by mean neutrophils, mean white blood cells, or mean platelet counts. In some embodiments, liver toxic side effects are evaluated by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the alpha-polyglutamylated folate antagonist compositions provided herein do not significantly decrease mean neutrophils, mean white blood cells, or mean platelet counts. In some embodiments, the alpha-polyglutamylated folate antagonist compositions provided herein do not significantly increase serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. In some embodiments, the alpha-polyglutamylated folate antagonist compositions provided herein do not significantly reduce serum albumin levels. In some embodiments, the in vivo assay comprises administering the alpha-polyglutamylated folate antagonist composition at 40 mg / kg or 80 mg / kg once a week for 4 weeks. In some embodiments, the alpha-polyglutamylated folate antagonist is a hexaglutamylated folate antagonist.

[0150] In some embodiments, the alpha-polyglutamylated folate antagonist composition does not contain a fluorine atom. In some embodiments, the alpha-polyglutamylated folate antagonist composition does not contain a 4-fluoroglutamyl group.

[0151] The compositions of alpha-polyglutamyl oxidized folic acid antagonists (αPANTIFOL) and their uses are further described in International Application No. PCT / US2017 / 046666, International Application No. PCT / US2017 / 046667, and U.S. Patent Application Nos. 62 / 630,820, 62 / 627,716, 62 / 627,731, 62 / 630,671, 62 / 630,825, 62 / 630,629, 62 / 630,634, 62 / 630,728, 62 / 630,637, 62 / 630,744, 62 / 583,432, 62 / 627,714, 62 / 627,741, and 62 / 627,703, respectively. The disclosures of each of these are hereby incorporated by reference in their entirety.

[0152] A. Alpha-polyglutamyl oxidized folic acid antagonist analogs and derivatives The present disclosure also encompasses alpha-polyglutamyl oxidized folic acid antagonist derivatives and analogs. The compositions and methods disclosed herein are contemplated for application to any and all derivatives or analogs of known polyglutamylated folic acid antagonists. In some embodiments, the analogs correspond to modified forms of the folic acid antagonists, in which case the glutamyl groups of the folic acid antagonists are not attached to the remainder of the folic acid antagonist molecule via gamma peptide bonds. In some embodiments, the analogs are variants of the folic acid antagonists, in which case the glutamyl groups in the folic acid antagonists are of the D form. In some embodiments, the polyglutamylated form of the folic acid antagonist, or the polyglutamyl oxidized folic acid antagonist analog or derivative, is not fluorinated.

[0153] In some embodiments, the folic acid antagonist is selected from the following: indoline ring and modified ornithine-containing methotrexate derivatives, indoline ring and modified glutamate-containing methotrexate derivatives, alkyl-substituted benzene ring C-containing methotrexate derivatives, benzoxazine moiety-containing methotrexate derivatives, benzothiazine moiety-containing methotrexate derivatives, 10-deazaaminopterin analogs, 5-deazaaminopterin methotrexate analogs, 5,10-dideazaaminopterin methotrexate analogs, indoline moiety-containing methotrexate derivatives, lipophilic amide methotrexate derivatives, L-threo-(2S,4S)-4-fluoroglutamic acid-containing methotrexate analogs, DL-3,3-difluoroglutamic acid-containing methotrexate analogs, methotrexate tetrahydroquinazoline analogs, N-(ac-aminoacyl)methotrexate derivatives, biotin methotrexate derivatives, D-glutamic acid methotrexate analogs, D-erythro,threo-4-fluoroglutamic acid methotrexate analogs, β,γ-methano methotrexate analogs, 10-deazaaminopterin (10-EDAM) analogs, γ-tetrazole methotrexate analogs, N-(L-α-aminoacyl)methotrexate derivatives, meta-isomers of aminopterin, ortho-isomers of aminopterin, hydroxymethylmethotrexate, γ-fluoromethotrexate, polyglutamyl methotrexate derivatives, gem-diphosphonate methotrexate analogs (see, for example, International Application No. 1988 / 06158, the contents of which are hereby incorporated by reference in their entirety), α-substituted methotrexate analogs, γ-substituted methotrexate analogs, 5-methyl-5-deazamethotrexate analogs (see, for example, U.S. Patent No. 4,725,Reference 687 (the entire contents of which are incorporated herein by reference), Nδ-acyl-Nα-(4-amino-4-deoxypteroyl)-L-ornithine derivatives, 8-deazamethotrexate analogs, asibicine methotrexate analogs, polymeric platinumol methotrexate derivatives, methotrexate-γ-dimyristoyl phosphatidylethanolamine, methotrexate polyglutamate analogs, poly-γ-glutamyl methotrexate derivatives, deoxyuridylate methotrexate derivatives, iodoacetyl lysine methotrexate analogs, 2,ω-diaminoalkanoic acid-containing methotrexate analogs, polyglutamate derivatives, 5-methyl-5-deaza analogs, quinazoline methotrexate analogs, pyrazine methotrexate analogs, cysteine or homocysteic acid methotrexate analogs (see, e.g., U.S. Patent No. 4,490,529 and EPA0142220, the entire contents of which are incorporated herein by reference), γ-tert-butyl methotrexate ester, fluorinated methotrexate analogs, folic acid methotrexate analogs, phosphonoglutamic acid analogs, poly(L-lysine) methotrexate complexes, dilysine or trilysin methotrexate derivatives, 7-hydroxymethotrexate, poly-γ-glutamyl methotrexate analogs, 3‘,5’-dichloromethotrexate, diazoketone or chloromethyl ketone methotrexate analogs, 10-propylgylaminopterin, alkyl methotrexate homologs, lectin derivatives of methotrexate, polyglutamate methotrexate derivatives, halogenated methotrexate derivatives, 8-alkyl-7,8-dihydro analogs, 7-methylmethotrexate derivatives, dichloromethotrexate, lipophilic methotrexate derivatives, 3‘,5’-dichloromethotrexate, deazaamethopterin analogs, and MX068, or stereoisomers thereof.,

[0154] In further embodiments, the alpha-polyglytamylated antifolate derivative or analog has a variant polyglutamate chain. In some embodiments, the polyglutamate chain comprises one or more natural or synthetic residues other than glutamate. In some embodiments, the polyglutamate chain comprises one or more glutamyl groups that do not contain an amide bond. In other embodiments, one or more glutamyl groups of the polyglutamate chain are derivatized.

[0155] B. αANTIFOL-PG Synthesis The antifolate polyglutamate compositions provided herein are obtained by the following synthetic methods known in the art. Procedures for synthesizing antifolates (including different pharmaceutically acceptable salts or acids (e.g., antifolate disodium) and crystalline and amorphous forms) and intermediates for synthesizing antifolates include, but are not limited to, U.S. Patent Nos. 2,512,572; 3,892,801; 3,989,703; 4,057,548; 4,067,867; 4,079,056; 4,080,325; 4,106,488; 4,136,101; 4,224,446; 4,306,064; 4,374,987; 4,421,913; 4,558,690; 4,662,359; and 4,767,859; and those described in Calvert, Semin. Oncol. 26:3-10 (1999).

[0156] The folic acid antagonist polyglutamate compositions provided herein are obtained by the following synthetic methods using available reagents and synthetic intermediates. The addition of glutamyl residues to the glutamyl residues of folic acid antagonists can be carried out using synthetic methods known in the art. In some embodiments, the glutamyl residues are sequentially added to the glutamyl residues of the folic acid antagonist. In further embodiments, the polyglutamate is added to the glutamyl residue of the folic acid antagonist using "click chemistry" methods or other bioconjugate chemistries known to those skilled in the art. Alternatively, a peptide of glutamyl residues of the desired length can be generated and added to a precursor without a glutamyl residue of the folic acid antagonist. The peptide can be prepared using methods known in the art. In some embodiments, the initial glutamyl residue is attached to Wang resin, and additional glutamyl residues are sequentially added by solid-phase peptide synthesis using F-moc chemistry. After the last glutamyl residue is added, the folic acid antagonist precursor is attached to the peptide and the molecule is cleaved from the resin.

[0157] C. Alpha Polyglutamine Oxidized Folic Acid Antagonist Complex Unexpectedly, the inventors have found that polyglutaminated folic acid antagonists such as polyglutaminated pemetrexed can form complexes with other compositions, including therapeutic agents containing cytotoxic compounds such as platinum-based compounds. Accordingly, in some embodiments, the present disclosure provides a complex of αPANTIFOL (e.g., αPANTIFOL disclosed herein) with a therapeutic agent or a salt or acid thereof. In some embodiments, the polyglutaminated folic acid antagonist is αPANTIFOL as described in Section II, or a salt or acid thereof. In some embodiments, the present disclosure provides a complex of αPANTIFOL as described in any one of items [1] to

[12] of the mode for carrying out the invention with a therapeutic agent or a salt or acid thereof. In some embodiments, the αPANTIFOL / complex comprises αPANTIFOL and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound such as a chemotherapeutic agent. In a further embodiment, the αPANTIFOL / complex comprises a platinum-based drug such as a platinum-based chemotherapeutic agent (e.g., cisplatin, carboplatin, and oxaliplatin). In other embodiments, the αPANTIFOL / complex comprises a taxane-based chemotherapeutic agent (e.g., paclitaxel and docetaxel). In other embodiments, the αPANTIFOL / complex comprises cyclodextrin. In a further embodiment, the αPANTIFOL / complex is encapsulated in liposomes. In some embodiments, the liposomes are Lp-αPANTIFOL as described in any one of items

[13] to

[72] of the mode for carrying out the invention.

[0158] In a further embodiment, the αPANTIFOL / therapeutic agent complex comprises one or more αPANTIFOLs comprising 2 to 150, 2 to 100, 2 to 75, 2 to 50, 2 to 24, 2 to 30, 2 to 20, 2 to 19, 2 to 15, 2 to 10, or 2 to 5 glutamyl groups. In some embodiments, the αPANTIFOL / therapeutic agent complex comprises one or more αPANTIFOLs comprising 3 to 10, 3 to 9, 3 to 8, or 3 to 7 glutamyl groups. In other embodiments, the αPANTIFOL / therapeutic agent complex comprises one or more αPANTIFOLs comprising 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 glutamyl groups. In a particular embodiment, the complex comprises one or more αPANTIFOLs comprising 3 to 10 glutamyl groups. In a further embodiment, the αPANTIFOL / therapeutic agent complex comprises one or more αPANTIFOLs comprising 3 to 7 glutamyl groups. In a further embodiment, the αPANTIFOL / therapeutic agent complex comprises one or more αPANTIFOLs comprising 5 glutamyl groups. In another embodiment, the αPANTIFOL / therapeutic agent complex comprises one or more αPANTIFOLs comprising 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 chemotherapeutic agent is a platinum-based drug. In another embodiment, the chemotherapeutic agent is a taxane-based drug. In a further embodiment, the molar ratio of αPANTIFOL / therapeutic agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of αPANTIFOL / therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 10:1. In some embodiments, the αPANTIFOL / therapeutic agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).In some embodiments, the molar ratio of αPANTIFOL / therapeutic agent in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In some embodiments, the molar ratio of αPANTIFOL / therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In some embodiments, the αPANTIFOL / therapeutic agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art). In some embodiments, the liposome is Lp-αPANTIFOL described in any one of items

[13] to

[72] of the form for carrying out the invention.

[0159] In alternative embodiments, the αPANTIFOL complex comprises αPANTIFOL and cyclodextrin. In some embodiments, the αPANTIFOL complex comprises αPANTIFOL as described in any one of items [1] to

[12] of the form for carrying out the invention. In some embodiments, the αPANTIFOL complex comprises a folic acid antagonist as described in Section II. In some embodiments, the molar ratio of αPANTIFOL (e.g., αPANTIFOL salt) / cyclodextrin in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPANTIFOL / cyclodextrin in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of αPANTIFOL / cyclodextrin in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPANTIFOL / cyclodextrin in the complex is 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, or 20:1. In some embodiments, the molar ratio of αPANTIFOL / 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 to 50):1, or >50:1. In other embodiments, the molar ratio of αPANTIFOL / cyclodextrin in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of αPANTIFOL / cyclodextrin in the complex is 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, or 1:20.In some embodiments, the molar ratio of αPANTIFOL / cyclodextrin in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In some embodiments, the αPANTIFOL / cyclodextrin complex is encapsulated in liposomes. In some embodiments, the liposomes are Lp-αPANTIFOL as described in any one of items

[13] to

[72] of the form for carrying out the invention.

[0160] In some embodiments, the present disclosure provides a composition comprising an αPANTIFOL / platinum-based chemotherapeutic agent complex. In some embodiments, the complex comprises αPANTIFOL as described in any one of items [1] to

[12] of the form for carrying out the invention. In some embodiments, the αPANTIFOL complex comprises the polyglutaminated folic acid antagonist described in Section II. In some embodiments, the platinum-based chemotherapeutic agent is selected from cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the αPANTIFOL / platinum-based chemotherapeutic agent complex comprises cisplatin, carboplatin, an analogue of oxaliplatin, or salts or acids thereof. In some embodiments, the molar ratio of αPANTIFOL / platinum-based agent in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPANTIFOL / platinum-based agent in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of αPANTIFOL / platinum-based agent in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPANTIFOL / platinum-based agent in the complex is 11:1, 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, or 20:1. In some embodiments, the molar ratio of αPANTIFOL / platinum-based agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of αPANTIFOL / platinum-based chemotherapeutic agent in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween.In some embodiments, the molar ratio of αPANTIFOL / platinum-based agent in the complex is 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, or 1:20. In some embodiments, the molar ratio of αPANTIFOL / platinum-based agent in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In some embodiments, the αPANTIFOL / platinum-based agent complex is encapsulated in liposomes. In some embodiments, the liposome is Lp-αPANTIFOL described in any one of items

[13] to

[72] of the form for carrying out the invention.

[0161] In further embodiments, the αPANTIFOL / platinum-based chemotherapeutic agent complex comprises cisplatin, carboplatin, an oxaliplatin analog, or a salt or acid thereof. In some embodiments, the complex comprises αPANTIFOL as described in any one of items [1] to

[12] of the form for carrying out the invention. In some embodiments, the αPANTIFOL complex comprises the polyglutamine oxidized folic acid antagonist described in Section II. In some embodiments, the molar ratio of αPANTIFOL / platinum-based analog in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPANTIFOL / platinum-based analog in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of αPANTIFOL / platinum-based drug in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPANTIFOL / platinum-based analog in the complex is 11:1, 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, or 20:1. In some embodiments, the molar ratio of αPANTIFOL / platinum-based analog in the complex is 11:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In some embodiments, the molar ratio of αPANTIFOL / platinum-based drug in the complex is 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, or 1:20. In some embodiments, the molar ratio of αPANTIFOL / platinum-based drug in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50.In some embodiments, the αPANTIFOL / platinum-based analog complex is encapsulated in liposomes. In some embodiments, the liposomes are Lp-αPANTIFOL as described in any one of items

[13] to

[72] of the form for carrying out the invention.

[0162] In a further embodiment, the present disclosure provides a complex comprising αPANTIFOL and cisplatin or a salt or acid thereof. In some embodiments, the complex comprises αPANTIFOL as described in any one of items [1] to

[12] of the form for carrying out the invention. In some embodiments, the αPANTIFOL complex comprises an antifolate as described in Section II. In some embodiments, the molar ratio of αPANTIFOL / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPANTIFOL / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of αPANTIFOL / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPANTIFOL / cisplatin (or a salt or acid of cisplatin) in the complex is 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, or 1:20. In some embodiments, the molar ratio of αPANTIFOL / cisplatin (or a salt or acid of cisplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In some embodiments, the molar ratio of αPANTIFOL / cisplatin (or a salt or acid of cisplatin) in the complex is 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, or 1:20.In some embodiments, the molar ratio of αPANTIFOL / cisplatin (or a salt or acid of cisplatin) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In some embodiments, the αPANTIFOL / cisplatin (or a salt or acid of cisplatin) complex is encapsulated in liposomes. In some embodiments, the liposomes are Lp-αPANTIFOL described in any one of items

[13] to

[72] of the form for carrying out the invention.

[0163] In another embodiment, the present disclosure provides a complex comprising αPANTIFOL and carboplatin or a salt or acid thereof. In some embodiments, the complex comprises αPANTIFOL as described in any one of items [1] to

[12] of the form for carrying out the invention. In some embodiments, the αPANTIFOL complex comprises the polyglutamated folic acid antimetabolite described in Section II of this specification. In some embodiments, the molar ratio of αPANTIFOL / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1 to 20:1, or any range therebetween. In a further embodiment, the molar ratio of αPANTIFOL / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of αPANTIFOL / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPANTIFOL / carboplatin (or a salt or acid of carboplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. In some embodiments, the molar ratio of αPANTIFOL / carboplatin (or a salt or acid of carboplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In some embodiments, the molar ratio of αPANTIFOL / carboplatin in the complex is 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, or 1:20.In some embodiments, the molar ratio of αPANTIFOL / carboplatin in the complex is 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 αPANTIFOL / carboplatin (or a salt or acid of carboplatin) complex is encapsulated in liposomes. In some embodiments, the liposomes are Lp-αPANTIFOL as described in any one of items

[13] to

[72] in the form section for carrying out the invention.

[0164] In another embodiment, the present disclosure provides a complex comprising αPANTIFOL and oxaliplatin or a salt or acid thereof. In...

Claims

【Claim 1】 A liposome composition comprising liposomes encapsulating an alpha-polyglutamylated folate antimetabolite and one or more non-polyglutamylated polyglutamylatable folate antimetabolites or non-polyglutamylatable folate antimetabolites, wherein the alpha-polyglutamylated folate antimetabolite contains 2 to 15 glutamyl groups having an alpha-carboxyl group bond; The polyglutamated folic acid metabolism antagonist is pterolactrexate, AG2034, GW1843, and LY309887, or their stereoisomers; RTX, and LMX, or their stereoisomers; 2-dMTX, 2-desamino-MTX; 2-CH3-MTX, 2-desamino-2-methyl-MTX; AMT, aminopterin; 2-dAMT, 2-desamino-AMT; 2-CH3-AMT, 2-desamino-2-methyl-AMT; 10-EdAM, 10-ethyl-10-deazaaminopterin; PT523, N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine; DDA THF (lometrexol), 5,10-dideaza-5,6,7,8,-tetrahydrofolic acid; 5-d(i)H4PteGlu, 5-deaza-5,6,7,8-tetrahydroiso-folic acid; N9-CH3-5-d(i)H4PteGlu, N9-methyl-5-deaza-5,6,7,8-tetrahydroiso-folic acid; 5-dPteHCysA, N alpha-(5-deazapteroyl)-L-homocysteic acid; 5-dPteAPBA, N alpha-(5-deazapteroyl)-DL-2-amino-4-phosphonobutyric acid; 5-dPteOrn, N alpha-(5-deazapteroyl)-L-ornithine; 5-dH4PteHCysA, N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-homocysteic acid; 5-dH4PteAPBA, N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dH4PteOro, N alpha-(5-dideaza-5,6,7,8-tetrahydropteroyl)-L-ornithine; CB3717, N10-propargyl-5,8-dideazafolic acid; ICI-198,583, 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid; 4-H-ICI-198,583, 4-deoxy-ICI-198,583: 4-OCH3-ICI-198,583, 4-methoxy-ICI-198,583 Glu-to-Val-ICI-198,583; valine-ICI-198,583; Glu-to-Sub-ICI-198,583, 2-aminosuberic acid-ICI-198,583; 7-CH3-ICI-198,583, 7-methyl-ICI-198,583; ZD1694, N-[5(N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl-methyl)amino)2-thienyl)]-L-glutamic acid; 2-NH2-ZD1694, 2-amino-ZD1694; BW1843U89, (S)-2[5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino-)-1-oxo-2-isoindolinyl]-glutaric acid; LY231514, N-(4-(2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-D]pyrimidin-5-yl)ethyl)-benzoyl]-L-glutamic acid; IAHQ, 5,8-dideazaisofolic acid; 2-dIAHQ, 2-desamino-IAHQ; 2-CH3-dIAHQ, 2-desamino-2-methyl-IAHQ; 5-d(i)PteGlu, 5-dideazaisofolic acid; N9-CH3-5-d(i)PteGlu, N9-methyl-5-dideazaisofolic acid; N9-CHO-5-d(i)PteGlu, N9-formyl-5-dideazaisofolic acid; AG337, 3,4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)quanazoline; and AG377, 2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline; or stereoisomers thereof; which is a polyglutamate of a folate antimetabolite selected from the group consisting of; (a) at least two of the glutamyl groups of the alpha-polyglutamylated folic acid antimetabolite are of the L-form, or (b) each of the glutamyl groups of the alpha-polyglutamylated folic acid antimetabolite is of the L-form, or (c) at least one of the glutamyl groups of the alpha-polyglutamylated folic acid antimetabolite is of the D-form, or (d) each of the glutamyl groups of the alpha-polyglutamylated folic acid antimetabolite other than the glutamyl group of the folic acid antimetabolite is of the D-form, or (e) at least two of the glutamyl groups of the alpha-polyglutamylated folic acid antimetabolite are of the L-form and at least one of the glutamyl groups is of the D-form; the liposome is pegylated, has a diameter of 30 nm to 175 nm, and has a zeta potential of 0 mV or less, a liposome composition.

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