Gamma polyglutamated antifolates and uses thereof

Abstract

To provide gamma polyglutamated antifolate compositions to overcome pharmacological challenges associated with antifolate therapy, the dose-limiting toxicities and treatment resistance.SOLUTION: Disclosed is a liposome composition comprising a gamma polyglutamated antifolate (Lp-γPANTIFOL composition), where the gamma polyglutamated antifolate comprises 2-10 glutamyl groups, the liposome is PEGylated, and the liposome composition comprises a targeting moiety that has a specific affinity to a surface antigen on a target cell of interest.SELECTED DRAWING: None

Description

Background Art

[0001] The present disclosure generally relates to compositions of gamma-polyglutamylated folate antagonists, such as liposomes containing delivery carriers such as gamma-polyglutamylated folate antagonist 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 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 polyglutamylatable folate antagonists are also transported across the cell membrane. Once taken up into cells, intracellular folic acid is converted to polyglutamic acid by the enzyme folylpolyglutamate synthase (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 at a lower pH than normal. 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 folic acid antagonists for DHFR; and (2) it facilitates the accumulation of polyglutamylated folic acid antagonists, which, unlike folic acid antagonists (monoglutamates), are not readily transported out of the cell by cellular efflux pumps.

[0004] Targeting folic acid metabolism and nucleotide biosynthesis is an established therapeutic strategy for cancer, but for folic acid antagonists, clinical efficacy is limited due to 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 folic acid antagonists into cells, (c) increased DHFR activity, (d) decreased activity of holylpolyglutamate synthase (FPGS), and (e) increased activity of gamma-glutamyl hydrolase (GGH), which cleaves the gamma-polyglutamate chain attached to folic acid and folic acid antagonists.

[0006] The problem with the long-term (>30 years) observation that higher levels of polyglutamates of various folic acid antagonists have much 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 gamma-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 a gamma-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 gamma-polyglutamylated folic acid antagonist (γPANTIFOL) compositions, as well as 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 a gamma-polyglutamylated folic acid antagonist; [2] The composition according to item [1], wherein the folic acid antagonist is selected from piritrexim, pralatrexate, AG2034, GW1843, and LY309887, or stereoisomers thereof; [3] The composition according to item [1], wherein the folic acid antagonist is selected from PMX, MTX, RTX, and LTX, or stereoisomers thereof; [4] The composition according to any one of items [1] to [3], 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, 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-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 2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline; or stereoisomers thereof; [5] The composition of 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], wherein the gamma-polyglutamylated folic acid antagonist contains 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups; [7] The composition according to any one of items [1] to [6], wherein the gamma-polyglutamyl oxidized folic acid metabolism antagonist is (a) a gamma-tetra-glutamyl oxidized folic acid metabolism antagonist, (b) a gamma-penta-glutamyl oxidized folic acid metabolism antagonist, or (c) a gamma-hexa-glutamyl oxidized folic acid metabolism antagonist, Composition; [8] The composition according to any one of items [1] to [7], wherein the gamma-polyglutamyl oxidized folic acid metabolism antagonist contains 1 to 10 glutamyl groups having a gamma-carboxyl group bond, Composition; [9] The composition according to any one of items [1] to [8], (a) at least two glutamyl groups of the gamma-polyglutamyl oxidized folic acid metabolism antagonist are of the L-type; (b) each glutamyl group of the gamma-polyglutamyl oxidized folic acid metabolism antagonist is of the L-type; (c) at least one glutamyl group of the gamma-polyglutamyl oxidized folic acid metabolism antagonist is of the D-type; (d) each glutamyl group of the gamma-polyglutamyl oxidized folic acid metabolism antagonist other than the glutamyl group of the folic acid metabolism antagonist is of the D-type; or (e) at least two glutamyl groups of the gamma-polyglutamyl oxidized folic acid metabolism antagonist are of the L-type and at least one glutamyl group is of the D-type, Composition:

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

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

[12] A liposome composition (Lp-γPANTIFOL) containing the gamma-polyglutamyl oxidized folic acid metabolism antagonist according to any one of items [1] to

[11] ;

[13] The Lp-γPANTIFOL composition according to item

[12] , wherein the polyglutamyl oxidized folic acid metabolism antagonist is an Lp-γPANTIFOL composition selected from the following: (a) AG2034, Piritrexim, Pralatrexate, GW1843, folic acid antagonists, and LY309887; or (b) PMX, MTX, RTX, and LTX, their stereoisomers;

[14] The Lp-γPANTIFOL composition according to item

[12] or

[13] , wherein the polyglutamylated folic acid antagonist is selected from the following Lp-γPANTIFOL compositions: 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 (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-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;

[15] An Lp-γPANTIFOL composition according to any one of items

[12] to

[14] , 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 6-R,S-BGC945 (ONX-0801), CB300638, and their stereoisomers such as BW1843U89;

[16] An Lp-γPANTIFOL composition according to any one of items

[12] to

[15] , wherein the liposome contains a gamma-polyglutamylated folic acid antagonist containing 4, 5, 2 to 10, 4 to 6, or more than 5 gamma-glutamyl groups;

[17] An Lp-γPANTIFOL composition according to any one of items

[12] to

[16] , wherein the liposome contains a gamma-tetraglutamylated folic acid antagonist;

[18] An Lp-γPANTIFOL composition according to any one of items

[12] to

[16] , wherein the liposome contains a gamma-pentaglutamylated folic acid antagonist;

[19] An Lp-γPANTIFOL composition according to any one of items

[12] to

[16] , wherein the liposome contains a gamma-hexaglutamylated folic acid antagonist;

[20] An Lp-γPANTIFOL composition according to any one of items

[12] to

[19] , wherein the gamma-polyglutamylated folic acid antagonist contains 1 to 10 glutamyl groups having a gamma-carboxyl group bond;

[21] An Lp-γPANTIFOL composition according to any one of items

[12] to

[20] , (a) At least two glutamyl groups of the gamma-polyglutamylated folic acid antagonist are of the L type; (b) Each glutamyl group of the gamma-polyglutamylated folic acid antagonist is of the L type; (c) At least one glutamyl group of the gamma-polyglutamylated folic acid antimetabolite is of the D-type; (d) Each glutamyl group of the gamma-polyglutamylated 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 gamma-polyglutamylated folic acid antimetabolite are of the L-type and at least one glutamyl group is of the D-type, Lp-γPANTIFOL composition:

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

[12] to

[21] , (a) At least two glutamyl groups of the gamma-polyglutamylated folic acid antimetabolite are of the L-type; (b) Each glutamyl group of the gamma-polyglutamylated folic acid antimetabolite is of the L-type; (c) At least one glutamyl group of the gamma-polyglutamylated folic acid antimetabolite is of the D-type; (d) Each glutamyl group of the gamma-polyglutamylated 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 gamma-polyglutamylated folic acid antimetabolite are of the L-type and at least one glutamyl group is of the D-type, Lp-γPANTIFOL composition:

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

[12] to

[22] , wherein the liposome is pegylated (PLp-γPANTIFOL), Lp-γPANTIFOL composition;

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

[12] to

[22] , wherein the liposome is not pegylated, Lp-γPANTIFOL composition;

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

[12] to

[24] , wherein the liposome has a diameter in the range of 20 nm to 200 nm, Lp-γPANTIFOL composition;

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

[12] to

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

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

[12] to

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

[28] An Lp-γPANTIFOL composition according to item

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

[29] An Lp-γPANTIFOL composition according to item

[27] or

[28] , 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;

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

[27] to

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

[31] An Lp-γPANTIFOL composition according to any one of items

[27] to

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

[32] The Lp-γPANTIFOL composition according to item

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

[33] The Lp-γPANTIFOL composition according to item

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

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

[12] to

[33] , wherein the liposome is anionic or neutral; the Lp-γPANTIFOL composition;

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

[33] , wherein the liposome is cationic; the Lp-γPANTIFOL composition;

[39] An Lp-γPANTIFOL composition according to any one of items

[12] to

[38] , wherein the liposome has an internal space containing a gamma polyglutamine oxidized folic acid antagonist and an aqueous pharmaceutically acceptable carrier;

[40] An Lp-γPANTIFOL composition according to item

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

[41] An Lp-γPANTIFOL composition according to item

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

[42] An Lp-γPANTIFOL composition according to item

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

[43] An Lp-γPANTIFOL composition according to any one of items

[39] to

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

[44] An Lp-γPANTIFOL composition according to any one of items

[39] to

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

[45] An Lp-γPANTIFOL composition according to any one of items

[39] to

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

[46] An Lp-γPANTIFOL composition according to any one of items

[39] to

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

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

[12] to

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

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

[12] to

[47] , wherein the liposome contains less than 500,000 or less than 200,000 gamma polyglutamylated folic acid antimetabolite molecules;

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

[12] to

[48] , wherein the liposome contains 10 to 100,000 or any range therebetween of gamma polyglutamylated folic acid antimetabolite molecules;

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

[12] to

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

[51] The Lp-γPANTIFOL composition according to item

[50] , 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;

[52] The Lp-γPANTIFOL composition according to item

[50] or

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

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

[50] to

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

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

[50] to

[53] , wherein the targeting moiety, as measured by BIACORE® analysis, is 0.5x10-10 to 10x10 -6An Lp-γPANTIFOL composition that binds to a surface antigen with an equilibrium dissociation constant (Kd) in the range of;

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

[50] to

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

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

[50] to

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

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

[50] to

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

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

[39] to

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

[59] The Lp-γPANTIFOL composition according to item

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

[60] The Lp-γPANTIFOL composition according to item

[58] or

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

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

[58] to

[60] , wherein the immunostimulant and the detectable marker are the same, the Lp-γPANTIFOL composition;

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

[58] to

[61] , further comprising a hapten, the Lp-γPANTIFOL composition;

[63] The Lp-γPANTIFOL composition according to item

[62] , wherein the hapten comprises one or more of fluorescein or beta-1,6-glucan, the Lp-γPANTIFOL composition:

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

[12] to

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

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

[64] ;

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

[49] ;

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

[12] to

[66] , further comprising carboplatin and / or pembrolizumab, the Lp-γPANTIFOL composition;

[68] A pharmaceutical composition comprising the liposomal gamma polyglutamine oxidized folic acid antagonist composition according to any one of items

[12] to

[67] ;

[69] A pharmaceutical composition comprising the gamma polyglutamine oxidized folic acid antagonist composition according to any one of items [1] to [7];

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

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

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

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

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

[70] to the subject;

[73] A method for treating or preventing a disease in a subject in need of such treatment or prevention, the method comprising administering the liposomal gamma-polyglutamylated folic acid antimetabolite composition according to any one of

[12] to

[69] to the subject;

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

[69] ;

[75] A method for killing hyperproliferative cells, the method comprising the step of contacting the hyperproliferative cells with the liposomal gamma-polyglutamylated folic acid antimetabolite composition according to any one of items

[12] to

[69] ;

[76] The method according to item

[74] or

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

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

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

[78] A method for treating cancer, the method comprising the step of administering an effective amount of the liposomal gamma-polyglutamylated folic acid antimetabolite composition according to any one of items

[12] to

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

[79] The method according to item

[77] or

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

[80] The method according to item

[77] or

[78] , wherein the cancer is selected from lung cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, ovarian cancer, and cervical cancer;

[81] The method according to item

[77] or

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

[82] The method according to item

[77] or

[78] , wherein the cancer is selected from colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma;

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

[50] to

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

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

[69] to the subject who is receiving or has received cancer therapy;

[85] Maintenance therapy for a subject who is receiving or has received cancer therapy, comprising administering an effective amount of the liposomal gamma-polyglutamyl oxidase metabolic antagonist composition according to any one of items

[12] to

[69] to the subject who is receiving or has received cancer therapy;

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

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

[87] Administering an effective amount of the liposomal gamma-polyglutamyl oxidized folic acid metabolic antagonist composition according to any one of items [8] to

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

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

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

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

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

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

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

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

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

[12] to

[69] ;

[90] A method for delivering a gamma-polyglutamylated folic acid metabolic antagonist to a tumor expressing a folic acid receptor on its surface, the method comprising the step of administering to a subject having a tumor an Lp-γPANTIFOL composition according to any one of items [1] to

[69] in an amount sufficient to deliver a therapeutically effective amount of the gamma-polyglutamylated folic acid metabolic antagonist to the tumor;

[91] A method for preparing a gamma-polyglutamylated folic acid metabolic antagonist composition comprising the liposomal gamma-polyglutamylated folic acid metabolic antagonist composition according to any one of items

[12] to

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

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

[12] to

[69] , the method comprising the steps of: forming a mixture comprising a liposomal component and a gamma-polyglutamylated folic acid metabolic antagonist in solution; homogenizing the mixture in solution to form liposomes; treating the mixture to form liposomes encapsulating and / or coating the gamma-polyglutamylated folic acid metabolic antagonist; and attaching a targeting moiety onto the surface of the liposomes, wherein the targeting moiety has specific affinity for at least one of folic acid receptor alpha (FR-α), folic acid receptor beta (FR-β) and folic acid receptor delta (FR-δ).

[93] The method according to item

[92] , 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-drying double emulsion, 3D printing, membrane contactor method, and agitation; and / or

[94] The method according to item

[92] , 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.

[0010] In some embodiments, the present disclosure provides a gamma-polyglutamyl oxidized folic acid antagonist (γPANTIFOL) composition, wherein at least two glutamyl residues of the gamma-polyglutamyl oxidized folic acid antagonist have its gamma-carboxyl group bond. In some embodiments, γPANTIFOL 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, the gamma-polyglutamyl oxidized folic acid antagonist is selected from (a) AG2034, pyrithioxime, pteropterin, GW1843, folic acid antagonist, and LY309887; or (b) PMX, MTX, RTX, and LTX, or their stereoisomers. In some embodiments, the gamma-polyglutamyl 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 (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, Nα-(5-Deaza-5,6,7,8-tetrahydroptenoyl)-DL-2-amino-4-phosphonobutanoic acid; 5-dH4PteOro, Nα-(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]-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-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. In some embodiments, the gamma polyglutamylated folate metabolism antagonist 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, γPANTIFOL comprises two or more L-type glutamyl groups. In some embodiments, γPANTIFOL comprises a D-type glutamyl group. In further embodiments, γPANTIFOL comprises a D-type glutamyl group and two or more L-type glutamyl groups.;

[0011] In one embodiment, the γPANTIFOL composition comprises a chain of three glutamyl groups attached to the glutamyl group in the folic acid antagonist (i.e., γ-tetraglutamylated folic acid antagonist). In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [2] of the section of the Summary of the Invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [3] of the section of the Summary of the Invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [4] of the section of the Summary of the Invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [5] of the section of the Summary of the Invention. In some embodiments, γPANTIFOL is the polyglutamylated folic acid antagonist described in the section of the Summary of the Invention. 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 some embodiments, the tetraglutamylated folic acid antagonist comprises two or more D-type glutamyl groups. In a further embodiment, the tetraglutamylated folic acid antagonist comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the tetraglutamylated folic acid antagonist comprises one, two, or three D-type glutamyl groups and three, two, or one L-type glutamyl groups, respectively.

[0012] In one embodiment, the γPANTIFOL composition comprises a chain of four γ-glutamyl groups attached to the glutamyl group in the folic acid antagonist (i.e., γ-pentaglutamylated folic acid antagonist). In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [2] of the Summary of the Invention section. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [3] of the Summary of the Invention section. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [4] of the Summary of the Invention section. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [5] of the Summary of the Invention section. In some embodiments, γPANTIFOL is the polyglutamylated folic acid antagonist described in the Summary of the Invention section. 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.

[0013] In one embodiment, the γPANTIFOL composition comprises a chain of five γ-glutamyl groups attached to the glutamyl group in the folic acid antagonist (i.e., γ-hexaglutamylated folic acid antagonist). In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [2] of the section of the Summary of the Invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [3] of the section of the Summary of the Invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [4] of the section of the Summary of the Invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [5] of the section of the Summary of the Invention. In some embodiments, γPANTIFOL is the polyglutamylated folic acid antagonist described in the section of the Summary of the Invention. In some embodiments, the hexaglutamylated folic acid antagonist comprises two or more L-type glutamyl groups. In some 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, respectively.

[0014] In further embodiments, the present disclosure provides compositions comprising delivery carriers such as liposomes filled with (i.e., encapsulated) and / or otherwise conjugated to a gamma-polyglutamylated folate antagonist, and methods of making a γPANTIFOL-filled / conjugated delivery carrier composition (DV-γPANTIFOL) and using the same to deliver a gamma-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 γPANTIFOL-filled / conjugated delivery carrier composition provides for the selective delivery of a higher cytotoxic payload (gamma-polyglutamylated folate antagonist) compared to the cytotoxicity of a folate antagonist administered in the monoglutamate state, resulting in improved efficacy and safety of delivery of the folate antagonist to cancer cells. In some embodiments, the gamma-polyglutamylated folate antagonist in DV-γPANTIFOL 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 a polyglutamylated folate antagonist as described in any of items [1] to

[11] of the Summary section of the invention. In some embodiments, the delivery carrier comprises a polyglutamylated folate antagonist as described in the Summary section of the invention. In some embodiments, the delivery carrier is a liposome as described in any of items

[12] to

[67] of the Summary section of the invention.

[0015] In a further embodiment, the present disclosure provides a composition (Lp-γPANTIFOL) comprising liposomes encapsulating (filling) a gamma polyglutamyl oxidized folic acid antagonist. In some embodiments, the gamma polyglutamyl oxidized folic acid antagonist in Lp-γPANTIFOL comprises 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 antagonist). In some embodiments, the gamma polyglutamyl oxidized folic acid antagonist encapsulated by the liposomes is selected from (a) AG2034, pyrithioxime, pteropterin, GW1843, folic acid antagonists, and LY309887; or (b) PMX, MTX, RTX, and LTX, or their stereoisomers. In some embodiments, the gamma polyglutamyl oxidized folic acid antagonist encapsulated by the liposomes 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, 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]-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. In some embodiments, the gamma-polyglutamylated folate antagonist encapsulated by the liposome is selected from methotrexate, raltitrexed, premetrexed, pemetrexed, lometrexol (LTX; 5,10-dideazatetrahydrofolic acid), cyclopenta[g]quinazoline having a dipeptide ligand, CB3717, CB300945, or 6-R,S-BGC945 (ONX-0801), CB300638, and BW1843U89, and stereoisomers thereof. In some embodiments, the gamma-polyglutamylated folate antagonist in Lp-γPANTIFOL contains two or more L-type glutamyl groups. In other embodiments, the gamma-polyglutamylated folate antagonist in Lp-γPANTIFOL contains a D-type glutamyl group. In further embodiments, the gamma-polyglutamylated folate antagonist in Lp-γPANTIFOL contains a D-type glutamyl group and two or more L-type glutamyl groups.;

[0016] In one embodiment, the Lp-γPANTIFOL composition comprises a gamma-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, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [2] of the section of the Summary of the Invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [3] of the section of the Summary of the Invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [4] of the section of the Summary of the Invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [5] of the section of the Summary of the Invention. In some embodiments, γPANTIFOL is the polyglutamylated folic acid antagonist described in the section of the Summary of the Invention. 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.

[0017] In one embodiment, the Lp-γPANTIFOL composition comprises a gamma polyglutamylated folic acid antagonist comprising a chain of four γ-glutamyl groups attached to the glutamyl group in the folic acid antagonist (e.g., a γ-pentaglutamylated folic acid antagonist). In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [2] of the section of the Summary of the Invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [3] of the section of the Summary of the Invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [4] of the section of the Summary of the Invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [5] of the section of the Summary of the Invention. In some embodiments, γPANTIFOL is the polyglutamylated folic acid antagonist described in the section of the Summary of the Invention. In some embodiments, the pentaglutamylated folic acid antagonist comprises two or more L-type glutamyl groups. In some embodiments, the gamma pentaglutamylated folic acid antagonist comprises a D-type glutamyl group. In a further embodiment, the pentaglutamylated folic acid antagonist comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0018] In one embodiment, the Lp-γPANTIFOL composition comprises a gamma-polyglutamylated folic acid antagonist comprising a chain of 5 gamma-glutamyl groups attached to the glutamyl group in the folic acid antagonist (e.g., gamma-hexaglutamylated folic acid antagonist). In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [2] of the section of the summary of the invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [3] of the section of the summary of the invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [4] of the section of the summary of the invention. In some embodiments, the polyglutamylated folic acid antagonist is the folic acid antagonist described in item [5] of the section of the summary of the invention. In some embodiments, γPANTIFOL is the polyglutamylated folic acid antagonist described in the section of the summary of the invention. In some embodiments, the gamma-hexaglutamylated folic acid antagonist comprises two or more L-type glutamyl groups. In other embodiments, the gamma-hexaglutamylated folic acid antagonist comprises a D-type glutamyl group. In further embodiments, the gamma-hexaglutamylated folic acid antagonist comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0019] In some embodiments, the Lp-γPANTIFOL composition is cationic. In some embodiments, the Lp-γPANTIFOL liposomes are cationic and have a diameter in the range of 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-γPANTIFOL liposomes are cationic and have a diameter in the range of 30 nm to 175 nm or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-γPANTIFOL liposomes have a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the cationic Lp-γPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% liposome-encapsulated gamma-tetraglutamyl oxidized folic acid antagonist. In some embodiments, the cationic Lp-γPANTIFOL comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% liposome-encapsulated gamma-pentaglutamyl oxidized folic acid antagonist. In other embodiments, the Lp-γPANTIFOL comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% liposome-encapsulated gamma-hexaglutamyl oxidized folic acid antagonist. In further embodiments, the gamma-polyglutamyl oxidized folic acid antagonist encapsulated by the liposomes is present in the HEPES buffer within the liposomes.

[0020] In other embodiments, the Lp-γPANTIFOL composition is anionic or neutral. In some embodiments, the Lp-γPANTIFOL composition is cationic. In some embodiments, the Lp-γPANTIFOL liposomes are anionic or neutral and have a diameter in the range of 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-γPANTIFOL liposomes are anionic or neutral and have a diameter in the range of 30 nm to 175 nm or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-γPANTIFOL 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-γPANTIFOL liposomes are anionic and have a diameter in the range of 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-γPANTIFOL liposomes are anionic and have a diameter in the range of 30 nm to 175 nm or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-γPANTIFOL 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-γPANTIFOL liposomes are neutral and have a diameter in the range of 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-γPANTIFOL liposomes are neutral and have a diameter in the range of 30 nm to 175 nm or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-γPANTIFOL 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-γPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% liposome-encapsulated gamma-tetraglutamyl oxidized folic acid antagonist. In some embodiments, the anionic or neutral Lp-γPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75 of liposome-encapsulated gamma-pentaglutamyl oxidized folic acid antagonist. In other embodiments, the Lp-γPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% liposome-encapsulated gamma-hexaglutamyl oxidized folic acid antagonist. In further embodiments, the gamma-polyglutamyl oxidized folic acid antagonist encapsulated by the liposome is present in the HEPES buffer within the liposome.

[0021] In further embodiments, the liposome gamma-polyglutamyl oxidized folic acid antagonist composition is pegylated (PLp-γPANTIFOL).

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

[0023] In other embodiments, the liposomal gamma polyglutamylated folic acid antimetabolite composition is targeted (TLp-γPANTIFOL). That is, the TLp-γPANTIFOL composition includes a targeting moiety having specific affinity for an epitope (surface antigen) on the target cell of interest. In some embodiments, TLp-γPANTIFOL or TPLp-γPANTIFOL is not bound to the liposome via a covalent bond. In other embodiments, the targeting moiety of TLp-γPANTIFOL or TPLp-γPANTIFOL is bound to one or both of the PEG and outer surface of the liposome. In some embodiments, the targeting moiety of TLp-γPANTIFOL or TPLp-γPANTIFOL is bound to the liposome via a covalent bond. The functions of the targeting moiety of the TLp-γPANTIFOL and / or TPLp-γPANTIFOL 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 (γPANTIFOL) 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 gamma polyglutamylated folate antimetabolite compositions (TLp-γPANTIFOL and TPLp-γPANTIFOL) provide further improvements over the efficacy and safety profiles of folate antimetabolites by specifically delivering gamma polyglutamylated (e.g., γ-pentaglutamylated and / or γ-hexaglutamylated) folate antimetabolites to target cells such as cancer cells. In further embodiments, the targeted liposomal gamma polyglutamylated folate antimetabolite compositions are pegylated (TPLp-γPANTIFOL). In some embodiments, the targeting moieties of TLp-γPANTIFOL and TPLp-γPANTIFOL are attached to one or both of the PEG and outer surface of the liposome. In some embodiments, the targeting moieties of TLp-γPANTIFOL and TPLp-γPANTIFOL are attached to the liposome via a covalent bond. γPANTIFOL). The functions of the targeting moiety of the TLp-γPANTIFOL and / or TPLp-γPANTIFOL compositions include, but are not limited to, targeting the liposome to the target cell of interest in vivo or in vitro; interacting with a surface antigen to which the targeting moiety has specific affinity; and delivering the liposome payload (γPANTIFOL) 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.

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

[0026] In certain embodiments, the TLp-γPANTIFOL or TPLp-γPANTIFOL 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 specifically bound by the targeting moiety specifically binds to 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 specifically bound by the targeting moiety is folate receptor alpha (FR-α). In some embodiments, the folate receptor specifically 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 Lp-γPANTIFOL 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 γPANTIFOL composition (e.g., Lp-γPANTIFOL, PLp-γPANTIFOL, NTLp-γPANTIFOL, NTPLp-γPANTIFOL, TLp-γPANTIFOL, or TPLp-γPANTIFOL) is cationic. In other embodiments, the liposomal γPANTIFOL composition (e.g., Lp-γPANTIFOL, PLp-γPANTIFOL, NTLp-γPANTIFOL, NTPLp-γPANTIFOL, TLp-γPANTIFOL or TPLp-γPANTIFOL) is anionic or neutral. In a further embodiment, the liposomes of the liposomal γPANTIFOL composition (e.g., Lp-γPANTIFOL, PLp-γPANTIFOL, NTLp-γPANTIFOL, NTPLp-γPANTIFOL, TLp-γPANTIFOL or TPLp-γPANTIFOL) 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 γPANTIFOL composition have a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the liposomal γPANTIFOL composition is pegylated (e.g., PLp-γPANTIFOL, NTPLp-γPANTIFOL, or TPLp-γPANTIFOL). In some embodiments, the liposomal γPANTIFOL composition comprises a targeting moiety (e.g., TLp-γPANTIFOL or TPLp-γPANTIFOL). In a further embodiment, the liposomal γPANTIFOL composition is pegylated and targeted (e.g., TPLp-γPANTIFOL). In some embodiments, the liposomal γPANTIFOL composition comprises a gamma-polyglutamylated folic acid antagonist comprising 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomal γPANTIFOL composition comprises a gamma-tetraglutamylated folic acid antagonist.In some embodiments, the liposomal γPANTIFOL composition comprises a gamma-pentaglutamyl oxidized folic acid antagonist. In some embodiments, the liposomal γPANTIFOL composition comprises a gamma-hexaglutamyl oxidized folic acid antagonist. In some embodiments, the liposomal composition comprises a gamma-polyglutamyl oxidized folic acid antagonist as set forth in any of [1] to

[11] of the Summary section of the invention. In some embodiments, the liposome comprises a liposomal composition as set forth in any of items

[11] to

[69] of the Summary section of the invention. In some embodiments, the composition comprises a gamma-polyglutamyl oxidized folic acid antagonist as set forth in the Summary section of the invention.

[0028] In a further embodiment, the liposomal γPANTIFOL composition (i.e., Lp-γPANTIFOL such as PLp-γPANTIFOL, NTLp-γPANTIFOL, NTPLp-γPANTIFOL, TLp-γPANTIFOL or TPLp-γPANTIFOL) comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% liposome-encapsulated gamma polyglutamyl oxidized folic acid antagonist. In some embodiments, the liposomal γPANTIFOL composition comprises 1% to 98.5% liposome-encapsulated gamma polyglutamyl oxidized folic acid antagonist. In a further embodiment, the liposomal γPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% liposome-encapsulated gamma polyglutamyl oxidized folic acid antagonist comprising 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomal γPANTIFOL composition comprises 1% to 98.5% liposome-encapsulated gamma polyglutamyl oxidized folic acid antagonist comprising 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomal γPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% liposome-encapsulated gamma tetraglutamyl oxidized folic acid antagonist. In some embodiments, the liposomal γPANTIFOL composition comprises 1% to 98.5% liposome-encapsulated gamma polyglutamyl oxidized folic acid antagonist. In some embodiments, the liposomal γPANTIFOL composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% liposome-encapsulated gamma pentaglutamyl oxidized folic acid antagonist. In some embodiments, the liposomal γPANTIFOL composition comprises 1% to 98.5% liposome-encapsulated gamma pentaglutamyl oxidized folic acid antagonist.In some embodiments, the liposomal γPANTIFOL 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 gamma-hexaglutamyl oxidized folate antagonist. In some embodiments, the liposomal γPANTIFOL composition comprises 1% to 98.5% of a liposome-encapsulated gamma-pentaglutamyl oxidized folate antagonist. In some embodiments, the liposomal composition comprises a gamma-polyglutamyl oxidized folate antagonist as described in any one of [1] to

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

[11] to

[69] of the Summary section of the invention. In some embodiments, the composition comprises a gamma-polyglutamyl oxidized folate antagonist as described in the Summary section of the invention or in the figures of this specification.

[0029] There is also provided a liposomal composition comprising γPANTIFOL-encapsulated liposomes. In some embodiments, the liposomal composition comprises a pegylated γPANTIFOL composition. In some embodiments, the liposomal composition comprises a γPANTIFOL composition linked or otherwise conjugated to a targeting moiety. In further embodiments, the liposomal composition comprises a pegylated γPANTIFOL composition linked or otherwise conjugated to a targeting moiety. In some embodiments, the liposomal composition comprises γPANTIFOL comprising 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomal composition comprises a gamma-tetraglutamyl folate antagonist. In some embodiments, the liposomal composition comprises a gamma-pentaglutamyl folate antagonist. In other embodiments, the liposomal composition comprises a gamma-hexaglutamyl folate antagonist. In some embodiments, the polyglutamylated folate antagonist is the folate antagonist described in item [2] of the Summary section of the invention. In some embodiments, the polyglutamylated folate antagonist is the folate antagonist described in item [3] of the Summary section of the invention. In some embodiments, the polyglutamylated folate antagonist is the folate antagonist described in item [4] of the Summary section of the invention. In some embodiments, the polyglutamylated folate antagonist is the folate antagonist described in item [5] of the Summary section of the invention. In some embodiments, γPANTIFOL is the polyglutamylated folate antagonist described in the Summary section of the invention.

[0030] In some embodiments, the liposomal composition comprises liposomal γPANTIFOL (e.g., Lp-γPANTIFOL, PLp-γPANTIFOL, NTLp-γPANTIFOL, NTPLp-γPANTIFOL, TLp-γPANTIFOL, and TPLp-γPANTIFOL). In some embodiments, the liposomal γPANTIFOL is pegylated (e.g., NTPLp-γPANTIFOL, and TPLp-γPANTIFOL). In some embodiments, the pharmaceutical composition comprises γPANTIFOL containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition comprises a gamma-tetraglutamyl methotrexate. In some embodiments, the pharmaceutical composition comprises a gamma-pentaglutamyl methotrexate. In other embodiments, the pharmaceutical composition comprises a gamma-hexaglutamyl methotrexate. In some embodiments, the polyglutamyl methotrexate is the methotrexate described in item [2] of the section of the Summary of the Invention. In some embodiments, the polyglutamyl methotrexate is the methotrexate described in item [3] of the section of the Summary of the Invention. In some embodiments, the polyglutamyl methotrexate is the methotrexate described in item [4] of the section of the Summary of the Invention. In some embodiments, the polyglutamyl methotrexate is the methotrexate described in item [5] of the section of the Summary of the Invention. In some embodiments, γPANTIFOL is the polyglutamyl methotrexate described in the section of the Summary of the Invention. In some embodiments, the liposomal γPANTIFOL comprises a targeting moiety having a specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TLp-γPANTIFOL or TPLp-γPANTIFOL). In a further embodiment, the liposomal composition comprises pegylated liposomal γPANTIFOL and further comprises a targeting moiety having a specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TPLp-γPANTIFOL). In some embodiments, the liposomal composition comprises liposomal γPANTIFOL that is cationic.In other embodiments, the liposome composition comprises liposomal γPANTIFOL that is anionic or neutral. In further embodiments, the liposome composition comprises liposomal γPANTIFOL having a diameter in the range of 20 nm to 200 nm, or any range therebetween. In further embodiments, liposomal γPANTIFOL has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

[0031] There is also provided a pharmaceutical composition comprising a gamma polyglutamylated folate antagonist (γPANTIFOL) comprising a delivery carrier such as liposomal γPANTIFOL. In some embodiments, the pharmaceutical composition comprises a pegylated γPANTIFOL composition. In some embodiments, the pharmaceutical composition comprises a γPANTIFOL composition linked to or otherwise conjugated to a targeting moiety. In further embodiments, the pharmaceutical composition comprises a pegylated γPANTIFOL composition linked to or otherwise conjugated to a targeting moiety. In some embodiments, the pharmaceutical composition comprises γPANTIFOL comprising 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition comprises a gamma tetraglutamylated folate antagonist. In some embodiments, the pharmaceutical composition comprises a gamma pentaglutamylated folate antagonist. In other embodiments, the pharmaceutical composition comprises a gamma hexaglutamylated folate antagonist. In some embodiments, the gamma polyglutamylated folate antagonist is the polyglutamylated folate antagonist described in any of items [1] to

[11] of the Summary section of the invention. In some embodiments, the polyglutamylated folate antagonist is the polyglutamylated folate antagonist described in the Summary section of the invention.

[0032] In some embodiments, the pharmaceutical composition comprises liposomal γPANTIFOL (e.g., Lp-γPANTIFOL, PLp-γPANTIFOL, NTLp-γPANTIFOL, NTPLp-γPANTIFOL, TLp-γPANTIFOL, and TPLp-γPANTIFOL). In some embodiments, the liposomal γPANTIFOL composition is pegylated (e.g., NTPLp-γPANTIFOL, and TPLp-γPANTIFOL). In some embodiments, liposomal γPANTIFOL 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-γPANTIFOL or TPLp-γPANTIFOL). In further embodiments, the pharmaceutical composition comprises pegylated liposomal γPANTIFOL 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-γPANTIFOL). In some embodiments, the pharmaceutical composition comprises liposomal γPANTIFOL that is cationic. In other embodiments, the pharmaceutical composition comprises liposomal γPANTIFOL that is anionic or neutral. In further embodiments, the pharmaceutical composition comprises liposomal γPANTIFOL having a diameter of 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposomal γPANTIFOL composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the pharmaceutical composition comprises γPANTIFOL comprising 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition comprises a gamma-tetraglutamyl folate antagonist. In some embodiments, the pharmaceutical composition comprises a gamma-pentaglutamyl folate antagonist. In other embodiments, the pharmaceutical composition comprises a gamma-hexaglutamyl folate antagonist. In some embodiments, the composition comprises a gamma-polyglutamyl folate antagonist as described in any of items [1] to

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

[11] to

[69] of the section of the summary of the invention.In some embodiments, the composition comprises the gamma polyglutamyl oxidized folic acid antimetabolite described in the Summary of the Invention section.

[0033] In further embodiments, the present disclosure provides a method of modulating cell activation, chemokine production, or metabolic activity, the method comprising contacting a cell with a composition comprising a gamma polyglutamyl oxidized folic acid antimetabolite (γPANTIFOL) 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, γPANTIFOL comprises 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the γPANTIFOL composition comprises a gamma tetraglutamyl oxidized folic acid antimetabolite. In some embodiments, the γPANTIFOL composition comprises a gamma pentaglutamyl oxidized folic acid antimetabolite. In other embodiments, the γPANTIFOL composition comprises a gamma hexaglutamyl oxidized folic acid antimetabolite. In some embodiments, the composition comprises the gamma polyglutamyl oxidized folic acid antimetabolite described in any of items [1]-

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

[11] -

[69] of the Summary of the Invention section. In some embodiments, the composition comprises the gamma polyglutamyl oxidized folic acid antimetabolite described in the Summary of the Invention section or in the figures of this specification.

[0034] In further embodiments, the present disclosure provides a method for regulating cell activation, chemokine production, or metabolic activity, the method comprising contacting a cell with liposomes comprising a gamma-polyglutamylated folate antagonist (γPANTIFOL) 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, γPANTIFOL comprises 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the γPANTIFOL composition comprises a gamma-tetraglutamylated folate antagonist. In some embodiments, the γPANTIFOL composition comprises a gamma-pentaglutamylated folate antagonist. In other embodiments, the γPANTIFOL composition comprises a gamma-hexaglutamylated folate antagonist. In some embodiments, the polyglutamylated folate antagonist comprises the folate antagonist described in item [2] of the Summary section of the invention. In some embodiments, the polyglutamylated folate antagonist is the folate antagonist described in item [3] of the Summary section of the invention. In some embodiments, the polyglutamylated folate antagonist is the folate antagonist described in item [4] of the Summary section of the invention. In some embodiments, the polyglutamylated folate antagonist is the folate antagonist described in item [5] of the Summary section of the invention. In some embodiments, γPANTIFOL is the polyglutamylated folate antagonist described in the Summary section of the invention.

[0035] In further embodiments, the present disclosure provides a method of killing cells, the method comprising contacting the cells with a composition comprising a gamma-polyglutamylated folic acid antagonist (γPANTIFOL) 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-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 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, central nervous system (CNS) cancer. In some embodiments, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained from lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained from breast cancer (e.g., HER2++ or triple-negative breast cancer). In some embodiments, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained from colorectal cancer. In some embodiments, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained from ovarian cancer. In some embodiments, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained from endometrial cancer. In some embodiments, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained from pancreatic cancer.In some embodiments, the cancer cells to be contacted are cells derived from primary cells or cell lines obtained from liver cancer. In some embodiments, the cancer cells to be contacted are cells derived from primary cells or cell lines obtained from head and neck cancer. In some embodiments, the cancer cells to be contacted are cells derived from primary cells or cell lines obtained from osteosarcoma. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, γPANTIFOL contains 4, 5, 2 to 10, 4 to 6, or more than 5 γ-glutamyl groups. In some embodiments, the γPANTIFOL composition contains a gamma-tetra-glutamyl oxidized folic acid antagonist. In some embodiments, the γPANTIFOL composition contains a gamma-penta-glutamyl oxidized folic acid antagonist. In other embodiments, the γPANTIFOL composition contains a gamma-hexa-glutamyl oxidized folic acid antagonist. In some embodiments, the gamma-poly-glutamyl oxidized folic acid antagonist is the poly-glutamyl oxidized folic acid antagonist described in any of items [1] to

[11] in the section of the summary of the invention. In some embodiments, the gamma-poly-glutamyl oxidized folic acid antagonist is the poly-glutamyl oxidized folic acid antagonist described in the items of the section of the summary of the invention.

[0036] In a further embodiment, the present disclosure provides a method of killing cells, the method comprising contacting the cells with liposomes comprising a gamma polyglutamylated folic acid antimetabolite (e.g., Lp-γPANTIFOL such as PLp-γPANTIFOL, NTLp-γPANTIFOL, NTPLp-γPANTIFOL, TLp-γPANTIFOL or TPLp-γPANTIFOL). In some embodiments, the cells to be contacted are mammalian cells. In a further embodiment, the cells to be contacted are human cells. In some embodiments, the cells to be contacted are hyperproliferative cells. In a further embodiment, the hyperproliferative cells to be contacted are cancer cells. In a further embodiment, 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 dysplasias 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 carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, central nervous system (CNS) cancer. In some embodiments, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained from lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained from breast cancer (e.g., HER2++ or triple negative breast cancer). In some embodiments, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained from colorectal cancer. In some embodiments, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained from ovarian cancer.In some embodiments, the cancer cells to be contacted are cells derived from primary cells or cell lines obtained from endometrial cancer. In some embodiments, the cancer cells to be contacted are cells derived from primary cells or cell lines obtained from pancreatic cancer. In some embodiments, the cancer cells to be contacted are cells derived from primary cells or cell lines obtained from liver cancer. In some embodiments, the cancer cells to be contacted are cells derived from primary cells or cell lines obtained from head and neck cancer. In some embodiments, the cancer cells to be contacted are cells derived from primary cells or cell lines obtained from osteosarcoma. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the liposome contains γPANTIFOL containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposome contains a gamma tetraglutamyl oxidized folic acid antagonist. In some embodiments, the liposome contains a gamma pentaglutamyl oxidized folic acid antagonist. In other embodiments, the liposome contains a gamma hexaglutamyl oxidized folic acid antagonist. In some embodiments, the gamma polyglutamyl oxidized folic acid antagonist is the polyglutamyl oxidized folic acid antagonist described in any of items [1] to

[11] in the section of the summary of the invention. In some embodiments, γPANTIFOL is the polyglutamyl oxidized folic acid antagonist described in the section of the summary of the invention. In some embodiments, the liposome is the liposome described in any of items

[12] to

[67] in the section of the summary of the invention.

[0037] 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 an effective amount of a delivery carrier (e.g., an antibody immunocomplex or a liposome) comprising a gamma-polyglutamylated folate antimetabolite. In some embodiments, the delivery carrier is an antibody-containing immunocomplex (e.g., comprising a full-length IgG antibody, a bispecific antibody, or a scFv). In some embodiments, the delivery carrier is a liposome (e.g., an Lp-γPANTIFOL such as PLp-γPANTIFOL, NTLp-γPANTIFOL, NTPLp-γPANTIFOL, TLp-γPANTIFOL, or TPLp-γPANTIFOL). In some embodiments, the delivery carrier being administered is pegylated. In some embodiments, the delivery carrier being administered is not pegylated. In a further embodiment, the delivery carrier being administered comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of cancer cells.In a further embodiment, the delivery 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 delivery carrier to be administered comprises γPANTIFOL containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the delivery carrier to be administered comprises a gamma-tetraglutamyl oxidized folic acid antagonist. In some embodiments, the delivery carrier to be administered comprises a gamma-pentaglutamyl oxidized folic acid antagonist. In some embodiments, the delivery carrier to be administered comprises an L-gamma-polyglutamyl oxidized folic acid antagonist. In some embodiments, the delivery carrier to be administered comprises 2, 3, 4, 5, or more than 5 L-gammaglutamyl groups. In some embodiments, the delivery carrier to be administered comprises a D-gamma-polyglutamyl oxidized folic acid antagonist. In some embodiments, the delivery carrier to be administered comprises 2, 3, 4, 5, or more than 5, or more than 5 D-gammaglutamyl groups. In some embodiments, the delivery carrier to be administered comprises L and D gamma-polyglutamyl oxidized folic acid antagonists. In some embodiments, the delivery carrier to be administered comprises 2, 3, 4, 5, or more than 5, or more than 5 L-gammaglutamyl groups and 2, 3, 4, 5, or more than 5, or more than 5 D-gammaglutamyl groups. In some embodiments, the cancer is, for example, a non-hematological tumor including lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma, brain cancer, central nervous system cancer, and melanoma; and, for example, a hematological tumor selected from leukemia, lymphoma and other B cell malignancies, multiple myeloma and other plasma cell dysplasias or cachexia.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, 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. In some embodiments, the delivery carrier administered comprises γPANTIFOL containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the delivery carrier administered comprises a gamma-tetraglutamyl folate antagonist. In some embodiments, the delivery carrier administered comprises a gamma-pentaglutamyl folate antagonist. In other embodiments, the delivery carrier administered comprises a gamma-hexaglutamyl folate antagonist. In some embodiments, the delivery carrier administered comprises a polyglutamylated folate antagonist as described in any of items [1]-

[11] of the section of the Summary of the Invention. In some embodiments, the delivery carrier comprises a polyglutamylated folate antagonist as described in the section of the Summary of the Invention. In some embodiments, the delivery carrier administered is a liposomal composition comprising a polyglutamylated folate antagonist as described in any of items [1]-

[11] of the section of the Summary of the Invention. In some embodiments, γPANTIFOL is a polyglutamylated folate 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 of items

[12] -

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

[0038] In a further embodiment, the present disclosure provides a method of treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of liposomes comprising a gamma-polyglutamyl oxidized folic acid antimetabolite (e.g., Lp-γPANTIFOL such as PLp-γPANTIFOL, NTLp-γPANTIFOL, NTPLp-γPANTIFOL, TLp-γPANTIFOL or TPLp-γPANTIFOL). In some embodiments, the liposomes are pegylated. In some embodiments, the liposomes are not pegylated. In a further embodiment, the liposomes comprise a targeting moiety having a specific affinity for an epitope of an antigen on the surface of cancer cells. In a further embodiment, the liposomes comprise 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. 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 targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the liposome comprises γPANTIFOL containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome comprises a gamma tetraglutamyl oxidized folate antagonist. In some embodiments, the liposome comprises a gamma pentaglutamyl oxidized folate antagonist. In other embodiments, the liposome comprises a gamma hexaglutamyl oxidized folate antagonist. In some embodiments, the polyglutamyl oxidized folate antagonist comprises a folate antagonist described in any of items [1]-

[11] in the Summary section of the invention. In some embodiments, γPANTIFOL is a polyglutamyl oxidized folate antagonist described in the Summary section of the invention. In some embodiments, the liposome composition comprises a liposome described in any of items

[12] -

[67] in the Summary section of the invention. In some embodiments, the liposome comprises an L-gamma polyglutamyl oxidized folate antagonist. In some embodiments, the liposome comprises 2, 3, 4, 5, or more than 5 L-gammaglutamyl groups. In some embodiments, the liposome comprises a D-gamma polyglutamyl oxidized folate antagonist. In some embodiments, the liposome comprises 2, 3, 4, 5, or more than 5 D-gammaglutamyl groups. In some embodiments, the administered liposome comprises 2, 3, 4, 5, or more than 5 L-gammaglutamyl groups. In some embodiments, the liposome comprises L- and D-gamma polyglutamyl oxidized folate antagonists. In some embodiments, the liposome comprises 2, 3, 4, 5, or more than 5, or more than 5 L-gammaglutamyl groups and 2, 3, 4, 5, or more than 5 D-gammaglutamyl 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 cells are 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.

[0039] 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 gamma polyglutamyl oxidized folate antimetabolite and liposomes comprising a targeting moiety having specific affinity for an epitope of an antigen on the cancer surface. In some embodiments, the liposomes comprise 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 liposome comprises a targeting moiety that specifically binds to a cell surface antigen(s) derived from or determined to be expressed on a particular subject's tumor, such as a neoantigen. In some embodiments, the targeting moiety comprises an antibody or an antigen-binding antibody fragment. In some embodiments, the liposome comprises γPANTIFOL containing 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the liposome comprises a gamma-tetra-glutamyl oxidized folic acid antagonist. In some embodiments, the liposome comprises a gamma-penta-glutamyl oxidized folic acid antagonist. In other embodiments, the polyglutamyl oxidized folic acid antagonist comprises a gamma-hexa-glutamyl oxidized folic acid antagonist. In some embodiments, the polyglutamyl oxidized folic acid antagonist is a folic acid antagonist described in any of items [1]-

[11] of the section of the Summary of the Invention. In some embodiments, γPANTIFOL is a polyglutamyl oxidized 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 of items

[12] -

[67] of the section of the Summary of the Invention. In some embodiments, the liposome comprises γPANTIFOL containing an L-type γ-glutamyl group. In some embodiments, the liposome comprises γPANTIFOL containing a D-type γ-glutamyl group. In some embodiments, the liposome comprises γPANTIFOL containing at least one L-type γ-glutamyl group and at least one D-type γ-glutamyl group. 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 cells are 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.

[0040] In some embodiments, the administered liposomal composition comprises pegylated liposomes (e.g., TPLp-γPANTIFOL). In some embodiments, the administered liposomal composition comprises non-pegylated liposomes. In some embodiments, the liposomes of the administered liposomal composition comprise γPANTIFOL containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise a gamma-tetraglutamyl-methotrexate. In some embodiments, the liposomes of the administered liposomal composition comprise a gamma-pentaglutamyl-methotrexate. In other embodiments, the liposomes of the administered liposomal composition comprise a gamma-hexaglutamyl-methotrexate. In some embodiments, the liposomes comprise a polyglutamylated methotrexate as described in any of items [1] to

[11] of the summary section of the invention. In some embodiments, γPANTIFOL is a polyglutamylated methotrexate as described in the summary section of the invention. In some embodiments, the liposomal composition comprises liposomes as described in any of items

[12] to

[67] of the summary section of the invention. In some embodiments, the liposomal 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, myeloma and other plasma cell dyscrasias or cachexia, and leukemia, lymphoma and other B-cell malignancies. In some embodiments, the liposomal composition is administered for treating 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 liposomal composition is administered for treating 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.

[0041] 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 that expresses a folate receptor on its cell surface an effective amount of a liposomal composition, the liposomal composition comprising liposomes comprising (a) a gamma-polyglutamylated folate antagonist (γPANTIFOL) 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-γPANTIFOL). In some embodiments, the liposomal composition to be administered comprises non-pegylated liposomes. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPANTIFOL comprising 4, 5, 2 to 10, 4 to 6, or more than 5 gamma-glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise a gamma-tetra-glutamylated folate antagonist. In some embodiments, the liposomes of the liposomal composition to be administered comprise a gamma-penta-glutamylated folate antagonist. In other embodiments, the liposomes of the liposomal composition to be administered comprise a gamma-hexa-glutamylated folate antagonist. In some embodiments, the liposomes comprise a polyglutamylated folate antagonist as described in any of items [1] to

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

[12] to

[67] of the summary section of the invention.In some embodiments, the liposomal 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, for example, hematological tumors such as leukemia, lymphoma and other B - cell malignancies, multiple myeloma and other plasma cell dyscrasias or cachexia. In some embodiments, the liposomal composition is administered to treat cancer selected from 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 liposomal composition is administered to treat lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the liposomal composition is administered to treat breast cancer (e.g., HER2 ++ or triple - negative breast cancer). In some embodiments, the liposomal composition is administered to treat colorectal cancer. In some embodiments, the liposomal composition is administered to treat ovarian cancer. In some embodiments, the liposomal composition is administered to treat endometrial cancer. In some embodiments, the liposomal composition is administered to treat pancreatic cancer. In some embodiments, the liposomal composition is administered to treat liver cancer. In some embodiments, the liposomal composition is administered to treat head and neck cancer. In some embodiments, the liposomal composition is administered to treat osteosarcoma.

[0042] In a further embodiment, the present disclosure provides a method for maintenance therapy of cancer, the method comprising administering to a subject who is undergoing or has undergone cancer therapy an effective amount of a liposomal composition (Lp-γPANTIFOL) comprising a gamma-polyglutamylated folate antagonist. In some embodiments, the liposomal composition to be administered is PLp-γPANTIFOL, NTLp-γPANTIFOL, NTPLp-γPANTIFOL, TLp-γPANTIFOL or TPLp-γPANTIFOL. In some embodiments, the liposomes of the liposomal composition to be administered comprise pegylated liposomes (e.g., PLp-γPANTIFOL, NTPLp-γPANTIFOL, or TPLp-γPANTIFOL). In some embodiments, the liposomal composition to be administered comprises targeted liposomes (e.g., TLp-γPANTIFOL or TPLp-γPANTIFOL). In some embodiments, the liposomal composition to be administered comprises liposomes that are pegylated and comprise a targeting moiety (e.g., TPLp-γPANTIFOL). In some embodiments, the liposomes of the liposomal composition to be administered comprise a gamma-polyglutamylated folate antagonist comprising 4, 5, 2-10, 4-6, or more than 5 gamma-glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise a gamma-tetra-glutamylated folate antagonist. In some embodiments, the liposomes of the liposomal composition to be administered comprise a gamma-penta-glutamylated folate antagonist. In some embodiments, the liposomes of the liposomal composition to be administered comprise a gamma-hexa-glutamylated folate antagonist. In some embodiments, the liposomal composition comprises liposomes comprising a gamma-polyglutamate as described in any of items [1]-

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

[12] -

[67] of the summary section of the invention.

[0043] In further embodiments, the present disclosure provides a method of treating a disorder of the immune system, the method comprising administering to a subject having or at risk of having a disorder of the immune system an effective amount of a liposomal composition comprising a gamma polyglutamylated folic acid antimetabolite (e.g., Lp-γPANTIFOL, PLp-γPANTIFOL, NTLp-γPANTIFOL, NTPLp-γPANTIFOL, TLp-γPANTIFOL or TPLp-γPANTIFOL). 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's disease, and psoriasis. In some embodiments, the liposomal composition administered comprises a pegylated liposome (e.g., PLp-γPANTIFOL, NTPLp-γPANTIFOL, or TPLp-γPANTIFOL). In some embodiments, the liposomal composition administered comprises a targeted liposome (e.g., TLp-γPANTIFOL or TPLp-γPANTIFOL) comprising a targeting moiety having specific affinity for a surface antigen on a target cell of interest (e.g., an immune cell). In further embodiments, the liposomal composition administered comprises a liposome that is pegylated and comprises a targeting moiety (e.g., TPLp-γPANTIFOL). In some embodiments, the liposomes of the liposomal composition administered comprise a gamma polyglutamylated folic acid antimetabolite comprising 4, 5, 2 to 10, 4 to 6, or more than 5 gamma-glutamyl groups. In some embodiments, the liposomes of the liposomal composition administered comprise a gamma-tetra-glutamylated folic acid antimetabolite. In some embodiments, the liposomes of the liposomal composition administered comprise a gamma-penta-glutamylated folic acid antimetabolite. In other embodiments, the liposomes of the liposomal composition administered comprise a gamma-hexa-glutamylated folic acid antimetabolite.In some embodiments, the liposome composition comprises liposomes containing gamma polyglutamate as described in any of items [1] to

[11] of the section of the Summary of the Invention. In some embodiments, γPANTIFOL is a polyglutaminated folic acid antimetabolite as described in the section of the Summary of the Invention. In some embodiments, the liposome composition comprises liposomes as described in any of items

[12] to

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

[0044] In further embodiments, 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 a gamma polyglutamyl oxidized folic acid antagonist (e.g., such as Lp-γPANTIFOL, PLp-γPANTIFOL, NTLp-γPANTIFOL, NTPLp-γPANTIFOL, TLp-γPANTIFOL or TPLp-γPANTIFOL). In some embodiments, the autoimmune disease is rheumatoid arthritis. In some embodiments, the autoimmune disease is a disease or disorder 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, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis. In some embodiments, the liposomal composition administered comprises a pegylated liposome (e.g., PLp-γPANTIFOL, NTPLp-γPANTIFOL, or TPLp-γPANTIFOL). In some embodiments, the liposomal composition administered comprises a targeted liposome (e.g., TLp-γPANTIFOL or TPLp-γPANTIFOL) comprising a targeting moiety having specific affinity for a surface antigen on a target cell of interest (e.g., an immune cell). In further embodiments, the liposomal composition administered comprises a liposome that is pegylated and comprises a targeting moiety (e.g., TPLp-γPANTIFOL). In some embodiments, the liposomes of the liposomal composition administered comprise a gamma polyglutamyl oxidized folic acid antagonist comprising 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the liposomal composition administered comprise a gamma tetraglutamyl oxidized folic acid antagonist.In some embodiments, the liposomes of the administered liposomal composition comprise a gamma pentaglutamyl oxidized folate antagonist. In other embodiments, the liposomes of the administered liposomal composition comprise a gamma hexaglutamyl oxidized folate antagonist. In some embodiments, the liposomes comprise a polyglutamyl oxidized folate antagonist as described in any of items [1] to

[11] of the section of the summary of the invention. In some embodiments, γPANTIFOL is a polyglutamyl oxidized folate antagonist as described in the section of the summary of the invention. In some embodiments, the liposomal composition comprises liposomes as described in any of items

[12] to

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

[0045] 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 a gamma-polyglutamyl oxidized folic acid antimetabolite (e.g., Lp-γPANTIFOL, PLp-γPANTIFOL, NTLp-γPANTIFOL, NTPLp-γPANTIFOL, TLp-γPANTIFOL or TPLp-γPANTIFOL). 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, gonococcal arthritis, viral arthritis), connective tissue inflammation, 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 liposomal composition administered comprises a pegylated liposome (e.g., PLp-γPANTIFOL, NTPLp-γPANTIFOL, or TPLp-γPANTIFOL). In some embodiments, the liposomal composition administered comprises a targeted liposome (e.g., TLp-γPANTIFOL or TPLp-γPANTIFOL) 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 a liposome that is pegylated and comprises a targeting moiety (e.g., TPLp-γPANTIFOL).In some embodiments, the liposomes of the administered liposomal composition comprise a gamma pentaglutamine oxidized folate antagonist containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise a gamma tetraglutamine oxidized folate antagonist. In some embodiments, the liposomes of the administered liposomal composition comprise a gamma pentaglutamine oxidized folate antagonist. In other embodiments, the liposomes of the administered liposomal composition comprise a gamma hexaglutamine oxidized folate antagonist. In some embodiments, the liposomes comprise a polyglutamine oxidized folate antagonist as described in any of items [1] to

[11] of the section of the Summary of the Invention. In some embodiments, γPANTIFOL is a polyglutamine oxidized folate antagonist as described in the section of the Summary of the Invention. In some embodiments, the liposomal composition comprises liposomes as described in any of items

[12] to

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

[0046] The present disclosure also provides a method for delivering a gamma-polyglutamyl oxidized folic acid antimetabolite to an inflammatory site of a subject, the method comprising administering to a subject having inflammation a composition comprising a gamma-polyglutamyl oxidized folic acid antimetabolite (L-γPANTIFOL) 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 via pro-inflammatory cytokine production). 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-γPANTIFOL). In some embodiments, the composition being administered comprises a gamma-polyglutamyl oxidized folic acid antimetabolite comprising 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the composition being administered comprises a gamma-tetra-glutamyl oxidized folic acid antimetabolite. In some embodiments, the composition being administered comprises a gamma-penta-glutamyl oxidized folic acid antimetabolite. In other embodiments, the composition being administered comprises a gamma-hexa-glutamyl oxidized folic acid antimetabolite. In some embodiments, γPANTIFOL is a polyglutamyl oxidized folic acid antimetabolite as described in any of items [1] to

[11] of the Summary section of the invention. In some embodiments, γPANTIFOL is a polyglutamyl oxidized folic acid antimetabolite as described in the Summary section of the invention. In some embodiments, the delivery carrier is a liposome as described in any of items

[12] to

[67] of the Summary section of the invention.

[0047] The present disclosure also provides a method for delivering a gamma-polyglutamylated folate antimetabolite to a tumor and / or cancer cell, the method comprising administering to a subject having a tumor a composition comprising a gamma-polyglutamylated folate antimetabolite (L-γPANTIFOL) 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-γPANTIFOL). In some embodiments, the composition to be administered comprises a gamma-polyglutamylated folate antimetabolite comprising 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the composition to be administered comprises a gamma-tetraglutamylated folate antimetabolite. In some embodiments, the composition to be administered comprises a gamma-pentaglutamylated folate antimetabolite. In other embodiments, the composition to be administered comprises a gamma-hexaglutamylated folate antimetabolite. In some embodiments, the composition to be administered is a folate antimetabolite described in any of items [1] to

[11] of the Summary section of the invention. In some embodiments, γPANTIFOL is a polyglutamylated folate antimetabolite described in the Summary section of the invention. In some embodiments, the composition to be administered comprises a liposome described in any of items

[12] to

[67] of the Summary section of the invention.

[0048] In a further embodiment, the present disclosure provides a method of preparing a liposomal composition comprising a liposomal gamma polyglutamate antifolate (γPANTIFOL) composition, the method comprising the steps of forming a mixture comprising liposomal components and a gamma polyglutamate antifolate in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes comprising a polyglutamate antifolate. In some embodiments, the gamma polyglutamate antifolate comprises 4, 5, 2 to 10, 4 to 6, or more than 5 gamma glutamyl groups. In some embodiments, γPANTIFOL comprises a gamma tetraglutamate antifolate. In some embodiments, γPANTIFOL comprises a gamma pentaglutamate antifolate. In other embodiments, γPANTIFOL comprises a gamma hexaglutamate antifolate. In some embodiments, γPANTIFOL is a polyglutamate antifolate as described in any of items [1] to

[11] of the section of the Summary of the Invention. In some embodiments, γPANTIFOL is a polyglutamate antifolate as described in the section of the Summary of the Invention. In some embodiments, the liposomal composition comprises liposomes as described in any of items

[12] to

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

[0049] In one embodiment, the present disclosure provides a kit comprising a folate antagonist gamma polyglutamate composition and / or a γPANTIFOL delivery carrier such as a liposome comprising γPANTIFOL and a γPANTIFOL immunocomplex (e.g., ADC) as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0050]

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Fig. 21A-F

Mode for Carrying Out the Invention

[0051] Generally, the present disclosure relates to gamma - polyglutamylated folate antimetabolite compositions. The compositions provide an advance over prior treatments for hyperproliferative diseases such as cancer. Methods for the manufacture, delivery, and use of gamma - polyglutamylated folate antimetabolite compositions are also provided. The gamma - 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.

[0052] 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.

[0053] When an embodiment is described herein using the term "comprising", other similar embodiments described in terms of "containing", "consisting of", and / or "consisting essentially of" are provided as well. However, when used as a transitional phrase in the claims, each should be interpreted separately and in an appropriate legal and factual context (e.g., in the claims, the transitional phrase "comprising" is considered a more open-ended phrase, "consisting of" is considered more exclusive, and "consisting essentially of" is considered intermediate between these).

[0054] 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 a plural referent is not intended.

[0055] 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" includes each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0056] Headings and subheadings are used for convenience only and / or for compliance with official rules, and do not limit the subject technology nor are they referred to in connection with the interpretation of the description of the subject technology. Features described under one heading or subheading of the subject disclosure may be combined with features described under other headings or subheadings in various embodiments. Further, not all features under a single heading or subheading are necessarily used together in some embodiments.

[0057] 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 various cations such as Na + 、Mg 2+ 、K + 、NH 4+ 、and / or Ca 2+ of any of them. In certain embodiments, the salt is a pharmaceutically acceptable salt. The antifol contains one L-gamma-glutamyl group and is thus considered to be monoglutaminylated for the purposes of the present disclosure.

[0058] The compounds of the present invention can exist as mixtures of stereoisomers, but they are preferably resolved as 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 achieving the desired activity.

[0059] However, as shown previously, the cyclopenta[g]quinazolines of the present invention contain at least three asymmetric carbon atoms. Of these, the asymmetric carbon atoms at six positions of 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 less preferred are mixtures in which one or both of these asymmetric carbon atoms are not resolved.

[0060] The folic acid antagonist can be any known one or a folic acid antagonist in which folic acid or polyglutaminated folic acid is oxidized and will be induced in the future. In some embodiments, the folic acid 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-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]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, or a stereoisomer selected therefrom;

[0061] In some embodiments, the folic acid antagonist is a member selected from the following: aminopterin, methotrexate, raltitrexed (also known as TOMUDEX®, ZD1694 (RTX)), premetrexed (BGC9331; also known as ZD9331), pemetrexed (also known as ALIMTA, LY231514), lometrexol (LTX) (5,10-dideazatetrahydrofolic acid), cyclopenta[g]quinazoline having a dipeptide ligand, CB3717, CB300945 (also known as BGC945), or 6-R,S-BGC945 (ONX-0801), CB300638 (also known as BGC638), and their stereoisomers such as BW1843U89.

[0062] The terms "polyglutamylated folic acid antagonist", "polyglutamylated ANTIFOL", "ANTIFOL-PG", "PANTIFOL" are used interchangeably herein and mean a folic acid antagonist composition containing at least one glutamyl group in addition to the glutamyl group in the folic acid antagonist (i.e., ANTIFOL-PGn, n≧1). References to the number of glutamyl groups in γPANTIFOL (ANTIFOL-PG) herein account for the glutamyl groups in the folic acid antagonist. For example, an ANTIFOL-PG composition containing 5 glutamyl residues in addition to the glutamyl group of ANTIFOL is referred to herein as a hexaglutamylated folic acid antagonist or folic acid antagonist hexaglutamate. The polyglutamate chain contains an N-terminal glutamyl group and one or more C-terminal glutamyl groups. The N-terminal glutamyl group of the polyglutamate chain is not bound to another glutamyl group via its amino group, but is bound to one or more glutamyl groups via its carboxylic acid group. In some embodiments, the N-terminal glutamyl group of the polyglutamylated folic acid antagonist is the glutamyl group of the folic acid antagonist. The C-terminal glutamyl group(s) of the polyglutamate chain are bound to another glutamyl group via their amino groups, but are not bound to another glutamyl group via their carboxylic acid groups.

[0063] The terms "alpha-glutamyl group", "alpha-glutamate", "alpha bond", and iterations thereof, when they relate to the bonding of glutamyl groups, mean a glutamyl group that includes an alpha-carboxyl group bond. In some embodiments, none of the glutamyl groups of the provided polyglutamated folate antagonists include an alpha bond.

[0064] The terms "gamma-glutamyl group", "gamma-glutamate", and "gamma bond", when they relate to the bonding of glutamyl groups, mean a glutamyl group that includes a gamma-carboxyl group bond. In some embodiments, the gamma bond is an amide bond between the gamma-carboxyl group of one glutamyl group and a second glutamyl group. The gamma 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 within a polyglutamate chain that is not present in the folate antagonist but is attached to the folate antagonist. In some embodiments, the gamma bond means an amide bond of a glutamyl group of a folate antagonist. References to gamma bonds include gamma bonds of glutamyl groups of folate antagonists unless specifically stated otherwise or it is clear from the context that something other than that is intended. In some embodiments, the gamma-glutamyl group is of the L-form. In some embodiments, the gamma-glutamyl group is of the D-form. As discussed herein, during folate antagonist therapy, the folate antagonist enters cells and is polyglutamated by the enzyme holylpoly-gamma-glutamate synthetase (FPGS), which adds L-glutamyl groups in series to the gamma-carboxyl group of glutamate within the L-glutamyl group of the folate antagonist. As a result, D-gamma-polyglutamated folate antagonist compositions are not formed intracellularly during folate antagonist therapy

[0065] The terms "gamma-polyglutamylated folate antimetabolite", "γ-polyglutamylated folate antimetabolite", "γPANTIFOL", "gamma-polyglutamylated folate antimetabolite", "polyglutamylated folate antimetabolite", "γANTIFOL-PG", and iterations thereof are used interchangeably herein and mean a folate antimetabolite composition containing at least one gamma-glutamyl group having a gamma-carboxy group bond in addition to the gamma-glutamyl group in the folate antimetabolite (e.g., ANTIFOL-PG n , where n ≧ 1 gamma-glutamyl groups). References herein to the number of glutamyl groups in γPANTIFOL (γANTIFOL-PG) include the gamma-glutamyl groups of the folate antimetabolite. For example, a γANTIFOL-PG composition containing five gamma-glutamyl groups in addition to the glutamyl groups of the folate antimetabolite may be referred to herein as a gamma-hexaglutamylated folate antimetabolite or gamma-folate antimetabolite hexaglutamate.

[0066] The terms "alpha-glutamyl group", "α-glutamyl group", and "alpha bond" refer to a glutamyl group containing an alpha-carboxyl group bond when these relate to the bonding of glutamyl groups.

[0067] As used herein, the term "isolated" means a composition in a form not found in nature. Isolated gamma-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 gamma-polyglutamine oxidized folate antagonists are substantially pure. An isolated composition is free or substantially free of naturally incorporated substances such as 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). Gamma-polyglutamine oxidized compositions can be formulated with a diluent or adjuvant and further isolated for practical purposes - for example, when used in a diagnostic or therapeutic agent, the gamma-polyglutamine oxidized composition is usually mixed with a pharmaceutically acceptable carrier or diluent. In some embodiments, the isolated gamma-polyglutamine oxidized composition (e.g., a delivery vehicle such as a gamma-polyglutamate and liposomes containing gamma-polyglutamate) contains less than 1% or less than 0.1% of unwanted DNA or protein contaminants. In some embodiments, the gamma-polyglutamate composition (e.g., a delivery vehicle such as a gamma-polyglutamate and liposomes containing gamma-polyglutamate) is "isolated".

[0068] As used herein, the term "targeting moiety" means a molecule that confers enhanced affinity for a selected target, such as a cell, cell type, tissue, organ, region of the body, or compartment, e.g., a compartment of a cell, tissue or organ. The targeting moiety can include a wide variety of entities. 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.

[0069] The term "specific affinity" or "specifically binds" means that a targeting moiety, such as an antibody or an antigen-binding antibody fragment, reacts or binds to an epitope, protein, or target molecule with a higher frequency, more rapidly, for a longer period, with greater affinity, or in some combination of these, than 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 term "specific affinity" or "specific binding" may 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 may specifically bind to two or more targets. In certain embodiments, multiple targets may be bound by the same targeting moiety.

[0070] 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 moiety), such as an antibody. When the antigen is a polypeptide, the epitope can be formed from both contiguous and non-contiguous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed from contiguous amino acids are usually retained 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 commonly at least 5 or 8 - 10 amino acids, in a unique spatial higher-order structure.

[0071] Expressions such as "binding affinity for a target" and "binding to a target", which are known in the art, and similar expressions, refer to the properties of a targeting moiety that can be directly measured by determining the affinity constant, e.g., the amount of targeting moiety that binds and dissociates at a given antigen concentration. Without limitation, intermolecular interactions can be characterized using other methods such as competitive analysis, equilibrium analysis, and microcalorimetry, and real-time interaction analysis based on surface plasmon resonance interactions (e.g., using a BIACORE® instrument). These methods are well known to those skilled in the art and are described, for example, in Neri et al., Tibtech 14:465-470 (1996), and Jansson et al., J. Biol. Chem. 272:8189-8197 (1997).

[0072] The term "delivery vehicle" generally means any composition that acts to assist, facilitate, or ease the entry of a gamma-polyglutamyl oxidized folic acid antimetabolite into cells. Such delivery vehicles are known in the art and include, without limitation, liposomes, lipospheres, polymers (e.g., polymer conjugates), peptides, proteins such as antibodies (e.g., immunoconjugates such as antibody-drug conjugates (ADCs) and antigen-binding antibody fragments and their derivatives), 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 substances, or lipid or liposome formulations, and combinations thereof. The delivery vehicle can be directly or indirectly bound to the targeting moiety. In some embodiments, the targeting moiety is selected from a polymer, protein, peptide, monoclonal antibody, or fatty acid lipid.

[0073] "Subject" means a vertebrate mammal, including but not limited to humans, dogs, cats, horses, goats, and primates such as monkeys. Thus, the present invention can also be used to treat diseases or conditions in non-human subjects. For example, cancer is one of the leading causes of death in companion animals (e.g., cats and dogs). In some embodiments of the present invention, the subject is a human. In the present disclosure, the terms "subject" and "patient" are used interchangeably and have the same meaning. Generally, the maximum dose, i.e., the maximum safe dose according to sound medical judgment, is preferably used.

[0074] As used herein, "effective amount" means an amount of a drug sufficient to produce a medically desirable result. The effective amount can vary depending on the desired outcome, the particular condition to be treated or prevented, the age and health status 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 practitioner. The "effective amount" can be determined experimentally and routinely in relation to the indicated purpose. In the case of cancer, an effective amount of a drug 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 to some extent and preferably stop); inhibit the metastasis of the tumor (i.e., slow down to some extent and preferably stop); 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, for example, by evaluating survival time, progression-free survival (PFS) period, response rate (RR), response duration, and / or quality of life.

[0075] The terms "hyperproliferative disorder", "proliferative disease", and "proliferative disorder" are used interchangeably herein and relate to unwanted or abnormal cell growth, such as neoplastic or hyperplastic growth, whether in vitro or in vivo, of unwanted or uncontrolled cells. In some embodiments, a proliferative disease is a cancer or tumor disease (including benign or cancerous) and / or any tumor metastasis, regardless of the location of the cancer, tumor and / or tumor metastasis. In some embodiments, a proliferative disease is a benign or malignant tumor. In some embodiments, a proliferative disease is a non-cancerous disease. In some embodiments, a proliferative disease is a hyperproliferative condition such as hyperplasia, fibrosis (particularly pulmonary, but also other types such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis, and smooth muscle proliferation in blood vessels such as stenosis or restenosis after angioplasty.

[0076] "Cancer", "tumor", or "malignant tumor" are used as synonymous terms and mean any of a number of diseases characterized by uncontrolled, abnormal growth of cells, local or spread (metastasis) to other parts of the body via the bloodstream and lymphatic system of infected cells, 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 γPANTIFOL compositions provided herein include, but are not limited to, 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, etc., non-hematological tumors; and, for example, leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias or cachexia, etc., hematological tumors. 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. Other types of cancers and tumors that can be treated with the γPANTIFOL 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 or organ or tissue system within the subject of the tumor, cancer or neoplasm, and the site, location, region or organ or tissue system within the subject 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 herein.

[0077] 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, a subject is "being treated successfully" by the methods provided herein if the subject exhibits, for example, a complete, partial, or temporary remission or elimination of symptoms associated with a disease or condition (e.g., cancer, inflammation, and rheumatoid arthritis). In certain embodiments, the term "treating" or "treatment" or "treat" means an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, that may not be distinguishable by the patient. In other embodiments, the term "treating" or "treatment" or "treat" means inhibiting the progression of a proliferative disorder, for example, physically by stabilization of distinguishable symptoms or physiologically by stabilization of physical parameters, or both. In other embodiments, the term "treating" or "treatment" or "treat" means a decrease or stabilization in size, tumor cell proliferation or survival, or cancer cell number. The γPANTIFOL composition can be used alone or in combination with additional therapeutic agents for treatment.

[0078] "Subject", "patient", and "animal" are used synonymously and mean mammalian patients 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; domestic 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.

[0079] As used herein, "treatment of a proliferative disorder" includes maintaining or reducing the tumor size of a subject with a proliferative disorder, inducing (partial or complete) tumor shrinkage, suppressing tumor growth, and / or extending lifespan. In one embodiment, the proliferative disorder is a solid tumor. Such tumors include, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma. In one embodiment, the proliferative disorder is a hematological tumor. Such hematological tumors include, for example, leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias or malignancies.

[0080] 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.

[0081] The terms "inflammation" and "inflammatory disease" are used interchangeably and refer to a disease or disorder characterized by or caused by inflammation. "Inflammation" refers to a local reaction to cellular injury characterized by capillary dilation, leukocyte infiltration, erythema, heat, and pain, which serves to initiate the mechanism for 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, rheumatoid 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, cellulitis, 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 primarily chronic inflammatory disease of the joints, usually polyarticular, characterized by inflammatory changes in the synovium and joint structures as well as muscle spasm and bone rarefaction.

[0082] As used herein, the term "therapeutic agent" means an agent, or a derivative 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 in the methods of the present disclosure include, but are not limited to, anti-restenosis agents, pro-proliferative or anti-proliferative agents, anti-inflammatory agents, anti-neoplastic agents, anti-mitotic agents, antiplatelet agents, anticoagulants, antifibrin agents, antithrombin agents, cell growth inhibitory agents, antibiotics and other anti-infective agents, anti-enzyme agents, antimetabolites, angiogenesis agents, cytoprotective agents, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists and / or cardioprotective agents. "Therapeutic agent" also means salts, acids, and free base forms of the above agents.

[0083] As used herein, the term "chemotherapeutic agent" means, when used in the context of cancer therapy, any agent that causes the death of cancer cells or inhibits the growth or spread of cancer cells. Examples of such chemotherapeutic agents include alkylating agents, antibiotics, antimetabolites, plant-derived drugs, and hormones. In some embodiments, the 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.

[0084] As used herein, the term "antimetabolite" means an antitumor agent that inhibits the utilization of metabolites or their prodrugs. 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 gamma-polyglutamylated 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.

[0085] 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.

[0086] The terms "pharmaceutically-acceptable carrier" and "pharmaceutically acceptable carrier" mean components other than the active ingredient in a pharmaceutical formulation that are non-toxic to the subject. 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 a human or other subject.

[0087] The present disclosure generally relates to compositions of gamma-polyglutamyl folate antagonists (γANTIFOL), and methods of making and using compositions for treating diseases including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV, malaria, and schistosomiasis.

[0088] In some embodiments, the present disclosure provides the following. [1] A composition comprising a gamma-polyglutamyl folate antagonist; [2] The composition according to [1], wherein the folic acid antagonist is selected from piritrexim, pralatrexate, AG2034, GW1843, and LY309887, and / or their stereoisomers; [3] The composition according to [1], wherein the folic acid antagonist is selected from PMX, MTX, RTX, and LTX, or their stereoisomers; [4] The composition according to any one of items [1] to [3], 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, 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-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.; A composition according to [5][1], wherein the folic acid antagonist is selected from methotrexate, raltitrexed, premetrexed, pemetrexed, lometrexol (LTX; 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; A composition according to any one of items [1] to [5] of [6], wherein the gamma-polyglutamylated folic acid antagonist contains 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups; [7] The composition according to any one of items [1] to [6], wherein the gamma-polyglutamyl oxidized folic acid metabolism antagonist is (a) a gamma-tetraglutamyl oxidized folic acid metabolism antagonist, or (b) a gamma-pentaglutamyl oxidized folic acid metabolism antagonist, or (c) a gamma-hexaglutamyl oxidized folic acid metabolism antagonist, Composition; [8] The composition according to any one of items [1] to [7], wherein the gamma-polyglutamyl oxidized folic acid metabolism antagonist contains 1 to 10 glutamyl groups having a gamma-carboxyl group bond, Composition; [9] The composition according to any one of items [1] to [8], wherein (a) at least two glutamyl groups of the gamma-polyglutamyl oxidized folic acid metabolism antagonist are of the L type; (b) each glutamyl group of the gamma-polyglutamyl oxidized folic acid metabolism antagonist is of the L type; (c) at least one glutamyl group of the gamma-polyglutamyl oxidized folic acid metabolism antagonist is of the D type; (d) each glutamyl group of the gamma-polyglutamyl oxidized folic acid metabolism antagonist other than the glutamyl group of the folic acid metabolism antagonist is of the D type; or (e) at least two glutamyl groups of the gamma-polyglutamyl oxidized folic acid metabolism antagonist are of the L type and at least one glutamyl group is of the D type, Composition:

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

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

[12] A liposome composition (Lp-γPANTIFOL) containing the gamma-polyglutamyl oxidized folic acid metabolism antagonist according to any one of items [1] to

[11] ;

[13] The Lp-γPANTIFOL composition according to item

[12] , wherein the polyglutamyl oxidized folic acid metabolism antagonist is an Lp-γPANTIFOL composition selected from the following: (a) AG2034, Piritrexim, Pralatrexate, GW1843, folic acid metabolism antagonists, and LY309887; or (b) PMX, MTX, RTX and LTX, and their stereoisomers;

[14] The Lp-γPANTIFOL composition according to item

[12] or

[13] , wherein the polyglutamated folic acid antagonist is selected from the following Lp-γPANTIFOL compositions: 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, 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]-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 their stereoisomers;

[15] An Lp-γPANTIFOL composition according to any one of items

[12] to

[14] , 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 6-R,S-BGC945 (ONX-0801), CB300638, and BW1843U89 and their stereoisomers such as;

[16] An Lp-γPANTIFOL composition according to any one of items

[12] to

[15] , wherein the liposome contains a gamma-polyglutamylated folic acid antagonist containing 4, 5, 2 to 10, 4 to 6, or more than 5 gamma-glutamyl groups;

[17] An Lp-γPANTIFOL composition according to any one of items

[12] to

[16] , wherein the liposome contains a gamma-tetraglutamylated folic acid antagonist;

[18] An Lp-γPANTIFOL composition according to any one of items

[12] to

[16] , wherein the liposome contains a gamma-pentaglutamylated folic acid antagonist;

[19] An Lp-γPANTIFOL composition according to any one of items

[12] to

[16] , wherein the liposome contains a gamma-hexaglutamylated folic acid antagonist;

[20] An Lp-γPANTIFOL composition according to any one of items

[12] to

[19] , wherein the gamma-polyglutamylated folic acid antagonist contains 1 to 10 glutamyl groups having a gamma-carboxyl group bond;

[21] An Lp-γPANTIFOL composition according to any one of items

[12] to

[20] , (a) At least two glutamyl groups of the gamma-polyglutamylated folic acid antagonist are of the L type; (b) Each glutamyl group of the gamma-polyglutamylated folic acid antagonist is of the L type; (c) At least one glutamyl group of the gamma-polyglutamyl oxidized folic acid antimetabolite is of the D-type; (d) Each glutamyl group of the gamma-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 gamma-polyglutamyl oxidized folic acid antimetabolite are of the L-type and at least one glutamyl group is of the D-type, Lp-γPANTIFOL composition:

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

[12] to

[21] , (a) At least two glutamyl groups of the gamma-polyglutamyl oxidized folic acid antimetabolite are of the L-type; (b) Each glutamyl group of the gamma-polyglutamyl oxidized folic acid antimetabolite is of the L-type; (c) At least one glutamyl group of the gamma-polyglutamyl oxidized folic acid antimetabolite is of the D-type; (d) Each glutamyl group of the gamma-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 gamma-polyglutamyl oxidized folic acid antimetabolite are of the L-type and at least one glutamyl group is of the D-type, Lp-γPANTIFOL composition:

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

[12] to

[22] , being a Lp-γPANTIFOL composition in which the liposome is pegylated (PLp-γPANTIFOL);

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

[12] to

[22] , being a Lp-γPANTIFOL composition in which the liposome is not pegylated;

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

[12] to

[24] , being a Lp-γPANTIFOL composition in which the liposome has a diameter in the range of 20 nm to 200 nm;

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

[12] to

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

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

[12] to

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

[28] The Lp-γPANTIFOL composition according to item

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

[29] The Lp-γPANTIFOL composition according to item

[27] or

[28] , 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;

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

[27] to

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

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

[27] to

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

[32] The Lp-γPANTIFOL composition according to item

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

[33] The Lp-γPANTIFOL composition according to item

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

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

[12] to

[33] , wherein the liposome is anionic or neutral; an Lp-γPANTIFOL composition;

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

[12] to

[33] , wherein the liposome has a zeta potential of zero or less; an Lp-γPANTIFOL composition;

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

[12] to

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

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

[12] to

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

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

[12] to

[33] , wherein the liposome is cationic; an Lp-γPANTIFOL composition;

[39] An Lp-γPANTIFOL composition according to any one of items

[12] to

[38] , wherein the liposome has an internal space containing a gamma polyglutamine oxidized folic acid antagonist and an aqueous pharmaceutically acceptable carrier;

[40] An Lp-γPANTIFOL composition according to item

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

[41] An Lp-γPANTIFOL composition according to item

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

[42] An Lp-γPANTIFOL composition according to item

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

[43] An Lp-γPANTIFOL composition according to any one of items

[39] to

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

[44] An Lp-γPANTIFOL composition according to any one of items

[39] to

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

[45] An Lp-γPANTIFOL composition according to any one of items

[39] to

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

[46] An Lp-γPANTIFOL composition according to any one of items

[39] to

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

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

[12] to

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

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

[12] to

[47] , wherein the liposome contains less than 500,000 or less than 200,000 gamma polyglutamylated folic acid antimetabolite molecules;

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

[12] to

[48] , wherein the liposome contains 10 to 100,000 or any range therebetween of gamma polyglutamylated folic acid antimetabolite molecules;

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

[12] to

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

[51] The Lp-γPANTIFOL composition according to item

[50] , 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;

[52] The Lp-γPANTIFOL composition according to item

[50] or

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

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

[50] to

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

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

[50] to

[53] , wherein the targeting moiety, as measured by BIACORE® analysis, is 0.5x10 -10 ~10x10 -6An Lp-γPANTIFOL composition that binds to a surface antigen with an equilibrium dissociation constant (Kd) in the range of;

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

[50] to

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

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

[50] to

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

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

[50] to

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

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

[39] to

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

[59] The Lp-γPANTIFOL composition according to item

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

[60] The Lp-γPANTIFOL composition according to item

[58] or

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

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

[58] to

[60] , wherein the immunostimulant and the detectable marker are the same, the Lp-γPANTIFOL composition;

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

[58] to

[61] , further comprising a hapten, the Lp-γPANTIFOL composition;

[63] The Lp-γPANTIFOL composition according to item

[62] , wherein the hapten comprises one or more of fluorescein or beta-1,6-glucan, the Lp-γPANTIFOL composition:

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

[12] to

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

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

[64] ;

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

[49] ;

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

[12] to

[66] , further comprising carboplatin and / or pembrolizumab, the Lp-γPANTIFOL composition;

[68] A pharmaceutical composition comprising the liposomal gamma polyglutamine oxidized folic acid antagonist composition according to any one of items

[12] to

[67] ;

[69] A pharmaceutical composition comprising the gamma polyglutamine oxidized folic acid antagonist composition according to any one of items [1] to [7];

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

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

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

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

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

[70] to the subject;

[73] A method for treating or preventing a disease in a subject in need of such treatment or prevention, the method comprising administering to the subject the liposomal gamma-polyglutamylated folic acid antimetabolite composition according to any one of

[12] to

[69] ;

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

[69] ;

[75] A method for killing hyperproliferative cells, the method comprising the step of contacting the hyperproliferative cells with the liposomal gamma-polyglutamylated folic acid antimetabolite composition according to any one of items

[12] to

[69] .

[76] The method according to item

[74] or

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

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

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

[78] A method for treating cancer, the method comprising the step of administering an effective amount of the liposomal gamma-polyglutamylated folic acid antimetabolite composition according to any one of items

[12] to

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

[79] The method according to item

[77] or

[78] , 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;

[80] The method according to item

[77] or

[78] , wherein the cancer is selected from lung cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, ovarian cancer, and cervical cancer;

[81] The method according to item

[77] or

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

[82] The method according to item

[77] or

[78] , wherein the cancer is selected from colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma;

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

[50] to

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

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

[69] to the subject;

[85] Maintenance therapy for a subject who is receiving or has received cancer therapy, comprising administering an effective amount of the liposomal gamma-polyglutamine oxidized metabolite antagonist composition according to any one of items

[12] to

[69] to the subject;

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

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

[87] Administering an effective amount of the liposomal gamma polyglutamyl oxidized folic acid metabolic antagonist composition according to any one of items [8] to

[69] to a subject having or at risk of having an immune system disorder, optionally, the immune system disorder being 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; a method for treating an immune system disorder;

[88] 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

[69] 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

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

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

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

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

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

[89] A method for treating an infectious disease, comprising the step of administering an effective amount of the liposomal gamma-polyglutamylated folic acid metabolic antagonist composition according to any one of items

[12] to

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

[90] A method for delivering a gamma-polyglutamylated folic acid metabolic antagonist to a tumor expressing a folate receptor on its surface, the method comprising the step of administering to a subject having a tumor an Lp-γPANTIFOL composition according to any one of items [1] to

[69] in an amount sufficient to deliver a therapeutically effective amount of the gamma-polyglutamylated folic acid metabolic antagonist to the tumor;

[91] A method for preparing a gamma-polyglutamylated folic acid metabolic antagonist composition comprising the liposomal gamma-polyglutamylated folic acid metabolic antagonist composition according to any one of items

[12] to

[69] , the method comprising the steps of: forming a mixture comprising a liposomal component and a gamma-polyglutamylated folic acid metabolic antagonist in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes comprising the gamma-polyglutamylated folic acid metabolic antagonist.

[92] A method for preparing a gamma-polyglutamylated folic acid metabolic antagonist comprising the liposomal gamma-polyglutamylated folic acid metabolic antagonist composition according to any one of items

[12] to

[69] , the method comprising the steps of: forming a mixture comprising a liposomal component and a gamma-polyglutamylated folic acid metabolic antagonist in solution; and treating the mixture to form liposomes comprising the gamma-polyglutamylated folic acid metabolic antagonist.

[93] The method according to item

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

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

[50] to

[69] , comprising the steps of: forming a mixture comprising liposome components and a gamma-polyglutamylated folic acid antimetabolite in a solution; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes encapsulating and / or coating the gamma-polyglutamylated folic acid antimetabolite; and imparting a targeting moiety onto 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).

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

[50] to

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

[96] The method according to item

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

[97] The method according to item

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

[98] The method according to any one of items

[95] to

[97] , wherein the treating step comprises one or more steps of changing the size of the liposomes by one or more steps selected from extrusion, high pressure microfluidization, and / or sonication; and / or A method according to any one of items

[91] to

[98] , wherein at least 1% of the starting material of the gamma-polyglutamylated folic acid antimetabolite is encapsulated or enclosed in liposomes.

[0089] I. Gamma-polyglutamylated folic acid antimetabolite (γPANTIFOL) Generally, the present disclosure relates to gamma-polyglutamylated folic acid antimetabolite (γPANTIFOL) compositions. The γPANTIFOL compositions contain at least one glutamyl group having a gamma-carboxyl group bond. These compositions are structurally different from the L-gamma-polyglutamylated form of the folic acid antimetabolite (Lγ1PANTIFOL) produced by the enzyme holylpolyglutamate synthase (FPGS) in cells during folic acid antimetabolite therapy.

[0090] In some embodiments, the γPANTIFOL composition contains 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 antimetabolite). In some embodiments, each glutamyl group in γPANTIFOL other than the glutamyl group of the folic acid antimetabolite has a gamma bond. In some embodiments, two or more glutamyl groups in γPANTIFOL have a gamma bond. In some embodiments, each glutamyl group in γPANTIFOL is of the L type. In some embodiments, each glutamyl group in γPANTIFOL other than the glutamyl group of the folic acid antimetabolite is of the D type. In some embodiments, γPANTIFOL contains two or more L-type glutamyl groups and one or more D-type glutamyl groups.

[0091] In some embodiments, the folic acid antimetabolite is selected from PMX, MTX, RTX, and LTX, or stereoisomers thereof.

[0092] 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-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.;

[0093] In some embodiments, the folic acid antagonist is selected from methotrexate, raltitrexed, premetrexed, pemetrexed, lometrexol (LTX; 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.;

[0094] 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 (I), 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, 2 to 10, 4 to 6, or greater than 5.

Chemical formula

[0095] 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 incorporated herein by reference in its entirety), α-substituted methotrexate analogs, γ-substituted methotrexate analogs, 5-methyl-5-deazamethotrexate analogs (see, 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, polymeric platinumol methotrexate derivatives, methotrexate - γ - dimyristoylphosphatidylethanolamine, methotrexate polyglutamate analogs, poly - γ - glutamyl methotrexate derivatives, deoxyuridylate methotrexate derivatives, iodoacetyllysine 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 - propargylaminopterin, 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 their stereoisomers.,

[0096] In some embodiments, the folic acid antagonist is of formula (IV):

Chemical formula

[0097] In some embodiments, the folic acid antagonist has 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.

[0098] In some embodiments, the folic acid antagonist has formula (V):

Chemical formula

[0099] In some embodiments, the folic acid antagonist has 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.

[0100] In some embodiments, the folic acid antagonist has formula (VI):

Chemical formula

[0101] In some embodiments, the folic acid antagonist has 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.

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

Chemical formula

[0103] In some embodiments, the folic acid antagonist has formula (VII), wherein (a) X = N; Y = NH2; and R = H; (b) X = N; H = 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.

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

Chemical formula

[0105] In some embodiments, the folic acid antagonist has formula (IX):

Chemical formula

[0106] 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.

[0107] In some embodiments, the folic acid antagonist has the formula (X):

Chemical formula

[0108] 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., a 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.

[0109] 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 particularly 2-fluoro-1,4-phenylene, or pyridine 2,5-diyl. In some embodiments, Ar is 1,4 phenylene or 2-fluoro-1,4 phenylene.

[0110] In other embodiments, the gamma polyglutamylated folic acid antagonist is cyclopenta[g]quinazoline as 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.

[0111] In some embodiments, the gamma polyglutamylated folate antagonist is diglutamylated. That is, the gamma polyglutamylated folate antagonist contains, in addition to the glutamyl group in the folate antagonist, one additional glutamyl group (γANTIFOL-PG1), and the additional glutamyl group is linked to the glutamyl group in the folate antagonist via a gamma bond. In some embodiments, each glutamyl group of the gamma diglutamylated folate antagonist is of the L-type. In other embodiments, the gamma diglutamylated folate antagonist contains a D-type glutamyl group.

[0112] In some embodiments, the gamma polyglutamylated folate antagonist is triglutamylated. That is, the gamma polyglutamylated folate antagonist contains, in addition to the glutamyl group in the folate antagonist, two gamma glutamyl groups (γANTIFOL-PG2). In some embodiments, each of the two additional glutamyl groups has a gamma bond. In other embodiments, one of the two glutamyl groups has a gamma bond and the other glutamyl group has a gamma bond. In some embodiments, each glutamyl group of the gamma triglutamylated folate antagonist is of the L-type. In other embodiments, the gamma triglutamylated folate antagonist contains a D-type glutamyl group. In further embodiments, each glutamyl group of the gamma triglutamylated folate antagonist other than the gamma glutamyl group in the folate antagonist is of the D-type. In further embodiments, the gamma triglutamylated folate antagonist contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0113] In some embodiments, the gamma polyglutamylated antifolate is tetraglutamylated and thus contains three γ-glutamyl groups in addition to the glutamyl group of the antifolate (γANTIFOL-PG3). In some embodiments, the gamma tetraglutamylated antifolate contains two or more L-type γ-glutamyl groups. In further embodiments, each γ-glutamyl group of the gamma tetraglutamylated antifolate is of the L-type. In other embodiments, the gamma tetraglutamylated antifolate contains a D-type γ-glutamyl group. In some embodiments, the gamma tetraglutamylated antifolate contains two D-type γ-glutamyl groups. In some embodiments, each glutamyl group of the gamma tetraglutamylated antifolate other than the glutamyl group in the antifolate is of the D-type. In further embodiments, the tetraglutamylated antifolate contains a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0114] In some embodiments, the gamma polyglutamylated antifolate is pentaglutamylated (γANTIFOL-PG4) and contains a chain of four γ-glutamyl groups attached to the glutamyl group in the antifolate. In some embodiments, the gamma pentaglutamylated antifolate contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of the gamma pentaglutamylated antifolate is of the L-type. In other embodiments, the gamma pentaglutamylated antifolate contains a D-type glutamyl group. In some embodiments, the gamma tetraglutamylated antifolate contains two or three D-type γ-glutamyl groups. In further embodiments, each γ-glutamyl group of the gamma pentaglutamylated antifolate other than the glutamyl group in the antifolate is of the D-type. In further embodiments, the pentaglutamylated antifolate contains a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0115] In some embodiments, the gamma polyglutamylated antifolate is hexaglutamylated (γANTIFOL-PG5) and includes a chain of five γ-glutamyl groups attached to the glutamyl group in the antifolate. In some embodiments, the gamma hexaglutamylated antifolate includes two or more L-type γ-glutamyl groups. In a further embodiment, each glutamyl group of the gamma hexaglutamylated antifolate is of the L-type. In other embodiments, the gamma hexaglutamylated antifolate includes a D-type γ-glutamyl group. In some embodiments, the gamma tetraglutamylated antifolate includes two, three, four, or five D-type γ-glutamyl groups. In a further embodiment, each glutamyl group of the gamma hexaglutamylated antifolate other than the glutamyl group in the antifolate is of the D-type. In a further embodiment, the hexaglutamylated antifolate includes a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0116] In some embodiments, the gamma polyglutamylated antifolate is heptaglutamylated (γANTIFOL-PG6) and thus includes a chain of six γ-glutamyl groups attached to the glutamyl group in the antifolate. In some embodiments, the gamma heptaglutamylated antifolate includes two or more L-type γ-glutamyl groups. In a further embodiment, each γ-glutamyl group of the gamma heptaglutamylated antifolate is of the L-type. In other embodiments, the gamma heptaglutamylated antifolate includes a D-type γ-glutamyl group. In some embodiments, the gamma tetraglutamylated antifolate includes two, three, four, five, or six D-type γ-glutamyl groups. In a further embodiment, each γ-glutamyl group of the gamma heptaglutamylated antifolate other than the glutamyl group in the antifolate is of the D-type. In a further embodiment, the heptaglutamylated antifolate includes a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0117] In some embodiments, the gamma polyglutamylated antifolate is octaglutamylated (γANTIFOL-PG7) and thus contains a chain of seven γ-glutamyl groups attached to the glutamyl group in the antifolate. In some embodiments, the gamma octaglutamylated antifolate contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of the gamma octaglutamylated antifolate is of the L-type. In other embodiments, the gamma octaglutamylated antifolate contains a D-type glutamyl group. In some embodiments, the gamma octaglutamylated antifolate contains two, three, four, five, six, or seven D-type γ-glutamyl groups. In further embodiments, each glutamyl group of the gamma octaglutamylated antifolate other than the glutamyl group in the antifolate is of the D-type. In further embodiments, the octaglutamylated antifolate contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0118] In some embodiments, the gamma polyglutamylated antifolate is nonaglutamylated (γANTIFOL-PG8) and contains a chain of eight γ-glutamyl groups attached to the glutamyl group in the antifolate. In some embodiments, the gamma nonaglutamylated antifolate contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of the gamma nonaglutamylated antifolate is of the L-type. In other embodiments, the gamma nonaglutamylated antifolate contains a D-type glutamyl group. In further embodiments, each glutamyl group of the gamma nonaglutamylated antifolate other than the glutamyl group in the antifolate is of the D-type. In further embodiments, the nonaglutamylated antifolate contains a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0119] In some embodiments, the gamma polyglutamylated antifolate is decaglutamylated (γANTIFOL-PG9) (i.e., it contains a chain of 9 γ-glutamyl groups attached to the glutamyl group in the antifolate). In some embodiments, the gamma decaglutamylated antifolate contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the gamma decaglutamylated antifolate is of the L-type. In other embodiments, the gamma decaglutamylated antifolate contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the gamma decaglutamylated antifolate other than the glutamyl group in the antifolate is of the D-type. In a further embodiment, the decaglutamylated antifolate contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0120] In some embodiments, the gamma polyglutamylated antifolate is undecaglutamylated (γANTIFOL-PG10) and contains a chain of 10 γ-glutamyl groups attached to the glutamyl group in the antifolate. In some embodiments, the gamma undecaglutamylated antifolate contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the gamma undecaglutamylated antifolate is of the L-type. In other embodiments, the gamma undecaglutamylated antifolate contains a D-type glutamyl group. In a further embodiment, each glutamyl group of the gamma undecaglutamylated antifolate other than the glutamyl group in 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.

[0121] In some embodiments, the gamma polyglutamylated antifolate is dodecaglutamylated (γANTIFOL-PG11) and contains a chain of 11 γ-glutamyl groups attached to the glutamyl group in the antifolate. In some embodiments, the gamma dodecaglutamylated antifolate contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the gamma dodecaglutamylated antifolate is of the L-type. In other embodiments, the gamma dodecaglutamylated antifolate contains a D-type glutamyl group. In a further embodiment, each glutamyl group in the gamma dodecaglutamylated antifolate other than the glutamyl group in 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.

[0122] In some embodiments, the gamma polyglutamylated antifolate is tridecaglutamylated (γANTIFOL-PG12) and contains a chain of 12 γ-glutamyl groups attached to the glutamyl group in the antifolate. In some embodiments, the gamma tridecaglutamylated antifolate contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the gamma tridecaglutamylated antifolate is of the L-type. In other embodiments, the gamma tridecaglutamylated antifolate contains a D-type glutamyl group. In a further embodiment, each glutamyl group in the gamma tridecaglutamylated antifolate other than the glutamyl group in the antifolate is of the D-type. In a further embodiment, the tridecaglutamylated antifolate contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0123] In some embodiments, the gamma polyglutamylated antifolate is tetradecaglutamylated (γANTIFOL-PG13) and comprises a chain of 13 γ-glutamyl groups attached to the glutamyl group in the antifolate. In some embodiments, the gamma tetradecaglutamylated antifolate comprises two or more L-type glutamyl groups. In further embodiments, each glutamyl group of the gamma tetradecaglutamylated antifolate is of the L-type. In other embodiments, the gamma tetradecaglutamylated antifolate comprises a D-type glutamyl group. In further embodiments, each glutamyl group of the gamma tetradecaglutamylated antifolate other than the glutamyl group in the antifolate is of the D-type. In further embodiments, the tetradecaglutamylated antifolate comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0124] In some embodiments, the gamma polyglutamylated antifolate is pentadecaglutamylated (γANTIFOL-PG14) and comprises a chain of 14 γ-glutamyl groups attached to the glutamyl group in the antifolate. In some embodiments, the gamma pentadecaglutamylated antifolate comprises two or more L-type glutamyl groups. In further embodiments, each glutamyl group of the gamma pentadecaglutamylated antifolate is of the L-type. In other embodiments, the gamma pentadecaglutamylated antifolate comprises a D-type glutamyl group. In further embodiments, each glutamyl group of the gamma pentadecaglutamylated antifolate other than the glutamyl group in the antifolate is of the D-type. In further embodiments, the pentadecaglutamylated antifolate comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0125] In some embodiments, the gamma polyglutamylated antifolate is hexadeca-glutamylated (γANTIFOL-PG15) and comprises a chain of 15 γ-glutamyl groups attached to the glutamyl groups in the antifolate. In some embodiments, the gamma hexadeca-glutamylated antifolate comprises two or more L-type glutamyl groups. In further embodiments, each glutamyl group of the gamma hexadeca-glutamylated antifolate is of the L-type. In other embodiments, the gamma hexadeca-glutamylated antifolate comprises a D-type glutamyl group. In further embodiments, each glutamyl group of the gamma hexadeca-glutamylated antifolate other than the glutamyl groups in the antifolate is of the D-type. In further embodiments, the hexadeca-glutamylated antifolate comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0126] In other embodiments, the gamma polyglutamylated antifolate is heptadeca-glutamylated (γANTIFOL-PG16) and comprises a chain of 16 γ-glutamyl groups attached to the glutamyl groups in the antifolate. In some embodiments, the gamma heptadeca-glutamylated antifolate comprises two or more L-type glutamyl groups. In further embodiments, each glutamyl group of the gamma heptadeca-glutamylated antifolate is of the L-type. In other embodiments, the gamma heptadeca-glutamylated antifolate comprises a D-type glutamyl group. In further embodiments, each glutamyl group of the gamma heptadeca-glutamylated antifolate other than the glutamyl groups in the antifolate is of the D-type. In further embodiments, the heptadeca-glutamylated antifolate comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0127] In some embodiments, the gamma polyglutamylated antifolate is octadeca-glutamylated (γANTIFOL-PG17) and comprises a chain of 17 γ-glutamyl groups attached to the glutamyl groups in the antifolate. In some embodiments, the gamma octadeca-glutamylated antifolate comprises two or more L-type glutamyl groups. In further embodiments, each glutamyl group of the gamma octadeca-glutamylated antifolate is of the L-type. In other embodiments, the gamma octadeca-glutamylated antifolate comprises a D-type glutamyl group. In further embodiments, each glutamyl group of the gamma octadeca-glutamylated antifolate other than the glutamyl groups in the antifolate is of the D-type. In further embodiments, the octadeca-glutamylated antifolate comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0128] In some embodiments, the gamma polyglutamylated antifolate is nonadeca-glutamylated (γANTIFOL-PG18) and comprises a chain of 18 γ-glutamyl groups attached to the glutamyl groups in the antifolate. In some embodiments, the gamma nonadeca-glutamylated antifolate comprises two or more L-type glutamyl groups. In further embodiments, each glutamyl group of the gamma nonadeca-glutamylated antifolate is of the L-type. In other embodiments, the gamma nonadeca-glutamylated antifolate comprises a D-type glutamyl group. In further embodiments, each glutamyl group of the gamma nonadeca-glutamylated antifolate other than the glutamyl groups in the antifolate is of the D-type. In further embodiments, the nonadeca-glutamylated antifolate comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0129] In some embodiments, the gamma polyglutamylated folate antagonist is eicosaglutamylated (γANTIFOL-PG19) and includes a chain of 19 γ-glutamyl groups attached to the glutamyl group in the folate antagonist. In some embodiments, the gamma eicosaglutamylated folate antagonist includes two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the gamma eicosaglutamylated folate antagonist is of the L-type. In other embodiments, the gamma eicosaglutamylated folate antagonist includes a D-type glutamyl group. In a further embodiment, each glutamyl group of the gamma eicosaglutamylated folate antagonist other than the glutamyl group in the folate antagonist is of the D-type. In a further embodiment, the eicosaglutamylated folate antagonist includes a D-type glutamyl group and two or more L-type glutamyl groups.

[0130] In some embodiments, the gamma polyglutamylated folate antagonist is heneicosaglutamylated (γANTIFOL-PG20) and includes a chain of 20 γ-glutamyl groups attached to the glutamyl group in the folate antagonist. In some embodiments, the gamma heneicosaglutamylated folate antagonist includes two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of the gamma heneicosaglutamylated folate antagonist is of the L-type. In other embodiments, the gamma heneicosaglutamylated folate antagonist includes a D-type glutamyl group. In a further embodiment, each glutamyl group of the gamma heneicosaglutamylated folate antagonist other than the glutamyl group in the folate antagonist is of the D-type. In a further embodiment, the heneicosaglutamylated folate antagonist includes a D-type glutamyl group and two or more L-type glutamyl groups.

[0131] In some embodiments, the gamma polyglutamylated folic acid antagonist comprises 4 to 7 glutamyl groups attached to the folic acid antagonist (i.e., γANTIFOL-PGn, n = 4 to 7), and each of the 4 to 7 attached glutamyl groups has a gamma bond. In some embodiments, each of the 4 to 7 attached glutamyl groups is of the L-form. In other embodiments, each of the 4 to 7 attached glutamyl groups is of the D-form. In other embodiments, the 4 to 7 attached glutamyl groups are of the L-form and the D-form.

[0132] In some embodiments, the gamma polyglutamylated folic acid antagonist (γPANTIFOL) comprises 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 folic acid antagonist, or any range therebetween. In some embodiments, each glutamyl group in γPANTIFOL other than the glutamyl group in the folic acid antagonist has a gamma bond. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in γPANTIFOL have a gamma bond. In some embodiments, γPANTIFOL comprises γ-glutamyl groups of the L-form and the D-form. In some embodiments, each glutamyl group in the polyglutamate structure of the polyglutamylated folic acid antagonist is of the L-form. In some embodiments, each glutamyl group in γPANTIFOL other than the glutamyl group in the 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, or 15 of the glutamyl groups in γPANTIFOL are of the L-form. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in γPANTIFOL are of the D-form.

[0133] In some embodiments, gamma-polyglutamyl oxidized folate antagonist (γPANTIFOL) contains a total of 2 to 20, 2 to 15, 2 to 10, 2 to 5, or any range therebetween of glutamyl groups, including the glutamyl group of the folate antagonist. In some embodiments, each glutamyl group in γPANTIFOL is of the L-type. In some embodiments, each glutamyl group in γPANTIFOL other than the glutamyl group in the folate antagonist 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 γPANTIFOL 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 γPANTIFOL are of the D-type.

[0134] In some embodiments, the gamma-polyglutamyl oxidized folate antagonist contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in addition to the glutamyl group of the folate antagonist.

[0135] 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 a gamma-polyglutamyl oxidized 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 the gamma-polyglutamyl oxidized folic acid antagonist is of the L-form. In other embodiments, each glutamyl group of the gamma-polyglutamyl oxidized folic acid antagonist other than the glutamyl groups of the folic acid antagonist is of the D-form. In alternative embodiments, at least two of the glutamyl groups of the gamma-polyglutamyl oxidized folic acid antagonist are of the L-form and at least one of the glutamyl groups in the gamma-polyglutamyl oxidized 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 the gamma-polyglutamyl oxidized 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 the gamma-polyglutamyl oxidized folic acid antagonist are of the D-form.

[0136] In further embodiments, the gamma-polyglutamyl oxidized 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 the gamma-polyglutamyl oxidized folic acid antagonist is of the L-form. In other embodiments, each glutamyl group of the gamma-polyglutamyl oxidized folic acid antagonist other than the glutamyl groups of the folic acid antagonist is of the D-form. In alternative embodiments, at least two of the glutamyl groups of the gamma-polyglutamyl oxidized folic acid antagonist are of the L-form and at least one of the glutamyl groups in the gamma-polyglutamyl oxidized folic acid antagonist is of the D-form.

[0137] In further embodiments, the provided composition comprises a gamma-polyglutamylated folate antagonist comprising a glutamyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 gamma linkages. In some embodiments, the gamma-polyglutamylated folate antagonist comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 L-type glutamyl groups. In some embodiments, the gamma-polyglutamylated folate 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 gamma-polyglutamylated folate 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.

[0138] In some embodiments, one or more additional glutamyl groups can be added to the gamma-polyglutamylated folate antagonist compositions provided herein, i.e., the composition can serve as a substrate for FPGS (folylpolyglutamate synthetase). Reagents and assays for measuring the ability of a gamma-polyglutamylated folate antagonist 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.

[0139] In some embodiments, the rate of uptake of the naked gammaPANTIFOL compositions disclosed herein (e.g., gammaPANTIFOL not bound to a delivery carrier) by liver cells is significantly reduced compared to the rate of uptake of folate antagonists under physiological conditions. In some embodiments, the rate of liver cell uptake of the naked gammaPANTIFOL composition is less than 30%, 20%, 15%, or 10% compared to the rate of the folate antagonist. In further embodiments, the rate of efflux (transport) of the gammaPANTIFOL compositions disclosed herein from liver cells occurs at a significantly slower rate (less than 30%, 20%, 15%, or 10%) compared to folate antagonists.

[0140] In some embodiments, the gamma-polyglutamyl oxidized folate antimetabolite compositions provided herein have higher cytotoxicity against hyperproliferative cells than folate antimetabolites. In some embodiments, the hyperproliferative cells are cancer cells. In some embodiments, the hyperproliferative cells are colorectal cancer cells, colon cancer cells, breast cancer cells, or ovarian cancer cells. In some embodiments, the cancer cells are mesothelioma cells or non-small cell lung cancer cells. In some embodiments, the cytotoxicity is measured in an in vitro assay. In some embodiments, the gamma-polyglutamyl oxidized folate antimetabolite is a hexaglutamyl oxidized folate antimetabolite.

[0141] In some embodiments, the gamma-polyglutamyl oxidized folate antimetabolite compositions provided herein have lower toxic side effects than folate antimetabolites. In some embodiments, the gamma-polyglutamyl oxidized folate antimetabolite compositions provided herein have less toxicity against non-hyperproliferative cells than folate antimetabolites. In some embodiments, the gamma-polyglutamyl oxidized folate antimetabolite compositions provided herein have less toxicity against 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 gamma-polyglutamyl oxidized folate antimetabolite is a hexaglutamyl oxidized folate antimetabolite.

[0142] In some embodiments, the gamma-polyglutamylated folate antagonist compositions provided herein have lower toxic side effects relative to folate antagonists. In some embodiments, the gamma-polyglutamylated folate antagonist compositions provided herein result in fewer or less severe toxic side effects than folate antagonists in an in vivo assay. In some embodiments, the in vivo assay is performed in an in vivo mouse model. In some embodiments, the gamma-polyglutamylated folate antagonist compositions provided herein result in fewer or less severe hematological or liver toxic side effects than folate antagonists. 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 gamma-polyglutamylated folate antagonist composition at 40 mg / kg or 80 mg / kg once a week for 4 weeks. In some embodiments, the gamma-polyglutamylated folate antagonist is a hexaglutamylated folate antagonist.

[0143] In some embodiments, treatment with the gamma-polyglutamyl oxidized folic acid 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 gamma-polyglutamyl oxidized folic acid antagonist compositions provided herein do not significantly reduce mean neutrophils, mean white blood cells, or mean platelet counts. In some embodiments, the gamma-polyglutamyl oxidized folic acid antagonist compositions provided herein do not significantly increase serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. In some embodiments, the gamma-polyglutamyl oxidized folic acid antagonist compositions provided herein do not significantly reduce serum albumin levels. In some embodiments, the in vivo assay comprises administering the gamma-polyglutamyl oxidized folic acid antagonist composition at 40 mg / kg or 80 mg / kg once a week for 4 weeks. In some embodiments, the gamma-polyglutamyl oxidized folic acid antagonist is a hexaglutamyl oxidized folic acid antagonist.

[0144] In some embodiments, the gamma-polyglutamyl oxidized folic acid antagonist composition does not contain a fluorine atom. In some embodiments, the gamma-polyglutamyl oxidized folic acid antagonist composition does not contain a 4-fluoroglutamyl group.

[0145] Compositions of gamma-polyglutamylated folate antagonists (γPANTIFOL) and their uses are further described in International Application No. PCT / US2017 / 046667 and U.S. Patent Application Nos. 62 / 630,824, 62 / 630,613, 62 / 630,713, 62 / 630,620, 62 / 627,733, 62 / 630,625, 62 / 630,652, 62 / 627,732, 62 / 636,289, 62 / 630,751, 62 / 630,821, 62 / 627,741, and 62 / 583,432, respectively. The disclosures of each of these are hereby incorporated by reference in their entirety.

[0146] A. Gamma-polyglutamylated folate antagonist analogs and derivatives The present disclosure also encompasses gamma-polyglutamylated folate antagonist derivatives and analogs. The compositions and methods disclosed herein are contemplated for application to any and all derivatives or analogs of known polyglutamylated folate antagonists. In some embodiments, the analog corresponds to a modified form of the folate antagonist, in which case the glutamyl group of the folate antagonist is not attached to the remainder of the folate antagonist molecule via a gamma-peptide bond. In some embodiments, the analog is an isomer of the folate antagonist, in which case the glutamyl group in the folate antagonist is of the D-form. In some embodiments, the polyglutamylated form of the folate antagonist, or the polyglutamylated folate antagonist analog or derivative, is not fluorinated.

[0147] 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, polyglutamylmethotrexate derivatives, gem-diphosphonate methotrexate analogs (see, e.g., International Application No. 1988 / 06158, the contents of which are incorporated herein by reference in their entirety), α-substituted methotrexate analogs, γ-substituted methotrexate analogs, 5-methyl-5-deazamethotrexate analogs (see, e.g., U.S. Patent No. 4,725,Reference 687, the contents of which are hereby incorporated by reference in their entirety), 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 contents of which are hereby incorporated by reference in their entirety), 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 their stereoisomers.,

[0148] In further embodiments, the gamma polyglutamyl oxidized folate antimetabolite 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.

[0149] B. γ ANTIFOL-PG Synthesis The folate antimetabolite polyglutamate compositions provided herein are obtained by the following synthetic methods known in the art. Procedures for synthesizing folate antimetabolites (including different pharmaceutically acceptable salts or acids (e.g., folate antimetabolite disodium) and crystalline and amorphous forms) and intermediates for synthesizing folate antimetabolites 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).

[0150] 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 the "click chemistry" method or other bioconjugate chemistry known to those skilled in the art. Alternatively, a peptide of glutamyl residues of a desired length can be generated and added to a precursor of pemetrexed that does not have a glutamyl residue. 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 pemetrexed precursor is attached to the peptide and the molecule is cleaved from the resin.

[0151] C. Gamma Polyglutamyl Oxidized Folic Acid Antagonist Complex Unexpectedly, the inventors have found that polyglutamylated antifolates such as folic acid antagonists (γPANTIFOL) 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 γPANTIFOL (e.g., γPANTIFOL as disclosed herein) with a therapeutic agent or a salt or acid thereof. In some embodiments, the present disclosure provides a complex of γPANTIFOL as described in any of items [1] to

[11] of the section "Modes for Carrying Out the Invention" with a therapeutic agent or a salt or acid thereof. In some embodiments, the γPANTIFOL / complex comprises γPANTIFOL and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound such as a chemotherapeutic agent. In further embodiments, the γPANTIFOL / complex comprises a platinum-based drug such as a platinum-based chemotherapeutic agent (e.g., carboplatin and cisplatin). In other embodiments, the γPANTIFOL / complex comprises a taxane-based chemotherapeutic agent (e.g., carboplatin and cisplatin). In other embodiments, the γPANTIFOL / complex comprises cyclodextrin. In further embodiments, the γPANTIFOL / complex is encapsulated in liposomes. In some embodiments, the liposomes are Lp-αPANTIFOL as described in any of items

[12] to

[67] .

[0152] In further embodiments, the γPANTIFOL / therapeutic agent complex comprises one or more γPANTIFOLs containing 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 γPANTIFOL / therapeutic agent complex comprises one or more γPANTIFOLs containing 3 to 10, 3 to 9, 3 to 8, or 3 to 7 glutamyl groups, or any range therebetween. In other embodiments, the γPANTIFOL / therapeutic agent complex comprises one or more γPANTIFOLs containing 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 glutamyl groups, or any range therebetween. In a particular embodiment, the complex comprises one or more γPANTIFOLs containing 3 to 10 glutamyl groups. In further embodiments, the γPANTIFOL / therapeutic agent complex comprises one or more γPANTIFOLs containing 3 to 7 glutamyl groups. In another embodiment, the γPANTIFOL / therapeutic agent complex comprises one or more γPANTIFOLs containing 5 glutamyl groups. In another embodiment, the γPANTIFOL / therapeutic agent complex comprises one or more γPANTIFOLs containing 6 glutamyl groups. In some embodiments, the therapeutic agent is a cytotoxic compound or a salt or acid thereof. In further embodiments, the therapeutic agent is a chemotherapeutic agent or a salt or acid thereof. In another embodiment, the chemotherapeutic agent is a platinum-based drug. In another embodiment, the chemotherapeutic agent is a taxane-based drug. In further embodiments, the molar ratio of γPANTIFOL / therapeutic agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / therapeutic agent in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the molar ratio of γPANTIFOL / therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In some embodiments, the γPANTIFOL / 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 as described in any of items

[12] -

[67] in the section of the form for carrying out the invention.

[0153] In alternative embodiments, the γPANTIFOL complex comprises γPANTIFOL and cyclodextrin. In some embodiments, the γPANTIFOL complex comprises γPANTIFOL as described in any of items [1] to

[11] of the section on forms for carrying out the invention. In some embodiments, the γPANTIFOL complex comprises a folic acid antagonist as described in Section I. In some embodiments, the molar ratio of γPANTIFOL (e.g., γPANTIFOL salt) / cyclodextrin in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of γPANTIFOL / cyclodextrin in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of γPANTIFOL / cyclodextrin in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / cyclodextrin complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art). In some embodiments, the liposomes are Lp-γPANTIFOL as described in any of items

[12] to

[67] of the section of the form for carrying out the invention.

[0154] In some embodiments, the present disclosure provides a composition comprising a γPANTIFOL / platinum-based chemotherapeutic agent complex. In some embodiments, the complex comprises γPANTIFOL as described in any of items [1] to

[11] of the section on forms for carrying out the invention. In some embodiments, the γPANTIFOL complex comprises the polyglutamylated folic acid antagonist described in Section I. In some embodiments, the platinum-based chemotherapeutic agent is selected from cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the γPANTIFOL / platinum-based chemotherapeutic agent complex comprises an analog of cisplatin, carboplatin, oxaliplatin, or salts or acids thereof. In some embodiments, the molar ratio of γPANTIFOL / platinum-based agent in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of γPANTIFOL / platinum-based agent in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPANTIFOL / platinum-based agent in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / platinum-based agent complex is encapsulated in liposomes. In some embodiments, the liposomes are Lp-αPANTIFOL as described in any of items

[12] to

[67] of the section of the form for carrying out the invention.

[0155] In a further embodiment, the γPANTIFOL / platinum-based chemotherapeutic agent complex comprises cisplatin, carboplatin, an oxaliplatin analog, or a salt or acid thereof. In some embodiments, the complex comprises γPANTIFOL as described in any of items [1] to

[11] of the section on forms for carrying out the invention. In some embodiments, the γPANTIFOL complex comprises the polyglutamine oxidized folic acid antagonist described in Section I. In some embodiments, the molar ratio of γPANTIFOL / platinum-based analog in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of γPANTIFOL / platinum-based analog in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of γPANTIFOL / platinum-based agent in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPANTIFOL / 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 γPANTIFOL / 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 - 50):1, or >50:1. In some embodiments, the molar ratio of γPANTIFOL / 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 γPANTIFOL / 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 - 50), or 1:>50.In some embodiments, the γPANTIFOL / platinum-based analog complex is encapsulated within liposomes. In some embodiments, the liposomes are Lp-αPANTIFOL as described in any of items

[12] to

[67] of the section on forms for carrying out the invention.

[0156] In a further embodiment, the present disclosure provides a complex comprising γPANTIFOL and cisplatin or a salt or acid thereof. In some embodiments, the complex comprises γPANTIFOL as described in any of items [1] to

[11] of the section on forms for carrying out the invention. In some embodiments, the γPANTIFOL complex comprises an antifolate as described in Section I. In some embodiments, the molar ratio of γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / cisplatin (or a salt or acid of cisplatin) complex is encapsulated in liposomes. In some embodiments, the liposomes are Lp-αPANTIFOL as described in any of items

[12] to

[67] of the section of the form for carrying out the invention.

[0157] In another embodiment, the present disclosure provides a complex comprising γPANTIFOL and carboplatin or a salt or acid thereof. In some embodiments, the complex comprises γPANTIFOL as described in any of items [1] to

[11] of the section of the form for carrying out the invention. In some embodiments, the γPANTIFOL complex comprises the polyglutamated folic acid antimetabolite described in Section I herein. In some embodiments, the molar ratio of γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 to 50):1, or >50:1. In some embodiments, the molar ratio of γPANTIFOL / 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 γPANTIFOL / 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 γPANTIFOL / 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 of items

[12] to

[67] of the section of forms for carrying out the invention.

[0158] In another embodiment, the present disclosure provides a complex comprising γPANTIFOL and oxaliplatin or a salt or acid thereof. In some embodiments, the complex comprises γPANTIFOL as described in any of items [1] to

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

[12] to

[67] of the section of the form for carrying out the invention.

[0159] In further embodiments, the present disclosure provides a complex comprising γPANTIFOL and a platinum-based chemotherapeutic agent (platinum) selected from nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or salts or acids thereof. In other embodiments, the γPANTIFOL / platinum-based chemotherapeutic agent complex comprises nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, NK121, CI973, DWA2114R, NDDP, or an analogue of nedaplatin, or salts or acids thereof. In some embodiments, the molar ratio of γPANTIFOL / platinum-based chemotherapeutic agent (platinum) (or a salt or acid of the platinum-based chemotherapeutic agent) in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the complex comprises γPANTIFOL as described in any of items [1] to

[11] of the section of the form for carrying out the invention. In some embodiments, the γPANTIFOL complex comprises the polyglutamated folic acid antimetabolite described in Section I. In further embodiments, the molar ratio of γPANTIFOL / platinum (or a salt or acid of platinum) in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPANTIFOL / platinum (or a salt or acid of platinum) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPANTIFOL / platinum (or a salt or acid of platinum) 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 γPANTIFOL / platinum (or a salt or acid of platinum) 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 γPANTIFOL / platinum (or a salt or acid of platinum) 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 γPANTIFOL / platinum (or a salt or acid of platinum) 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 γPANTIFOL / platinum (or its salt or acid or analog) complex is encapsulated in liposomes. In some embodiments, the liposomes are Lp-αPANTIFOL as described in any of items

[12] to

[67] of the section of the form for carrying out the invention.

[0160] In some embodiments, the present disclosure provides a composition comprising a γPANTIFOL / taxane chemotherapeutic agent (taxane) complex. In some embodiments, the complex comprises γPANTIFOL as described in any of items [1] to

[11] of the section of the form for carrying out the invention. In some embodiments, the γPANTIFOL complex comprises the polyglutamated folic acid antagonist described in Section I. In some embodiments, the taxane chemotherapeutic agent is selected from paclitaxel (PTX), docetaxel (DTX), larotaxel (LTX), and cabazitaxel (CTX), or salts or acids thereof. In some embodiments, the molar ratio of γPANTIFOL / taxane (or a salt or acid of the taxane) in the complex ranges from 1 to 20:1, or any range therebetween. In a further embodiment, the molar ratio of γPANTIFOL / taxane (or a salt or acid of the taxane) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of γPANTIFOL / taxane (or a salt or acid of the taxane) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPANTIFOL / taxane (or a salt or acid of the taxane) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. In some embodiments, the molar ratio of γPANTIFOL / taxane (or a salt or acid of the taxane) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In some embodiments, the molar ratio of γPANTIFOL / taxane (or a salt or acid of the taxane) 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 γPANTIFOL / taxane (or a salt or acid of taxane) 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 a further embodiment, the γPANTIFOL / taxane (or a salt or acid of taxane) pharmaceutical complex is encapsulated in liposomes. In some embodiments, the liposomes are Lp-αPANTIFOL as described in any of items

[12] to

[67] of the section of the form for carrying out the invention.

[0161] In a further embodiment, the present disclosure provides a complex comprising γPANTIFOL and paclitaxel (PTX) or a salt or acid thereof. In other embodiments, the γPANTIFOL / paclitaxel (or a salt or acid of paclitaxel) chemotherapeutic agent complex comprises an analog of paclitaxel (PTX), or a salt or acid thereof. In some embodiments, the complex comprises γPANTIFOL as described in any of items [1] to

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

[12] to

[67] in the section of the form for carrying out the invention.

[0162] In further embodiments, the present disclosure provides a complex comprising γPANTIFOL and docetaxel (DTX) or a salt or acid thereof. In other embodiments, the γPANTIFOL / docetaxel complex comprises an analog of docetaxel (DTX) or a salt or acid thereof. In some embodiments, the complex comprises γPANTIFOL as described in any of items [1] to

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

[12] to

[67] of the section of the form for carrying out the invention.

[0163] In a further embodiment, the present disclosure provides a complex comprising γPANTIFOL and larotaxel (LTX) or a salt or acid thereof. In some embodiments, the complex comprises γPANTIFOL as described in any of items [1] to

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

[12] to

[67] of the section of the form for carrying out the invention.

[0164] In a further embodiment, the present disclosure provides a complex comprising γPANTIFOL and cabazitaxel (CTX) or a salt or acid thereof. In some embodiments, the complex comprises γPANTIFOL as described in any of items [1] to

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

[12] to

[67] of the section of the form for carrying out the invention.

[0165] In a further embodiment, the present disclosure provides a complex comprising γPANTIFOL and another antimetabolite, or a salt or acid thereof. In some embodiments, the complex comprises γPANTIFOL as described in any of items [1] to

[11] of the section of the forms for carrying out the invention. In some embodiments, the γPANTIFOL complex comprises the polyglutamated folic acid antimetabolite described in Section I. An antimetabolite is a chemical substance that is similar to a metabolite required for normal biochemical reactions but has a structure with sufficient differences to interfere with one or more normal functions of cells such as cell division. In some embodiments, the present disclosure provides a complex comprising γPANTIFOL and a folic acid antimetabolite (ANTIFOL), or a salt or acid thereof. In some embodiments, the present disclosure provides a complex comprising γPANTIFOL, and an antimetabolite selected from gemcitabine, fluorouracil, capecitabine, antifolate (e.g., folic acid antimetabolite, raltitrexed), tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, and any pharmaceutically acceptable salt or acid (single or plural) or derivative thereof. In some embodiments, the molar ratio of γPANTIFOL / antimetabolite (or salt or acid of the antimetabolite, or prodrug) in the complex ranges from 1 to 20:1, or any range therebetween. In a further embodiment, the molar ratio of γPANTIFOL / antimetabolite (or salt or acid of the antimetabolite, or prodrug) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of γPANTIFOL / antimetabolite (or salt or acid of the antimetabolite, or prodrug) in the complex ranges from 2 to 8:1, or any range therebetween.In some embodiments, the molar ratio of γPANTIFOL / antimetabolite (or a salt or acid of the antimetabolite, or a prodrug) 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 γPANTIFOL / antimetabolite (or a salt or acid of the antimetabolite, or a prodrug) 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 γPANTIFOL / antimetabolite (or a salt or acid of the antimetabolite, or a prodrug) 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 γPANTIFOL / antimetabolite (or a salt or acid of the antimetabolite, or a prodrug) 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 a further embodiment, the γPANTIFOL / antimetabolite (or a salt or acid of the antimetabolite, or a prodrug) complex is encapsulated in liposomes. In some embodiments, the liposomes are Lp-αPANTIFOL as described in any of items

[12] to

[67] of the section of the form for carrying out the invention.

[0166] In a further embodiment, the present disclosure provides a complex of γPANTIFOL (e.g., γPANTIFOL as disclosed herein) and cyclodextrin. Cyclodextrin (CD) is a group of cyclic oligosaccharides that have been shown to improve the physicochemical properties of many drugs through complex formation. CD is a cyclic oligosaccharide composed of several D-glucose units linked by α-(1,4) bonds. This cyclic structure provides a hydrophobic inner cavity and gives CD a truncated cone shape. Many hydroxyl groups are located on the ends of the ring, which makes CD both lipophilic and water-soluble. As a result, CD can form complexes with a wide variety of hydrophobic agents, thereby changing the physicochemical properties of these complexed agents. In some embodiments, the complex comprises γPANTIFOL as described in any of items [1] to

[11] of the section of the form for carrying out the invention.

[0167] The term "cyclodextrin" or "CD" generally means a parent or derivatized cyclic oligosaccharide that can form a complex with an antimetabolite-PG and contains a variable number of (α-1,4)-linked D-glucopyranoside units, unless otherwise specified. Each cyclodextrin glucopyranoside subunit has secondary hydroxyl groups at the 2 and 3 positions and a primary hydroxyl group at the 6 position. The term "parent", "undenivatized", or "inactive" cyclodextrin means a cyclodextrin having the basic formula C6H 12 O6 and a glucose structure and no additional chemical substituents (e.g., α-cyclodextrin consisting of 6 D-glucopyranoside units, β-cyclodextrin consisting of 7 D-glucopyranoside units, and γ-cyclodextrin consisting of 8 D-glucopyranoside units). The physical and chemical properties of the parent cyclodextrin can be modified by derivatizing the hydroxyl groups with other functional groups. Any substance located in the inner phase of cyclodextrin is said to be "complexed" with cyclodextrin or to form a complex (inclusion complex) with cyclodextrin.

[0168] As used herein, there are no particular restrictions on the cyclodextrin component of the γPANTIFOL / cyclodextrin complex, as long as the cyclodextrin can form a complex with γPANTIFOL. In certain embodiments, the cyclodextrin is derivatized to have ionizable (e.g., weakly basic and / or weakly acidic) functional groups to facilitate complex formation with γPANTIFOL and / or liposomal encapsulates.

[0169] Modification of hydroxyl groups of cyclodextrin, such as hydroxyl groups directed from the inner phase of cyclodextrin toward the opposite side using ionizable chemical groups, is known to facilitate the addition of cyclodextrin and a therapeutic agent complexed with cyclodextrin. In some embodiments, the cyclodextrin of the γPANTIFOL / cyclodextrin complex has hydroxyl groups substituted with at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ionizable chemical groups. The term "charged cyclodextrin" means a cyclodextrin having a hydroxyl group substituted with one or more of its charged moieties. Such moieties can include a charged group per se or an organic moiety (e.g., a C1-C6 alkyl or C1-C6 alkyl ether moiety) substituted with one or more charged moieties.

[0170] In some embodiments, the "ionizable" or "charged" portion of the CD derivative is weakly ionizable. The weakly ionizable portion is a weakly basic or weakly acidic portion. The weakly basic functional group (W) has a pKa by CH3-W in the range of about 6.0 to 9.0, 6.5 to 8.5, 7.0 to 8.0, 7.5 to 8.0, and any range therebetween (including the endpoints). Similarly, the weakly acidic functional group (X) has a logarithmically recorded dissociation constant (pKa) by CH3-X in the range of about 3.0 to 7.0, 4.0 to 6.5, 4.5 to 6.5, 5.0 to 6.0, 5.0 to 5.5, and any range therebetween (including the endpoints). Representative anionic portions include, but are not limited to, carboxylate, carboxymethyl, succinyl, sulfonyl, phosphate, sulfoalkyl ether, sulfate carbonate, thiocarbonate, dithiocarbonate, phosphate, phosphonate, sulfonate, nitrate, and borate groups. Representative cationic portions include, but are not limited to, amino, guanidine, and quaternary ammonium groups.

[0171] In another embodiment, the derivatized cyclodextrin is a "polyanion" or a "polycat ion". A polyanion is a derivatized cyclodextrin having two or more negatively charged groups, resulting in a net negative ionic charge of three or more units. A polycation is a derivatized cyclodextrin having two or more positively charged groups, resulting in a net positive ionic charge of three or more units.

[0172] In another embodiment, the derivatized cyclodextrin is a "chargeable amphiphile". "Chargeable" means that the amphiphile has a pK in the range of pH 4 to pH 8 or 8.5. The chargeable amphiphile can thus be a weak acid or base. "Amphoteric" as used herein means a derivatized cyclodextrin having ionizable groups with both anionic and cationic characteristics, (a) at least one, optionally both, of the cationic and anionic amphiphiles being chargeable and having at least one charge group with a pK between 4 and 8 to 8.5, (b) the cationic charge being dominant at pH 4, and (c) the anionic charge being dominant at pH 8 to 8.5.

[0173] In some embodiments, the “ionizable” or “charged” derivatized cyclodextrins are generally polyionic, amphiphilic, or otherwise, weakly ionizable (i.e., having a pKai of about 4.0 - 8.5, 4.5 - 8.0, 5.0 - 7.5, 5.5 - 7.5, 6.0 - 6.5, and any range therebetween (including both ends)).

[0174] Any one, some, or all of the hydroxyl groups of any one, some, or all of the α-D-glucopyranoside units of any cyclodextrin can be modified to an ionizable chemical group as described herein. Since each cyclodextrin hydroxyl group has different chemical reactivity, the reaction with the modifying moiety can produce a mixture of positional and optical isomers. Alternatively, the reaction can be made to form a homogeneous product of pre-modified α-D-glucopyranoside units by a specific chemistry.

[0175] Aggregate substitution occurring in cyclodextrin derivatives in a mixture is described by a term called degree of substitution. For example, 6-ethylenediamino-β-cyclodextrin having a degree of substitution of 7 would be composed of a distribution of isomers of 6-ethylenediamino-β-cyclodextrin where the number of ethylenediamino groups per 6-ethylenediamino-β-cyclodextrin molecule is 7. The degree of substitution of a cyclodextrin derivative mixture can be routinely measured using mass spectrometry or nuclear magnetic resonance spectroscopy.

[0176] In one embodiment, at least one hydroxyl moiety oriented in the opposite direction from within the cyclodextrin is replaced by an ionizable chemical group. For example, at least one of the α-D-glucopyranoside units of C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and all three of the C2-C3-C6 hydroxyls is replaced by an ionizable chemical group. Any such combination of hydroxyls can be combined with any of the degrees of substitution described herein, in addition to being able to similarly combine at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, up to all of the alpha-D-glucopyranoside units in the modified cyclodextrin. One such derivative is sulfalkyl ether cyclodextrin (SAE-CD). The sulfobutyl ether derivative of beta-cyclodextrin (SBE-β-CD) has been shown to have significantly improved water solubility compared to the parent cyclodextrin.

[0177] Additional cyclodextrin derivatives that can complex with a therapeutic agent in the disclosed liposome compositions include sugammadex or Org-25969, in which case the 6-hydroxy group on γ-CD was replaced by a carboxythioacetate ether bond and hydroxybutenyl-β-CD. Alternative cyclodextrins include 2,6-di-O-methyl-β-CD (DIMEB), 2-hydroxylpropyl-3-cyclodextrin (HP-β-CD), randomly methylated-β-cyclodextrin (RAMEB), sulfobutyl ether β-cyclodextrin (SBE-β-CD), and sulfobutyl ether-γ-cyclodextrin (SBEγCD), sulfobutylated-β-cyclodextrin sodium salt, (2-hydroxypropyl)-alpha-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, 2,6-di-O-methyl)-β-cyclodextrin (DIMEB-50 heptakis), 2,3,6-tri-O-methyl)-β-cyclodextrin (TRIMEB heptakis), methyl-β-cyclodextrin, octakis(6-deoxy-6-iodo)-γ-cyclodextrin, and octakis(6-deoxy-6-bromo)-γ-cyclodextrin.

[0178] In some embodiments, the cyclodextrin has high solubility in water to facilitate greater capture of the cyclodextrin in the liposome inner phase. In some embodiments, the aqueous solubility of the cyclodextrin is at least 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, or more. In some embodiments, the water solubility of the cyclodextrin is in the range of 10 - 150 mg / mL, 20 - 100 mg / mL, 20 - 75 mg / mL, and within any range between these (including both ends).

[0179] In some embodiments, a large binding constant between cyclodextrin and γPANTIFOL and / or other therapeutic agents complexed with cyclodextrin is preferred and can be obtained by selecting the number of glucose units in the cyclodextrin based on the size of the therapeutic agent (see, e.g., Albers et al., Crit. Rev. Therap. Drug Carrier Syst. 12:311 - 337 (1995); Stella et al., Toxicol. Pathol. 36:30 - 42 (2008)). When the binding constant is pH - dependent, the cyclodextrin can be selected such that the binding constant is large at the pH of the inner phase of the liposome. As a result, the solubility (apparent solubility) of the therapeutic agent in the presence of cyclodextrin can be further improved. In some embodiments, the binding constant between cyclodextrin and the therapeutic agent is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. In some embodiments, the binding constant between cyclodextrin and the therapeutic agent is in the range of 100 - 1,200, 200 - 1,000, 300 - 750, and any range therebetween.

[0180] In some embodiments, the cyclodextrin of the γPANTIFOL / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is not derivatized.

[0181] In some embodiments, the cyclodextrin of the γPANTIFOL / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is derivatized. In further embodiments, the cyclodextrin derivative of the complex has the structure of Formula I:

Chemical Formula

[0182] In some embodiments, the cyclodextrin derivatization of the γPANTIFOL / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is of formula II:

Chemical formula

[0183] In some embodiments, the cyclodextrin derivatization of the γPANTIFOL / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is the cyclodextrin disclosed in U.S. Patent Nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, 6,869,939; and International Publication No. 02005 / 117911. The content of each of these patent documents is hereby incorporated by reference into this specification preferentially.

[0184] In some embodiments, the cyclodextrin derivative of the γPANTIFOL / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is a sulfoalkyl ether cyclodextrin. In some embodiments, the cyclodextrin derivative of the complex is a sulfobutyl ether-3-cyclodextrin such as CAPTISOL® (CyDex Pharma.Inc., Lenexa, Kansas). Methods for preparing sulfobutyl ether-3-cyclodextrin and other sulfoalkyl ether cyclodextrins are known in the art.

[0185] In some embodiments, the cyclodextrin derivative of the γPANTIFOL / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is of Formula III:

Chemical Formula

[0186] In a further embodiment, the γPANTIFOL / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0187] D. γPANTIFOL Delivery Carrier In alternative embodiments, the present disclosure provides γPANTIFOL delivery systems and their use for delivering the payload of γPANTIFOL to cells (single or plural) in vitro or in vivo. In some embodiments, γPANTIFOL is complexed with or incorporated into a delivery carrier. Such delivery carriers are known in the art and include, but are not limited to, liposomes, lipospheres, polymers, peptides, proteins, antibodies (e.g., ADCs such as antibody-γPANTIFOL conjugates), cell components, cyclic oligosaccharides (e.g., cyclodextrins), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), lipoprotein particles, and combinations thereof. In certain embodiments, the delivery carrier is a liposome. In other certain embodiments, the delivery carrier is an antibody or an antigen-binding antibody fragment. In some embodiments, the γPANTIFOL delivery system comprises γPANTIFOL as described in any of items [1] to

[11] of the section of the form for carrying out the invention.

[0188] E. Liposomes In some embodiments, the present disclosure provides a liposomal composition comprising liposomes encapsulating (filling) a gamma-polyglutamylated folic acid antagonist (e.g., γPANTIFOL disclosed herein). In some embodiments, the liposomal composition comprises γPANTIFOL as described in any of items [1] to

[11] of the section of the form for carrying out the invention. In some embodiments, the liposomal composition comprises a polyglutamylated folic acid antagonist as described in Section I. In some embodiments, the liposomal composition comprises liposomes as described in any of items

[12] to

[67] of the section of the form for carrying out the invention. In some embodiments, the liposomes in the liposomal composition comprise γPANTIFOL containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups (including the glutamyl groups of the folic acid antagonist). In some embodiments, the gamma-polyglutamylated folic acid antagonist in Lp-γPANTIFOL comprises two or more L-type glutamyl groups. In other embodiments, the gamma-polyglutamylated folic acid antagonist in Lp-γPANTIFOL comprises a D-type glutamyl group. In further embodiments, the gamma-polyglutamylated folic acid antagonist in Lp-γPANTIFOL comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the gamma-polyglutamylated folic acid antagonist in Lp-γPANTIFOL comprises two or more glutamyl groups having a gamma-carboxyl bond. In some embodiments, the liposomal composition comprises liposomes comprising a gamma-pentaglutamylated folic acid antagonist. In further embodiments, the liposomes comprise an L-gamma-pentaglutamylated folic acid antagonist, a D-gamma-pentaglutamylated folic acid antagonist, or an L- and D-gamma-pentaglutamylated folic acid antagonist. In some embodiments, the liposomal com...

Claims

【Claim 1】 A liposomal composition comprising liposomes encapsulating a gamma-polyglutamylated folic acid antagonist and one or more non-gamma-polyglutamylated polyglutamylatable folic acid antagonists or non-polyglutamylatable folic acid antagonists, wherein the gamma-polyglutamylated folic acid antagonist contains 2 to 15 glutamyl groups having a gamma-carboxyl group bond; The polyglutamylated 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-dPteH CysA, 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-dH4PteH CysA, 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, 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)quinazoline; and AG377, 2,4-diamino-6[N-(4-(phenylsulfonyl)benzyl)ethyl)amino]quinazoline, or stereoisomers thereof; which is a polyglutamate of a folic acid antagonist selected from the group consisting of; (a) at least two of the glutamyl groups of the gamma-polyglutamylated folic acid antimetabolite are of the L-type, or (b) each of the glutamyl groups of the gamma-polyglutamylated folic acid antimetabolite is of the L-type, or (c) at least one of the glutamyl groups of the gamma-polyglutamylated folic acid antimetabolite is of the D-type, or (d) each of the glutamyl groups of the gamma-polyglutamylated folic acid antimetabolite other than the glutamyl group of the folic acid antimetabolite is of the D-type, or (e) at least two of the glutamyl groups of the gamma-polyglutamylated folic acid antimetabolite are of the L-type and at least one of the glutamyl groups is of the D-type; the liposome is pegylated, has a diameter of 30 nm to 175 nm, and has a zeta potential of 0 mV or less, liposome composition.

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