Gamma polyglutamated methotrexate and uses thereof

Abstract

To provide gamma polyglutamated methotrexate compositions to treat hyperproliferative disorders (e.g., cancer) and disorders of the immune system (e.g., inflammation and autoimmune diseases such as rheumatoid arthritis).SOLUTION: There is provided a liposomal composition comprising gamma polyglutamated methotrexate and a liposome encapsulating one or more non-polyglutamated, polyglutamatable antifolate compositions or non-polyglutamatable antifolates, wherein the liposome has a diameter of 50-150 nm.SELECTED DRAWING: None

Description

[Background technology]

[0001] The present disclosure relates generally to gamma polyglutamated methotrexate compositions, including delivery vehicles such as liposomes containing gamma polyglutamated methotrexate compositions, and methods of making and using the compositions to treat diseases, including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0002] Methotrexate has become widely used clinically worldwide as an essential component of combination therapy for the treatment of acute lymphoblastic leukemia (ALL), lymphoma, and solid tumors. It is also the core drug of the most widely applied disease-modifying antirheumatic drugs (DMARDs) in the treatment of patients with rheumatoid arthritis (RA). It is used as a single agent or in combination with other DMARDs (e.g., sulfasalazine and hydroxychloroquine). The use of MTX is essential for most treatment strategies, including biologics (e.g., anti-TNFα and anti-CD20 monoclonal antibodies). It is used in the treatment of breast cancer, advanced head and neck cancer, lung cancer, and gastric cancer, osteosarcoma, non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, and villous adenoma. Non-FDA approved cancer uses of methotrexate include nonleukemic meningeal carcinomatosis, soft tissue sarcomas (densemoid tumors, aggressive fibromatosis), bladder cancer, central nervous system (CNS) lymphoma, and graft-versus-host disease prophylaxis.

[0003] MTX is also used for non-cancer conditions such as psoriasis and rheumatoid arthritis, inflammatory bowel disease (IBD), systemic inflammation, atherosclerosis, cardiovascular disease (CVD), coronary artery disease, and gestational trophoblastic disease. Some non-FDA approved non-cancer uses include Crohn's disease, dermatomyositis / polymyositis, ectopic pregnancy, systemic lupus erythematosus, and Takayasu's arteritis.

[0004] Methotrexate is a folic acid analogue that differs from folic acid by the substitution of an amino group for the hydroxyl group at the 4-position of the pteridine ring. This slight structural change results in MTX's ability to inhibit the active catalytic site of dihydrofolate reductase (DHFR), which catalyzes the production of tetrahydrofolate (THF) from dihydrofolate (DHF). As a result, methotrexate interferes with the synthesis of tetrahydrofolate (THF), which serves as the primary one-carbon carrier for enzymatic processes involved in the de novo synthesis of thymidylate, purine nucleotides, and the amino acids serine and methionine. Inhibition of these metabolic processes disrupts the formation of DNA, RNA, and important cytoplasmic proteins.

[0005] Folate is an essential cofactor mediating the transfer of one-carbon units involved in nucleotide biosynthesis and DNA repair, homocysteine remethylation (Hcy), and methylation of DNA, proteins, and lipids. The only circulating form of folate in the blood is monoglutamate, and folate monoglutamate is the only form of folate transported across cell membranes, as are monoglutamate-type polyglutamate-able antifolate drugs such as methotrexate. Once inside cells, intracellular folate is converted to polyglutamate by the enzyme folylpolygammaglutamate synthase (FPGS).

[0006] Methotrexate is transported into cells by the reduced folate carrier (RFC) system and folate receptors (FR) α and β, as well as by the proton-coupled folate transporter (PCFT), which is most active in lower-than-normal pH environments. RFC is the major methotrexate transporter at physiological pH and is widely expressed in normal and diseased cells. Therefore, methotrexate is often subject to dose-limiting toxicity, a major obstacle in cancer chemotherapy. Once inside cells, methotrexate is polyglutamylated by FPGS, which can add up to six L-glutamyl groups to the L-gamma carboxyl group bond to methotrexate. L-gamma polyglutamation of methotrexate by FPGS serves at least two major therapeutic purposes: (1) it greatly increases the affinity and inhibitory activity of methotrexate for DHFR; and (2) it facilitates the accumulation of polyglutamylated methotrexate, which, unlike methotrexate (monoglutamate), is not readily transported out of cells by cellular efflux pumps.

[0007] Targeting folate metabolism and nucleotide biosynthesis is a well-established therapeutic strategy for cancer, but clinical efficacy of methotrexate has been limited due to a lack of tumor selectivity and the existence of new and acquired drug resistance. Like other antifolates, methotrexate acts on DNA and RNA synthesis, resulting in significant toxic effects on rapidly dividing cells, such as malignant and myeloid cells. Myelosuppression is usually the dose-limiting toxicity of methotrexate therapy, limiting its clinical application.

[0008] Resistance to methotrexate therapy is usually associated with one or more of the following: (a) increased cellular efflux pump activity, (b) decreased transport of MTX into cells, (c) increased DHFR activity, (d) decreased folylpoly-gamma-glutamate synthetase (FPGS) activity, and (e) increased gamma-glutamyl hydrolase (GGH) activity, which cleaves gamma-polyglutamate chains attached to folate and antifolate drugs.

[0009] A problem with the long-standing (>30 years) observation that higher polyglutamate levels of various antifolates have much greater potency than lower glutamate levels has been that the scientific community has relied on intracellular FPGS-mediated mechanisms to convert low levels of glutamate to their higher level forms. The present invention provides a means to deliver higher levels of polyglutamate forms of antifolates directly into cells without relying on cellular machinery to achieve this goal.

[0010] The provided gamma polyglutamated methotrexate compositions offer a strategy to overcome the pharmacological challenges associated with dose-limiting toxicity and therapeutic resistance associated with methotrexate therapy. The provided methods deliver novel gamma polyglutamated forms of methotrexate to cancer cells while (1) minimizing / reducing exposure to normal tissue cells, (2) optimizing / improving the cytotoxic effects of methotrexate-based drugs against cancer cells, and (3) minimizing / reducing the effects of efflux pumps and other resistance mechanisms that limit the therapeutic efficacy of methotrexate. Summary of the Invention

[0011] The present disclosure relates generally to gamma polyglutamated methotrexate (MTX) compositions and methods of making and using the compositions to treat diseases, including hyperproliferative diseases such as cancer, inflammation and immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0012] In some embodiments, the present disclosure provides: [1] A composition comprising gamma polyglutamated methotrexate; [2] The composition according to item [1], wherein the gamma polyglutamylated methotrexate contains 1 to 10 glutamyl groups having gamma carboxyl group bonds; [3] The composition according to item [1] or [2], wherein the gamma polyglutamated methotrexate contains 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups with gamma carboxyl group bonds; [4] The composition according to any one of items [1] to [3], wherein the gamma polyglutamated methotrexate is gamma tetraglutamated methotrexate; [5] The composition according to any one of items [1] to [3], wherein the gamma polyglutamated methotrexate is gamma pentaglutamated methotrexate; [6] The composition according to any one of items [1] to [3], wherein the gamma polyglutamated methotrexate is gamma hexaglutamated methotrexate; [7] The composition according to any one of items [1] to [6], wherein the composition is: (a) gamma polyglutamated methotrexate contains an L-glutamyl group with two or more gamma carboxyl group bonds; (b) gamma polyglutamylated methotrexate, wherein each glutamyl group is in the L-form and has a gamma carboxyl group bond; (c) at least one glutamyl group of the gamma polyglutamated methotrexate is in the D-form and has a gamma carboxyl group bond; (d) each glutamyl group of the gamma polyglutamylated methotrexate other than the glutamyl group of methotrexate is in the D-form and has a gamma carboxyl group bond; or (e) the gamma polyglutamated methotrexate contains two or more L-glutamyl groups and at least one D-glutamyl group with a gamma carboxyl linkage; [8] The composition according to item [4], wherein (a) each glutamyl group is in the L-configuration and has a gamma carboxyl linkage, or (b) each glutamyl group other than the glutamyl group of methotrexate is in the D-configuration and each glutamyl group has a gamma carboxyl linkage; [9] The composition according to item [5], wherein (a) each glutamyl group is in the L-configuration and has a gamma carboxyl linkage, or (b) each glutamyl group other than the glutamyl group of methotrexate is in the D-configuration and each glutamyl group has a gamma carboxyl linkage;

[10] The composition according to item [6], wherein (a) each glutamyl group is in the L-configuration and has a gamma carboxyl linkage, or (b) each glutamyl group other than the glutamyl group of methotrexate is in the D-configuration and each glutamyl group has a gamma carboxyl linkage;

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

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

[12] A liposome composition (Lp-γPMTX) containing gamma polyglutamated methotrexate according to any one of items [1] to

[11] ;

[13] The Lp-γPMTX composition according to item

[12] , wherein the gamma polyglutamylated methotrexate contains two or more L-glutamyl groups;

[14] The Lp-γPMTX composition according to item

[12] or

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

[15] The Lp-γPMTX composition according to item

[12] or

[13] , wherein at least one glutamyl group of the gamma polyglutamylated methotrexate is in the D-form;

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

[12] to

[15] , wherein the liposome contains gamma polyglutamated methotrexate containing 1 to 10 glutamyl groups having gamma carboxyl group bonds;

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

[12] to

[16] , wherein the liposome contains gamma polyglutamated methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups;

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

[12] to

[17] , wherein the liposome contains gamma-tetraglutamated methotrexate;

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

[12] to

[17] , wherein the liposome contains gamma-pentaglutamated methotrexate;

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

[12] to

[17] , wherein the liposome contains gamma hexaglutamated methotrexate;

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

[12] to

[20] , wherein the liposome is not PEGylated (PγLp-γPMTX);

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

[12] to

[20] , wherein the liposome is PEGylated (PγLp-γPMTX);

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

[12] to

[22] , wherein the liposome contains at least 1% by weight of gamma-polyglutamated methotrexate, or at least 1% of the gamma-polyglutamated MTX starting material is encapsulated (enclosed) in the Lp-γPMTX during the process of producing the Lp-γPMTX;

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

[12] to

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

[25] The Lp-γPMTX 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;

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

[12] to

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

[28] The Lp-γPMTX composition according to item

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

[29] The Lp-γPMTX composition according to item

[27] or

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

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

[27] to

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

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

[27] to

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

[32] The Lp-γPMTX composition according to item

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

[33] The Lp-γPMTX composition according to item

[32] , wherein the steric stabilizer is PEG, and the PEG has a number-average molecular weight (Mn) of 200 to 5,000 daltons;

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

[33] , wherein the liposome is cationic;

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

[12] to

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

[40] The Lp-γPMTX composition according to item

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

[41] The Lp-γPMTX composition according to item

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

[42] The Lp-γPMTX composition according to item

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

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

[39] to

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

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

[39] to

[43] , comprising 5% dextrose suspended in a HEPES buffer solution in the inner space of the liposome;

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

[39] to

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

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

[39] to

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

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

[12] to

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

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

[12] to

[47] , wherein the liposome contains less than 500,000 or less than 200,000 gamma polyglutamated methotrexate molecules;

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

[12] to

[48] , wherein the liposome contains 10 to 100,000 gamma polyglutamated methotrexate molecules or any range therebetween;

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

[50] or

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

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

[50] to

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

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

[39] to

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

[59] The Lp-γPMTX 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-γPMTX 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, resolvin (e.g., D n-6DPA Or D n-3DPAresolvin D, resolvin E, or T-series resolvins such as OXPAC, PGPC, and toll-like receptor (TLR) modulators such as erythropoietin lipids (e.g., E5564);

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

[58] to

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

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

[58] to

[61] , further comprising a hapten;

[63] The Lp-γPMTX composition according to item

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

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

[12] to

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

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

[64] ;

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

[49] ;

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

[12] to

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

[68] A pharmaceutical composition comprising the liposomal gamma polyglutamated methotrexate composition according to any one of items

[12] to

[67] ;

[69] A pharmaceutical composition comprising the gamma polyglutamated methotrexate 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 treating 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 thereof, the method comprising administering to the subject the composition according to any one of items [1] to

[70] ;

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

[12] to

[69] ;

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

[69] ;

[75] A method for killing hyperproliferative cells, comprising contacting the hyperproliferative cells with the liposomal gamma polyglutamated methotrexate composition according to any one of items

[12] to

[69] ;

[76] The method according to item

[74] or

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

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

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

[78] A method for treating cancer, comprising administering an effective amount of the liposomal gamma polyglutamated methotrexate 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 selected from the group consisting of non-hematological tumors, including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors, such as leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias;

[80] The method according to item

[77] or

[78] , wherein the cancer is a member selected from the group consisting of lung cancer, breast cancer, colon cancer, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, ovarian cancer and cervical cancer;

[81] The method according to item

[77] or

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

[82] The method according to item

[77] or

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

[83] A method for treating cancer, comprising administering an effective amount of the Lp-γPMTX 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 thereof;

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

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

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

[12] to

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

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

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

[88] Treatment of: (a) A method for treating 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 the infectious disease; (b) A method for treating an infectious disease, a cardiovascular disease, a metabolic 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, wherein the inflammation is optionally 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, wherein optionally the skin disease is psoriasis;

[89] A method for treating an infectious disease, comprising administering an effective amount of the liposomal gamma polyglutamated methotrexate composition according to any one of items

[12] to

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

[90] A method for delivering gamma polyglutamated methotrexate to a tumor expressing a folate receptor on its surface, comprising administering to a subject having a tumor the Lp-γPMTX composition described in any one of items [1] to

[69] in an amount that delivers a therapeutically effective amount of gamma polyglutamated methotrexate to the tumor;

[91] A method for preparing a gamma polyglutamated methotrexate composition comprising the liposomal gamma polyglutamated methotrexate composition according to any one of items

[12] to

[69] , the method comprising the steps of: forming a mixture in a solution containing liposome components and a gamma polyglutamated antifolate; homogenizing the mixture in the solution to form liposomes; and treating the mixture to form liposomes containing gamma polyglutamated methotrexate;

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

[12] to

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

[93] The method according to item

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

[94] The method according to item

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

[0013] In some embodiments, the present disclosure provides a gamma polyglutamylated methotrexate (γPMTX) composition, wherein at least two glutamyl residues of the gamma polyglutamylated methotrexate have gamma carboxyl linkages. In some embodiments, the γPMTX contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl groups of methotrexate). In some embodiments, the γPMTX contains two or more L-glutamyl groups. In other embodiments, the γPMTX contains D-glutamyl groups. In further embodiments, the γPMTX contains a D-glutamyl group and two or more L-glutamyl groups.

[0014] In one embodiment, the γPMTX composition comprises a chain of three glutamyl groups bound to a glutamyl group in methotrexate (i.e., tetraglutamated methotrexate). In some embodiments, the tetraglutamated MTX comprises two or more L-glutamyl groups. In other embodiments, the tetraglutamated MTX comprises a D-glutamyl group. In a further embodiment, the tetraglutamated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0015] In one embodiment, the gamma-PMTX composition comprises a chain of four gamma-glutamyl groups bound to a glutamyl group in methotrexate (e.g., gamma-pentaglutamated methotrexate). In some embodiments, the gamma-pentaglutamated MTX comprises two or more L-glutamyl groups. In other embodiments, the gamma-pentaglutamated MTX comprises a D-glutamyl group. In a further embodiment, the gamma-pentaglutamated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0016] In one embodiment, the γ-PMTX composition comprises a chain of five γ-glutamyl groups bound to a glutamyl group in methotrexate (e.g., γ-hexaglutamated methotrexate). In some embodiments, the gamma hexaglutamated MTX comprises two or more L-glutamyl groups. In other embodiments, the gamma hexaglutamated MTX comprises a D-glutamyl group. In a further embodiment, the gamma hexaglutamated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0017] In further embodiments, the present disclosure provides compositions comprising delivery vehicles, such as liposomes, loaded (e.g., encapsulated) and / or otherwise bound to gamma polyglutamated methotrexate, as well as methods for making and using γPMTX-loaded / bound delivery vehicle compositions (DV-γPMTX) to deliver gamma polyglutamated methotrexate 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 and malaria. In some embodiments, the gamma polyglutamated methotrexate in the DV-γPMTX contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, more than 5, or more than 20 glutamyl groups (including the glutamyl groups of methotrexate). The DV-γPMTX-loaded / bound delivery carrier composition provides selective delivery of a more cytotoxic payload (polyglutamylated methotrexate) compared to the cytotoxicity of methotrexate (MTX) administered in its monoglutamate state, thereby improving the efficacy and safety of methotrexate delivery to cancer cells.

[0018] In further embodiments, the present disclosure provides a composition (Lp-γPMTX) comprising liposomes encapsulating (loaded with) gamma polyglutamated methotrexate. In some embodiments, the gamma polyglutamated methotrexate in the Lp-γPMTX comprises 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 20 glutamyl groups (including the glutamyl group of methotrexate). In some embodiments, the gamma polyglutamated methotrexate in the Lp-γPMTX comprises two or more L-glutamyl groups. In other embodiments, the gamma polyglutamated methotrexate in the Lp-γPMTX comprises a D-glutamyl group. In a further embodiment, the gamma polyglutamated methotrexate in the Lp-γPMTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0019] In one embodiment, the Lp-γPMTX composition comprises gamma polyglutamylated MTX comprising a chain of three glutamyl groups bound to the glutamyl group of methotrexate (i.e., tetraglutamylated methotrexate). In some embodiments, the tetraglutamylated MTX comprises two or more L-glutamyl groups. In other embodiments, the tetraglutamylated MTX comprises a D-glutamyl group. In a further embodiment, the tetraglutamylated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0020] In one embodiment, the Lp-γPMTX composition comprises gamma polyglutamylated MTX comprising a chain of four γ-glutamyl groups bound to the glutamyl group of methotrexate (e.g., γ-pentaglutamylated methotrexate). In some embodiments, the gamma pentaglutamylated MTX comprises two or more L-glutamyl groups. In other embodiments, the gamma pentaglutamylated MTX comprises a D-glutamyl group. In a further embodiment, the gamma pentaglutamylated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0021] In one embodiment, the Lp-γPMTX composition comprises gamma polyglutamylated MTX comprising a chain of five gamma-glutamyl groups bound to the glutamyl group of methotrexate (e.g., gamma-hexaglutamylated methotrexate). In some embodiments, the gamma hexaglutamylated MTX comprises two or more L-glutamyl groups. In other embodiments, the gamma hexaglutamylated MTX comprises a D-glutamyl group. In a further embodiment, the gamma hexaglutamylated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0022] In some embodiments, the Lp-γPMTX composition is cationic. In some embodiments, the Lp-γPMTX liposomes are cationic and have a diameter ranging from 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-γPMTX liposomes are cationic and the composition has a diameter ranging from 80 nm to 120 nm, or any range therebetween. In some embodiments, the cationic Lp-γPMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w, or more than 75% w / w of gamma-polyglutamylated MTX. In some embodiments, during the process of making Lp-γPMTX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the gamma polyglutamylated MTX starting material is encapsulated (enclosed) in cationic Lp-γPMTX. In a further embodiment, the gamma polyglutamylated methotrexate encapsulated by the liposomes is present in a HEPES buffer solution within the liposomes.

[0023] In other embodiments, the Lp-γPMTX composition is anionic or neutral. In some embodiments, the Lp-γPMTX liposomes are anionic or neutral and have diameters in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-γPMTX liposomes are anionic or neutral and the composition has diameters in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the Lp-γPMTX liposomes are anionic and have diameters in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-γPMTX liposomes are anionic and the composition has diameters in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the Lp-γPMTX liposomes are neutral and have diameters ranging from 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In some embodiments, the anionic or neutral Lp-γPMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w, or more than 75% w / w of gamma polyglutamylated MTX. In some embodiments, during the Lp-γPMTX production process, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the gamma-polyglutamylated MTX starting material is encapsulated (encapsulated) in anionic or neutral Lp-γPMTX. In some embodiments, the anionic or neutral Lp-γPMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of gamma-tetraglutamylated MTX.In some embodiments, the anionic or neutral Lp-γPMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w, or more than 75% w / w of gamma-pentaglutamated MTX. In some embodiments, the anionic or neutral Lp-γPMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w, or more than 75% w / w of gamma-hexaglutamated MTX. In further embodiments, the liposomally encapsulated gamma-polyglutamated methotrexate is present in a HEPES buffer solution within the liposomes.

[0024] In a further embodiment, the liposomal gamma polyglutamated methotrexate composition is pegylated (PLp-γPMTX).

[0025] In some embodiments, the liposomal gamma polyglutamated methotrexate composition is non-targeted (NTLp-γPMTX). That is, the NTLp-γPMTX composition does not have specific affinity for an epitope expressed on the surface of a target cell of interest (e.g., an epitope on a surface antigen). In further embodiments, the non-targeted liposomal gamma polyglutamated methotrexate composition is pegylated (NTPLp-γPMTX).

[0026] In other embodiments, the liposomal gamma polyglutamated methotrexate composition is targeted (TLp-γPMTX). That is, the TLp-γPMTX composition includes a targeting moiety that has specific affinity for an epitope (surface antigen) on a target cell of interest. In some embodiments, the targeting moiety of the TLp-γPMTX or TPLp-γPMTX is not covalently attached to the liposome. In other embodiments, the targeting moiety of the TLp-γPMTX or TPLp-γPMTX is attached to one or both of the PEG and the exterior surface of the liposome. The targeted liposomal gamma polyglutamated methotrexate compositions (TLp-γPMTX and TPLp-γPMTX) provide further improvements over the efficacy and safety profile of methotrexate by specifically delivering gamma polyglutamated (e.g., γ-pentaglutamated and / or γ-hexaglutamated) methotrexate to target cells, such as cancer cells. In some embodiments, the non-targeted liposomal gamma polyglutamated methotrexate composition is PEGylated (TPLp-γPMTX). In some embodiments, the targeting moiety of TLp-γPMTX or TPLp-γPMTX is bound to one or both of the PEG and the outer surface of the liposome. In some embodiments, TLp-γPMTX or TPLp-γPMTX is bound to the liposome via a covalent bond. The function of the targeting moiety of the TLp-γPMTX and / or TPLp-γPMTX composition includes, but is not limited to, targeting the liposome to a target cell of interest in vivo or in vitro; interacting with a surface antigen to which the targeting moiety has specific affinity; and delivering the liposomal payload (γPMTX) 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.

[0027] In some embodiments, the targeting moiety of TLp-γPMTX or TPLp-γPMTX 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-γPMTX or TPLp-γPMTX has specific affinity for an epitope that is selectively expressed on target cells, such as tumor cells, compared to normal or non-tumor cells. In some embodiments, the targeting moiety has specific affinity for an epitope on a tumor cell surface antigen that is present on tumor cells but absent or inaccessible on non-tumor cells. In some embodiments, the targeting moiety has a specific affinity of 0.5x10 as measured by BIACORE® analysis. -10 ~10x10 -6 It binds to the epitope of interest with an equilibrium dissociation constant (Kd) in the range of

[0028] In certain embodiments, the targeting moiety of TLp-γPMTX or TPLp-γPMTX comprises a polypeptide that specifically binds to a folate receptor. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the folate receptor bound by the targeting moiety is specific for one or more folate receptors selected from the group consisting of folate receptor alpha (FR-α, FOLR1), folate receptor beta (FR-β, FOLR2), and folate receptor delta (FR-δ, FOLR4). In some embodiments, the folate receptor bound by the targeting moiety is folate receptor alpha (FR-α). In some embodiments, the folate receptor bound by the targeting moiety is folate receptor beta (FR-β). In some embodiments, the targeting moiety specifically binds to FR-α and FR-β.

[0029] In further embodiments, the Lp-γPMTX 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 γPMTX composition (e.g., Lp-γPMTX, PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, or TPLp-γPMTX) is cationic. In other embodiments, the liposomal γPMTX composition (e.g., Lp-γPMTX, PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, or TPLp-γPMTX) is anionic or neutral. In further embodiments, the liposomes of the liposomal γPMTX composition (e.g., Lp-γPMTX, PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, or TPLp-γPMTX) have diameters ranging from 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, 50 nm to 150 nm, or any range therebetween. In some embodiments, the liposomes of the liposomal γPMTX composition have diameters ranging from 30 nm to 175 nm or 50 nm to 150 nm, or any range therebetween. In further embodiments, the liposomes of the liposomal γPMTX composition have diameters ranging from 80 nm to 120 nm, or any range therebetween. In some embodiments, the liposomal γPMTX composition is pegylated (e.g., PLp-γPMTX, NTPLp-γPMTX, or TPLp-γPMTX). In some embodiments, the liposomal γPMTX composition comprises a targeting moiety (e.g., TLp-γPMTX or TPLp-γPMTX). In further embodiments, the liposomal γPMTX composition is pegylated and targeted (e.g., TPLp-γPMTX). In some embodiments, the liposomal γPMTX composition comprises gamma polyglutamated methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomal γPMTX composition comprises gamma tetraglutamated methotrexate.In some embodiments, the liposomal γPMTX composition comprises gamma-pentaglutamated methotrexate. In other embodiments, the liposomal γPMTX composition comprises gamma-hexaglutamated methotrexate.

[0030] In some embodiments, the liposome composition comprises gamma polyglutamated methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of gamma polyglutamated MTX. In some embodiments, the Lp-γPMTX composition comprises gamma polyglutamated methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups and 1% to 98.5% w / w of gamma polyglutamated MTX. In some embodiments, the liposomes comprise gamma polyglutamylated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups, and during the process of making Lp-γPMTX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the gamma polyglutamylated methotrexate starting material is encapsulated (encapsulated) in Lp-γPMTX.

[0031] In some embodiments, the liposome composition comprises gamma-tetraglutamated methotrexate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% w / w of gamma-tetraglutamated MTX. In some embodiments, the Lp-γPMTX composition comprises gamma-tetraglutamated methotrexate and 1% to 98.5% w / w of gamma-tetraglutamated MTX. In some embodiments, the liposomes comprise gamma-tetraglutamated methotrexate, and during the process of making Lp-γPMTX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of the gamma-tetraglutamated MTX starting material is encapsulated (encapsulated) in Lp-γPMTX.

[0032] In some embodiments, the liposome composition comprises gamma-pentaglutamated methotrexate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% w / w of gamma-pentaglutamated MTX. In some embodiments, the Lp-γPMTX composition comprises gamma-pentaglutamated methotrexate and 1% to 98.5% w / w of gamma-pentaglutamated MTX. In some embodiments, the liposomes contain gamma-pentaglutamated methotrexate, and during the process of producing Lp-γPMTX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the gamma-pentaglutamated MTX starting material is encapsulated (encapsulated) in the Lp-γPMTX. In some embodiments, the liposome composition consists of gamma-hexaglutamated methotrexate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of gamma-hexaglutamated MTX. In some embodiments, the Lp-γPMTX composition comprises gamma hexaglutamated methotrexate and 1% to 98.5% w / w gamma hexaglutamated MTX. In some embodiments, the liposomes comprise gamma hexaglutamated methotrexate, and during the process of making Lp-γPMTX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the gamma pentaglutamated MTX starting material is encapsulated (encapsulated) in the Lp-γPMTX.

[0033] Also provided are liposome compositions comprising γPMTX-encapsulated liposomes. In some embodiments, the liposome composition comprises a PEGylated γPMTX composition. In some embodiments, the liposome composition comprises a γPMTX composition linked or otherwise bound to a targeting moiety. In further embodiments, the liposome composition comprises a γPMTX composition that is PEGylated and linked or otherwise bound to a targeting moiety. In some embodiments, the liposome composition comprises γPMTX comprising 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome composition comprises gamma-tetraglutamated methotrexate. In some embodiments, the liposome composition comprises gamma-pentaglutamated methotrexate. In other embodiments, the liposome composition comprises gamma-hexaglutamated methotrexate.

[0034] In some embodiments, the liposome composition comprises liposomal γPMTX (e.g., Lp-γPMTX, PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, and TPLp-γPMTX). In some embodiments, the liposomal γPMTX is pegylated (e.g., NTPLp-γPMTX, TPLp-γPMTX). In some embodiments, the liposomal γPMTX comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell of interest, such as a cancer cell (e.g., TLp-γPMTX or TPLp-γPMTX). In further embodiments, the liposome composition comprises pegylated liposomal γPMTX and further comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell of interest, such as a cancer cell (e.g., TPLp-γPMTX). In some embodiments, the liposome composition comprises cationic liposomal γPMTX. In other embodiments, the liposome composition comprises anionic or neutral liposomal γPMTX. In further embodiments, the liposome composition comprises liposomal γPMTX having a diameter of 20 nm to 500 nm, 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposomal γPMTX has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

[0035] Pharmaceutical compositions comprising gamma polyglutamated methotrexate (γPMTX) in a delivery vehicle, such as liposomal γPMTX, are also provided. In some embodiments, the pharmaceutical composition comprises a pegylated γPMTX composition. In some embodiments, the pharmaceutical composition comprises a γPMTX composition linked or otherwise attached to a targeting moiety. In further embodiments, the pharmaceutical composition comprises a γPMTX composition that is pegylated and linked or otherwise attached to a targeting moiety. In some embodiments, the pharmaceutical composition comprises γPMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition comprises gamma tetraglutamated methotrexate. In some embodiments, the pharmaceutical composition comprises gamma pentaglutamated methotrexate. In other embodiments, the pharmaceutical composition comprises gamma hexaglutamated methotrexate.

[0036] In some embodiments, the pharmaceutical composition comprises liposomal γPMTX (e.g., Lp-γPMTX, PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, and TPLp-γPMTX). In some embodiments, the liposomal γPMTX composition is pegylated (e.g., NTPLp-γPMTX and TPLp-γPMTX). In some embodiments, the liposomal γPMTX comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell of interest, such as a cancer cell (e.g., TLp-γPMTX or TPLp-γPMTX). In further embodiments, the pharmaceutical composition comprises a pegylated liposomal γPMTX composition and further comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell of interest, such as a cancer cell (e.g., TPLp-γPMTX). In some embodiments, the pharmaceutical composition comprises cationic liposomal γPMTX. In other embodiments, the pharmaceutical composition comprises anionic or neutral liposomal γPMTX. In a further embodiment, the pharmaceutical composition comprises liposomal γPMTX having a diameter of 20 nm to 500 nm, or 20 nm to 500 nm, or any range therebetween. In a further embodiment, the liposomal γPMTX composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

[0037] In further embodiments, the present disclosure provides a method of killing a cell, the method comprising contacting the cell with a composition comprising a gamma polyglutamated methotrexate (γPMTX) composition (e.g., γPMTX disclosed herein). In some embodiments, the contacted cell is a mammalian cell. In further embodiments, the contacted cell is a human cell. In some embodiments, the contacted cell is a hyperproliferative cell. In further embodiments, the hyperproliferative cell is a cancer cell. In further embodiments, the contacted cancer cell is a primary cell or a cell from a cell line obtained / derived from a cancer selected from the group consisting of non-hematologic tumors including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematologic tumors such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. In further embodiments, the contacted cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from the group consisting of breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, and chorioadenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (dsemoid, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In still further embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from colorectal cancer, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the γPMTX contains 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, γPMTX comprises γ-glutamyl groups in the D form. In some embodiments, γPMTX comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D form. In some embodiments, γPMTX comprises γ-glutamyl groups in the L form.In some embodiments, γPMTX contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, γPMTX contains L- and D-type γ-glutamyl groups. In some embodiments, γPMTX contains 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the γPMTX composition contains gamma tetraglutamated methotrexate. In some embodiments, the γPMTX composition contains gamma pentaglutamated methotrexate. In other embodiments, the γPMTX composition contains gamma hexaglutamated methotrexate.

[0038] In further embodiments, the present disclosure provides a method of killing a cell, the method comprising contacting the cell with a liposome comprising gamma polyglutamylated methotrexate (e.g., Lp-γPMTX, such as PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, or TPLp-γPMTX). In some embodiments, the contacted cell is a mammalian cell. In further embodiments, the contacted cell is a human cell. In some embodiments, the contacted cell is a hyperproliferative cell. In still further embodiments, the contacted hyperproliferative cell is a cancer cell. In further embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from the group consisting of non-hematologic tumors including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematologic tumors such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. In further embodiments, the contacted cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from the group consisting of breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, and chorioadenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (dsemoid, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma. In still further embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from colorectal cancer, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the liposome comprises γPMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups.In some embodiments, the liposomes comprise gamma tetraglutamated methotrexate. In some embodiments, the liposomes comprise gamma pentaglutamated methotrexate. In other embodiments, the liposomes comprise gamma hexaglutamated methotrexate.

[0039] In some embodiments, the liposome comprises γPMTX containing 4, 5, 2 to 10, 4 to 6, or more than 5 γ-glutamyl groups. In some embodiments, the liposome comprises γPMTX containing γ-glutamyl groups in the D-form. In some embodiments, the liposome comprises γPMTX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form. In some embodiments, the liposome comprises γPMTX containing γ-glutamyl groups in the L-form. In some embodiments, the liposome comprises γPMTX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposome comprises γPMTX containing γ-glutamyl groups in both the L- and D-forms. In some embodiments, the liposome comprises γPMTX containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the liposome comprises gamma-pentaglutamated methotrexate. In other embodiments, the liposome comprises gamma-hexaglutamated methotrexate.

[0040] In further embodiments, the present disclosure provides a method of treating cancer, the method comprising administering an effective amount of a delivery vehicle (e.g., an immunoconjugate or liposome) comprising gamma polyglutamated methotrexate to a subject having or at risk of having cancer. In some embodiments, the delivery vehicle is an antibody-containing immunoconjugate (e.g., comprising a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery vehicle is a liposome (e.g., Lp-γPMTX, such as PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, or TPLp-γPMTX). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is not pegylated. In further embodiments, the administered delivery vehicle comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a cancer cell.In further embodiments, the delivery vehicle comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α, folate receptor-β, or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6 , FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD10 5, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C 242 antigens, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery vehicle comprises a targeting moiety that specifically binds to a cell surface antigen, such as a neoantigen, identified as being derived from or expressed on a specific target cancer (tumor). In some embodiments, the targeting moiety has specific affinity for an epitope of a cell surface antigen, such as a neoantigen, identified as being derived from or expressed on a specific target tumor. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the administered delivery vehicle comprises γPMTX containing 4, 5, 2 to 10, 4 to 6, or more than 5 γ-glutamyl groups. In some embodiments, the delivery vehicle comprises γPMTX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the delivery vehicle comprises γPMTX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the delivery vehicle comprises γPMTX comprising 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the administered delivery vehicle comprises gamma tetraglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises gamma pentaglutamated methotrexate. In other embodiments, the administered delivery vehicle comprises gamma hexaglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises L-gamma polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises D-gamma polyglutamated methotrexate. In further embodiments, the administered delivery vehicle comprises L- and D-gamma polyglutamated methotrexate. In some embodiments, the cancer is selected from the group consisting of non-hematologic tumors, including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma, brain cancer, central nervous system cancer, and melanoma; and hematologic tumors, such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia.In some embodiments, the cancer is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, chorioadenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (dsemoid, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma. In still further embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from colorectal cancer, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer.

[0041] In further embodiments, the present disclosure provides methods of treating cancer, the methods comprising administering an effective amount of a liposome comprising gamma polyglutamated methotrexate (e.g., Lp-γPMTX, such as PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, or TPLp-γPMTX) to a subject having or at risk of having cancer. In some embodiments, the liposome is PEGylated. In some embodiments, the liposome is not PEGylated. In further embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a cancer cell.In further embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α, folate receptor-β, or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6 , FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD10 5, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C 242 antigens, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.This also includes the use of cancer stem cell targeting moieties, such as those targeting CD34, CD133 and CD44, CD138, and CD15. In some embodiments, the liposomes contain a targeting moiety with specific affinity for an epitope of a cell surface antigen identified as originating from or expressed on a particular target tumor, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or antigen-binding antibody fragment. In some embodiments, the liposomes contain γPMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition contain γPMTX containing γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition contain γPMTX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPMTX containing L- and D-type γ-glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise γPMTX containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the administered liposome composition comprises tetraglutamylated γPMTX. In some embodiments, the administered liposome composition comprises pentaglutamylated γPMTX. In some embodiments, the administered liposome composition comprises hexaglutamylated γPMTX. In some embodiments, the cancer is selected from the group consisting of 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, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, and hematological tumors (e.g., leukemia or lymphoma). In still further embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from colorectal cancer, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer.

[0042] In a further embodiment, the present disclosure provides a method of treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a liposome composition comprising gamma polyglutamated methotrexate and a liposome comprising a targeting moiety having specific affinity for an epitope of an antigen on the surface of the cancer.In some embodiments, the liposome comprises a targeting moiety that has specific affinity for an epitope of a cell surface antigen selected from the group consisting of GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α, folate receptor-β, or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6 , FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD10 5, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C 242 antigens, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the liposome comprises a targeting moiety that has specific affinity for an epitope of a cell surface antigen identified as derived from or expressed on a particular target cancer (tumor), such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the liposome comprises γPMTX containing 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the liposome comprises γPMTX containing γ-glutamyl groups in the L-form. In some embodiments, the liposome comprises γPMTX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposome comprises γPMTX containing γ-glutamyl groups in the D-form. In some embodiments, the liposome comprises γPMTX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form. In some embodiments, the liposomes comprise gamma tetraglutamated methotrexate. In some embodiments, the liposomes comprise gamma pentaglutamated methotrexate. In some embodiments, the liposomes comprise gamma hexaglutamated methotrexate.

[0043] In some embodiments, the administered liposome composition comprises PEGylated liposomes (e.g., TPLp-γPMTX). In some embodiments, the administered liposome composition comprises non-PEGylated liposomes. In some embodiments, the administered liposomes of the liposome composition comprise γPMTX containing 4, 5, 2 to 10, 4 to 6, or more than 5 gamma-glutamyl groups. In some embodiments, the administered liposomes of the liposome composition comprise γPMTX containing γ-glutamyl groups in the D-form. In some embodiments, the administered liposomes of the liposome composition comprise γPMTX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form. In some embodiments, the administered liposomes of the liposome composition comprise γPMTX containing γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPMTX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPMTX containing γ-glutamyl groups in both the L- and D-forms. In some embodiments, the liposomes of the administered liposome composition comprise γPMTX containing 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D-form. In some embodiments, the liposomes of the administered liposome composition comprise gamma-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise gamma-pentaglutamated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise gamma-hexaglutamated methotrexate. In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of lung cancer (e.g., non-small cell), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, myeloma, leukemia, and lymphoma.

[0044] In further embodiments, the present disclosure provides a method for treating cancer, the method comprising administering an effective amount of a liposome composition to a subject having or at risk of having a cancer that expresses a folate receptor on its cell surface, the liposome composition comprising (a) gamma polyglutamated methotrexate (γPMTX) and (b) a liposome comprising a targeting moiety having specific binding affinity for a folate receptor. In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α) and folate receptor beta (FR-β). In some embodiments, the administered liposome composition comprises PEGylated liposomes (e.g., TPLp-γPMTX). In some embodiments, the administered liposome composition comprises non-PEGylated liposomes. In some embodiments, the administered liposomes of the liposome composition comprise γPMTX containing 4, 5, 2 to 10, 4 to 6, or more than 5 γ-glutamyl groups. In some embodiments, the administered liposomes of the liposome composition comprise γPMTX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the administered liposomes of the liposome composition comprise γPMTX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise γPMTX containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise gamma pentaglutamated methotrexate.In other embodiments, the liposomes of the administered liposome composition comprise gamma hexaglutamated methotrexate. In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of non-hematologic tumors, including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematologic tumors, such as, for example, leukemia, lymphoma, and other B-cell malignancies, myeloma, and other plasma cell dysplasias or cachexia. In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, chorioadenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (dsemoid, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma.

[0045] In further embodiments, the present disclosure provides a method for maintenance therapy of cancer, the method comprising administering an effective amount of a liposome composition comprising liposomes containing gamma polyglutamated methotrexate (Lp-γPMTX) to a subject undergoing or who has undergone cancer therapy. In some embodiments, the administered liposome composition is PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, or TPLp-γPMTX. In some embodiments, the administered liposome composition comprises PEGylated liposomes (e.g., PLp-γPMTX, NTPLp-γPMTX, or TPLp-γPMTX). In some embodiments, the administered liposome composition comprises targeted liposomes (e.g., TLp-γPMTX or TPLp-γPMTX). In some embodiments, the administered liposome composition comprises PEGylated liposomes comprising a targeting moiety (e.g., TPLp-γPMTX). In some embodiments, the liposomes of the administered liposome composition comprise gamma polyglutamated methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 γ-glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise γPMTX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise γPMTX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise γPMTX containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise gamma-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise gamma-pentaglutamated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise gamma-hexaglutamated methotrexate.

[0046] In further embodiments, the present disclosure provides methods for treating an immune system disorder, the method comprising administering an effective amount of a liposome composition comprising liposomes comprising gamma polyglutamated methotrexate (e.g., Lp-γPMTX, PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, or TPLp-γPMTX) to a subject having or at risk of having an immune system disorder. In some embodiments, the liposome composition is administered to treat an autoimmune disease. In a further embodiment, the liposome composition is administered to treat rheumatoid arthritis. In another embodiment, the liposome composition is administered to treat inflammation. In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., PLp-γPMTX, NTPLp-γPMTX, or TPLp-γPMTX). In some embodiments, the administered liposome composition comprises a targeted liposome (e.g., TLp-γPMTX or TPLp-γPMTX) comprising a targeting moiety with specific affinity for a surface antigen on a target cell (e.g., an immune cell) of interest. In further embodiments, the administered liposome composition comprises a liposome (e.g., TPLp-γPMTX) that is pegylated and comprises a targeting moiety. In some embodiments, the administered liposome comprises gamma-pentaglutamated methotrexate comprising 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the administered liposome comprises γPMTX comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise γPMTX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPMTX containing 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D-form.In some embodiments, the liposomes of the administered liposome composition comprise gamma-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise gamma-pentaglutamated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise gamma-hexaglutamated methotrexate.

[0047] The present disclosure also provides a method for delivering gamma polyglutamated methotrexate to tumor and / or cancer cells, the method comprising administering to a subject having a tumor a composition comprising gamma polyglutamated methotrexate (L-γPMTX) and a targeting moiety having specific binding affinity for an epitope on a surface antigen of a tumor or cancer cell. In some embodiments, the administered targeting moiety is conjugated to a delivery vehicle. In some embodiments, the delivery vehicle is an antibody or an antigen-binding fragment of an antibody. In further embodiments, the delivery vehicle is a liposome. In further embodiments, the antibody, antigen-binding antibody fragment, or liposome is a pegylated liposome (e.g., TPLp-γPMTX). In some embodiments, the administered composition comprises gamma polyglutamated methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the administered composition comprises gamma tetraglutamated methotrexate. In some embodiments, the composition administered comprises gamma-pentaglutamated methotrexate, hi other embodiments, the composition administered comprises gamma-hexaglutamated methotrexate.

[0048] In further embodiments, the present disclosure provides a method for making a liposome composition comprising a liposomal gamma polyglutamated methotrexate (γPMTX) composition, the method comprising: forming a mixture in solution comprising liposome components and γ polyglutamated methotrexate; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes comprising polyglutamine methotrexate. In some embodiments, the γ polyglutamated methotrexate comprises 4, 5, 2 to 10, 4 to 6, or more than 5 γ-glutamyl groups. In some embodiments, the γPMTX composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the γPMTX composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the γPMTX composition comprises 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the γPMTX composition comprises gamma-pentaglutamated methotrexate. In some embodiments, the γPMTX composition comprises gamma-tetraglutamated methotrexate. In other embodiments, the γPMTX composition comprises gamma-hexaglutamated methotrexate.

[0049] In one embodiment, the present disclosure provides a kit comprising a gamma polyglutamated methotrexate composition and / or a γPMTX delivery vehicle, such as a liposome, comprising γPMTX and a γPMTX immunoconjugate (e.g., an ADC described herein). [Brief explanation of the drawings]

[0050] [Figure 1-1]1A-1L show the chemical formulas of methotrexate (FIG. 1A), representative gamma methotrexate polyglutamates: methotrexate diglutamate (FIG. 1B), methotrexate triglutamate (FIG. 1C and 1D), methotrexate tetraglutamate (FIG. 1E and 1F), methotrexate pentaglutamate (FIG. 1G and 1H), methotrexate hexaglutamate (FIG. 1I and 1J), methotrexate heptaglutamate (FIG. 1K and 1L), methotrexate octaglutamate (FIG. 1M and 1N), representative gamma methotrexate polyglutamate (FIG. 1O), and representative methotrexate analogs (FIG. 1P and 1Q). [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 2] An example of the dose-response relationship, expressed as the percentage of surviving cells after 48 hours of treatment, is shown for free pemetrexed L-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6), pemetrexed, and the folate receptor alpha-targeted antibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6-FR1Ab) in NCI H2342 non-small cell lung cancer (NSCLC), adenocarcinoma subtype. [Figure 3] An example of the dose-response relationship at 48 hours for free pemetrexed L-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6), pemetrexed, and the folate receptor alpha-targeting antibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6-FR1Ab) in HT-29 (colon cancer) is shown. [Figure 4]Figure 1 shows the effect of free pemetrexed L-gamma hexaglutamate (hexa gG6) and liposomal pemetrexed L-gamma hexaglutamate (liposomal hexa gG6) on the proliferation of colon cancer SW260 cells after exposure to 256 nM of the corresponding drug over a 48-hour period. Non-targeted and targeted liposomal pemetrexed hexa gG6 can enter cells more efficiently than free pemetrexed hexa gG6 to inhibit the proliferation of colon cancer SW260 cells. [Figure 5] Figure 1 shows the relative efficacy of liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6) and its enantiomer, liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), to pemetrexed after 48 hours of exposure to the cancer cell lines SW620 (CRC), HT-29 (colon cancer), H1806 (triple-negative breast cancer), OAW28 (ovarian cancer), H292 (NSCLC, adenocarcinoma subtype), and H2342 (NSCLC, adenocarcinoma subtype). [Figure 6] 1 shows the therapeutic effects of liposomal pemetrexed gamma-L hexaglutamate (Lps Hexa gG6), liposomal pemetrexed gamma-D hexaglutamate (Lps Hexa gDG6), and pemetrexed on HCC1806 triple-negative breast cancer cells after 48 hours of exposure. [Figure 7] 1 shows the therapeutic effect of liposomal pemetrexed gamma-L hexaglutamate (Lps Hexa gG6), liposomal pemetrexed gamma-D hexaglutamate (Lps Hexa gDG6) compared to pemetrexed on OAW28 ovarian cancer cells after 48 hours of exposure. [Figure 8] 1 shows the therapeutic effects of liposomal pemetrexed gamma-L hexaglutamate (Lps Hexa gG6), liposomal pemetrexed gamma-D hexaglutamate (Lps Hexa gDG6), and pemetrexed on H292 non-small cell lung cancer cells after 48 hours of exposure. [Figure 9]This figure shows the therapeutic effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed at various dose levels ranging from 16 to 128 nM on H292 non-small cell lung cancer cells after 48 hours of exposure. At each dose range tested, the liposomal pemetrexed gG6 formulation exhibited superior inhibition of H292 non-small cell lung cancer cells compared to pemetrexed. [Figure 10] This figure shows the therapeutic effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed at various dose levels ranging from 16 to 128 nM on HCC1806 triple-negative breast cancer cells after 48 hours of exposure. Within each tested dose range, the liposomal pemetrexed gG6 formulation was superior to pemetrexed in inhibiting HCC1806 triple-negative breast cancer cells. [Figure 11] This figure shows the therapeutic effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal gamma-D hexaglutamate (liposomal gDG6), and pemetrexed on OAW28 ovarian cancer cells after 48 hours of exposure at a range of concentrations. At a dose of 128 nM, pemetrexed appears to be more effective than the liposomal pemetrexed gG6 liposomal formulation, but at doses of 32 nM and 64 nM, the liposomal formulation has superior therapeutic effects to pemetrexed. At 16 nM, the therapeutic effect of liposomal pemetrexed gG6 is similar to that of pemetrexed. [Figure 12] Figure 1 shows the toxicity of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed at 64 nM, 128 nM, and 264 nM toward differentiated human neutrophils. The figure shows that liposomal pemetrexed gG6 is significantly less toxic than pemetrexed toward differentiated human neutrophils. [Figure 13] The effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal gamma-D hexaglutamate (liposomal gDG6), and the corresponding pemetrexed agents on neutrophils (differentiated from CD34+ cells) after 48 hours of exposure at various dose levels ranging from 16 to 128 nM. [Figure 14] This figure shows the effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed at 16 nM, 32 nM, 64 nM, and 128 nM of the corresponding drug on AML12 liver cells after 48 hours of exposure. Remarkably, none of the liposomal drug tested at the dose levels appeared to be toxic to AML12 liver cells after treatment with liposomal pemetrexed gG6. In contrast, pemetrexed treatment resulted in a reduction in AML12 liver cell counts by approximately 40% at all doses examined. [Figure 15]

[0023] Figure 1 shows the effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed at 16 nM, 32 nM, 64 nM, and 128 nM of the corresponding drug on CCD841 colonic epithelial cells after 48 hours of exposure. At all concentrations tested, pemetrexed results in a reduction of CCD841 colonic epithelial cell counts of about 50% or more, compared to a reduction of about 20% or less after treatment with each of the liposomal compositions tested. [Figure 16] The structures of the polyglutamate antifolate, cisplatin (CDDP), and two possible gG6-cisplatin complexes are shown. The pH-dependent formation of inter- and / or intra-chain coordination between the carboxyl groups of the polyglutamate antifolate and cisplatin may lead to its degradation into separate molecules of gG6 and cisplatin upon encountering the acidic pH of the lysosome (pH 3-5) and in the presence of intracellular chloride ions. [Figure 17]Hematological parameters: Effect of liposomal aG6 treatment in mice at 40 mg / kg and 80 mg / kg once weekly for 4 weeks on white blood cell (WBC) count, neutrophil count, and platelet count. No significant decrease in mean neutrophil, mean white blood cell, or mean platelet count was observed. [Figure 18] This figure shows the effect of liposomal aG6 treatment in mice at 40 mg / kg and 80 mg / kg weekly doses for 4 weeks on hemoglobin and reticulocyte count indices. There is a minimal decrease in mean hemoglobin concentration at higher dose levels. Concomitantly, there is a slight increase in mean reticulocyte count indices. [Figure 19] The effects of liposomal aG6 treatment in mice at 40 mg / kg and 80 mg / kg once weekly for 4 weeks on liver markers, including serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT), in addition to serum albumin, are shown. No apparent increase in liver aminotransferase mean AST or ALT levels was observed, and furthermore, no changes in mean albumin levels were observed. [Figure 20] Relative tumor volumes in immunocompromised female Nu / J mice (6-8 weeks old) inoculated with NCI-H292 (non-small cell lung cancer) cells and treated intravenously with control, pemetrexed, or liposomal aG6 at 167 mg / kg once every three weeks are shown. These preliminary data show that liposomal aG6 results in reduced tumor control compared to pemetrexed. [Figure 21]Figures 21A-F show the efficacy of liposomal pemetrexed alpha-L triglutamine over 48 hours against H2342 (NSCLC, adenocarcinoma subtype) (Figure 21A), H292 (NSCLC, adenocarcinoma subtype) (Figure 21B), HT-29 (colon cancer) (Figure 21C), HCC1806 (triple-negative breast cancer) (Figure 21D), MCF7 (ER+ breast cancer) (Figure 21E), and OAW28 (ovarian cancer) (Figure 21F). Figure 1 shows the dose-response relationship for liposomal pemetrexed alpha-L pentaglutamate (liposome aG3), liposomal pemetrexed alpha-L octaglutamate (liposome aG7), and the combination of liposomal pemetrexed alpha-L hexaglutamate (liposome aG6) and alpha-L dodecaglutamate (liposome aG12) (liposome aG6 and aG12). Cell viability was measured using the CellTiter-Glo® (CTG) luminescent cell viability assay, essentially as described in Example 1. As shown in all cell lines, the potency of each polyglutamated pemetrexed liposome composition significantly exceeded that of the liposome carrier and empty liposome control. DETAILED DESCRIPTION OF THE INVENTION

[0051] Generally, the present disclosure relates to gamma polyglutamated methotrexate compositions. The compositions offer an advancement over existing treatments for hyperproliferative diseases, such as cancer. Methods for producing, delivering, and using the gamma polyglutamated methotrexate compositions are also provided. The gamma polyglutamated compositions have uses including, but not limited to, the treatment or prevention of hyperproliferative diseases, such as cancer, immune system disorders, such as inflammation and rheumatoid arthritis, and infectious diseases, such as HIV and malaria.

[0052] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0053] Whenever an embodiment is described herein with the term "comprising," other similar embodiments are also provided that are described with the terms "containing," "consisting of," and / or "consisting essentially of." However, when used as transitional phrases in the claims, each should be construed separately and in the appropriate legal and factual context. (E.g., in the claims, the transitional phrase "comprising" is considered to be more open-ended, while the transitional phrase "consisting of" is considered more exclusive, and "consisting essentially of" is considered intermediate.)

[0054] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise stated or unless it is clearly clear from the context that plural reference is not intended.

[0055] The term "and / or" as used in phrases such as "A and / or B" is intended herein to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in phrases such as "A, B and / or C" encompasses each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

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

[0057] Unless otherwise indicated, the terms "methotrexate" and "MTX" are used interchangeably and include salt, acid, and / or free base forms of methotrexate (e.g., methotrexate disodium). Compositions containing MTX salts may also contain various cations, e.g., Na + , Mg 2+ , K. + , NH4 + , and / or Ca 2+ In certain embodiments, the salt is typically a pharmaceutically acceptable salt. In further specific embodiments, the MTX salt may comprise Na + Methotrexate typically contains one L-gamma glutamyl group and is therefore considered to be monoglutamated for the purposes of this disclosure.

[0058] The terms "polyglutamylated methotrexate," "polyglutamylated MTX," "MTX-PG," and "PMTX" are used interchangeably herein to refer to methotrexate compositions containing at least one glutamyl group in addition to the glutamyl groups in methotrexate (i.e., MTX-PGn, n≧1). References to the number of glutamyl groups in γPMTX (MTX-PG) herein include the glutamyl groups in methotrexate. For example, an MTX-PG composition containing five glutamyl residues in addition to the glutamyl groups in MTX is referred to herein as hexaglutamylated methotrexate or methotrexate 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 through its amino group, but is bound to one or more glutamyl groups through its carboxylic acid group. In some embodiments, the N-terminal glutamyl group of polyglutamylated methotrexate is the glutamyl group of methotrexate. The C-terminal glutamyl group(s) of the polyglutamate chain is / are bound to another glutamyl group through their amino group, but is / are not bound to another glutamyl group through their carboxylic acid group.

[0059] The terms "gamma glutamyl group," "gamma glutamyl group," and "gamma bond," when referring to a glutamyl group bond, refer to a glutamyl group containing a gamma carboxyl group bond. The gamma bond can be a bond between a glutamyl group and a glutamyl group in methotrexate, or between a glutamyl group and a second glutamyl group, such as a glutamyl group in a polyglutamate chain bound to methotrexate, other than the one present in methotrexate (e.g., a glutamyl group in a polyglutamate chain bound to methotrexate). In some embodiments, the gamma bond is an amide bond between the gamma carboxyl group of one glutamyl group and a second glutamyl group. In some embodiments, the gamma bond refers to an amide bond of a glutamyl group in methotrexate. In some embodiments, the gamma bond is an amide bond between the gamma carboxyl group of one glutamyl group and a second glutamyl group. Reference to gamma bonds includes gamma bonds of the glutamyl groups of methotrexate unless otherwise specified or the context clearly indicates otherwise. In some embodiments, the gamma glutamyl groups are L-type. In some embodiments, the gamma glutamyl groups are D-type. As discussed herein, during methotrexate therapy, methotrexate enters cells and is polyglutamated by the enzyme folylpoly-gamma glutamate synthase (FPGS), which sequentially adds L-glutamyl groups to the gamma carboxyl groups of the glutamates in the methotrexate glutamyl groups of methotrexate. Thus, D-gamma polyglutamated methotrexate compositions are not formed intracellularly during methotrexate therapy.

[0060] The terms "gamma polyglutamated methotrexate," "gamma-polyglutamated methotrexate," "gamma PMTX," "gamma polyglutamated methotrexate," "polyglutamated MTX," "gamma MTX-PG," and repeats thereof are used interchangeably herein to refer to methotrexate compositions containing at least one gamma glutamyl group with a gamma carboxyl group linkage in addition to the gamma glutamyl groups in methotrexate (i.e., MTX-PGn, n≧1 gamma glutamyl group). References to the number of glutamyl groups in γ PMTX (γ MTX-PG) herein count the glutamyl groups in methotrexate. For example, a γ MTX-PG composition containing five gamma glutamyl groups in addition to the glutamyl groups in MTX is referred to herein as gamma hexaglutamated methotrexate or gamma methotrexate hexaglutamate.

[0061] The terms "alpha glutamyl group," "α-glutamyl group," and "alpha linkage," when referring to a glutamyl group linkage, refer to a glutamyl group that includes an alpha carboxyl group linkage.

[0062] As used herein, the term "isolated" refers to a composition in a form not found in nature. Isolated gamma polyglutamated compositions include those that have been purified to the extent that they are no longer in the form found in nature. In some embodiments, isolated gamma polyglutamated methotrexate is substantially pure. Isolated compositions are free or substantially free of naturally incorporated substances, such as proteins and other cellular components, such as nucleic acids, that may potentially be found in nature or in the environment in which they are produced (e.g., cell culture). Gamma polyglutamated compositions can be formulated with a diluent or adjuvant and further isolated for practical purposes—for example, when used as a diagnostic or therapeutic agent, gamma polyglutamated compositions are typically mixed with a pharmaceutically acceptable carrier or diluent. In some embodiments, isolated gamma polyglutamated compositions (e.g., gamma polyglutamates and delivery vehicles such as liposomes comprising gamma polyglutamates) contain less than 1% or less than 0.1% undesired DNA or protein content. In some embodiments, gamma polyglutamate compositions (e.g., gamma polyglutamates and delivery vehicles such as liposomes comprising gamma polyglutamates) are "isolated."

[0063] As used herein, the term "targeting moiety" refers to a molecule that confers enhanced affinity to a selected target, e.g., a cell, cell type, tissue, organ, body region, or compartment, e.g., a cell, tissue, or organ compartment. Targeting moieties can include a wide variety of substances. 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.

[0064] The terms "specific affinity," "specifically binds," and "enhanced affinity" mean that a targeting moiety, such as an antibody or antigen-binding antibody fragment, reacts with or binds to an epitope, protein, or target molecule more frequently, more rapidly, for a longer period of time, with greater affinity, or some combination thereof, than to another substance, including a protein unrelated to the antigen containing the target epitope. Due to sequence identity between homologous proteins in different species, a particular affinity, in some embodiments, includes binding substances that recognize epitopes and / or target molecules on proteins in more than one species. Similarly, due to homology within a particular region of the polypeptide sequence of different proteins, the terms "specific affinity" or "specific binding" can include binding substances that recognize epitopes present on more than one protein and / or target molecule. It is understood that, in certain embodiments, a targeting moiety that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific affinity" does not necessarily require (although it can include) exclusive binding, e.g., binding to an epitope on only one target. Thus, a targeting moiety may, in certain embodiments, specifically bind to an epitope present on more than one target. In certain embodiments, multiple targets may be bound by the same targeting moiety that specifically binds to epitopes present on multiple targets.

[0065] The term "epitope" refers to a portion of an antigen that can be recognized and specifically bound by a targeting moiety (i.e., binding moiety) such as an antibody. When the antigen is a polypeptide, epitopes can be formed from both contiguous and non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturation, whereas epitopes formed by tertiary folding are typically lost upon protein denaturation. An epitope typically comprises at least three amino acids, more commonly at least five or eight to ten amino acids, in a unique spatial conformation.

[0066] Expressions known in the art, such as " target binding affinity ", " target binding ", " enhanced affinity " and similar expressions refer to the affinity constant, which can be directly measured by determining the amount of targeting moiety that binds and dissociates at a given antigen concentration.Other methods can be used to characterize intermolecular interactions, including but not limited to, competitive analysis, equilibrium analysis and microcalorimetry, and real-time interaction analysis based on surface plasmon resonance interaction (for example, using BIACORE® device).These methods are well known to those skilled in the art and are described, for example, in Neri et al., Tibtech 14:465-470 (1996), and Jansson et al., J.Biol.Chem.272:8189-8197 (1997).

[0067] The term "delivery vehicle" generally refers to any composition that acts to support, promote, or facilitate the entry of gamma polyglutamated methotrexate into cells. Such delivery vehicles are known in the art and include, but are not limited to, liposomes, lipospheres, polymers (e.g., polymer conjugates), peptides, proteins such as antibodies (e.g., immunoconjugates such as antibody-drug conjugates (ADCs) and antigen-binding antibody fragments and their derivatives), cellular components, cyclic oligosaccharides (e.g., cyclodextrins), micelles, microparticles (e.g., microspheres), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), hydrogels, lipoprotein particles, viral sequences, viral agents, or lipid or liposomal formulations, and combinations thereof. The delivery vehicle can be directly or indirectly conjugated to a targeting moiety. In some examples, the targeting moiety is selected from a macromolecule, a protein, a peptide, a monoclonal antibody, or a fatty acid lipid.

[0068] "Subject" means a human or vertebrate mammal, including, but not limited to, a dog, a cat, a horse, a goat, and a primate, such as a monkey. Thus, the present invention can also be used to treat a disease or condition in a non-human subject. For example, cancer is one of the leading causes of death in companion animals (e.g., cats and dogs). In some embodiments of the present invention, the subject is a human. In this disclosure, the terms "subject" and "patient" are used interchangeably and have the same meaning. Generally, it is preferred to use a maximum dose, i.e., the maximum safe dose according to sound medical judgment.

[0069] As used herein, "effective amount" refers to the administration of a drug sufficient to produce a medically desired result. An effective amount may vary depending on the desired outcome, the specific condition being treated or prevented, the age and health of the subject being treated, the severity of the condition, the duration of treatment, the nature of concurrent or concomitant therapy (if any), the specific route of administration, and similar factors within the knowledge and professional opinion of a health practitioner. An "effective amount" may be determined empirically and routinely in connection with the stated purpose. In the case of cancer, an effective amount of a drug may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow to some extent, and preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder. Depending on the extent to which a drug may prevent and / or kill existing cancer cells, the drug may be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by assessing survival time, progression-free survival (PFS), response rate (RR), duration of response, and / or quality of life.

[0070] The terms "hyperproliferative disorder," "proliferative disease," and "proliferative disorder" are used interchangeably herein and refer to unwanted or uncontrolled cell proliferation of unwanted, excessive, or abnormal cells, such as neoplastic or hyperplastic growth, whether in vitro or in vivo. In some embodiments, the proliferative disorder is a cancer or tumor disease (including benign or cancerous) and / or any tumor metastasis, regardless of the location of the cancer, tumor, and / or tumor metastasis. In some embodiments, the proliferative disorder is a benign or malignant tumor. In some embodiments, the proliferative disorder is a non-cancerous disease. In some embodiments, the proliferative disorder is a hyperproliferative condition such as hyperplasia, fibrosis (particularly pulmonary, but also other types of fibrosis, such as renal fibrosis), angiogenesis, psoriasis, smooth muscle proliferation in blood vessels, such as atherosclerosis, and post-angioplasty stenosis or restenosis.

[0071] "Cancer," "tumor," or "malignant tumor" are used interchangeably and refer to any of a number of cell types or diseases characterized by uncontrolled, abnormal proliferation of cells, spread of infected cells locally or to other parts of the body via the bloodstream and lymphatic system (metastasis), and / or numerous characteristic structural and / or molecular features known to be associated with these cell types or diseases. As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. A "cancerous tumor," or "malignant cell," is understood to be a cell that has specific structural characteristics, lacks differentiation, and is capable of invasion and metastasis. Cancers that can be treated using the γPMTX compositions provided herein include, but are not limited to, non-hematological tumors, including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors, such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma.

[0072] Other types of cancers and tumors that can be treated with the γPMTX compositions are described herein or are known in the art. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0073] Terms such as "treating," "treatment," or "treat" refer to both (a) therapeutic measures that cure, slow, lessen the symptoms, and / or halt the progression of a diagnosed condition or disorder, and (b) prophylactic or preventative measures that prevent and / or delay the onset of the targeted disease or condition. Thus, subjects in need of treatment include those already with the cancer, disorder, or disease, those at risk of developing the cancer or condition, and those in whom the infection or condition is to be prevented. The subject has been identified, using well-known medical and diagnostic techniques, as being "at risk of having" cancer, an infectious disease, an immune system disorder, a hyperproliferative disease, or another disease or disorder referred to herein. In certain embodiments, a subject has been successfully "treated" by the methods provided herein if, for example, the subject exhibits total, partial, or temporary remission or elimination of symptoms associated with the disease or condition (e.g., cancer, inflammation, and rheumatoid arthritis). In certain embodiments, the term "treating" or "treatment" or "treat" refers to the improvement of at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which may not necessarily be discernible by the patient. In other embodiments, the term "treating" or "treatment" or "treat" refers to the inhibition of progression of a proliferative disorder, either physically, e.g., by stabilization of a discernible symptom, or physiologically, e.g., by stabilization of a physical parameter, or both. In other embodiments, the term "treating" or "treatment" or "treat" refers to the reduction or stabilization of size, tumor cell growth or survival, or cancer cell number. For treatment, the γ-PMTX composition can be used alone or in combination with an additional therapeutic agent.

[0074] "Subject," "patient," and "animal" are used interchangeably and refer to mammals, such as human patients and non-human primates, as well as laboratory animals, such as rabbits, rats, mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as chickens, amphibians, and reptiles. As used herein, "mammal" refers to any member of the class Mammalia, including, but not limited to, humans and non-human primates, e.g., chimpanzees and other apes and monkey species; livestock animals, such as cows, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, such as rodents, e.g., mice, rats, guinea pigs, and other members of the class Mammalia. In certain embodiments, the subject is a human.

[0075] As used herein, "treatment of a proliferative disorder" includes maintaining or reducing tumor size, inducing tumor regression (partial or complete), inhibiting tumor growth, and / or extending the lifespan of a subject with a proliferative disorder. In one embodiment, the proliferative disorder is a solid tumor. Such tumors include, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma. In one embodiment, the proliferative disorder is a hematological tumor. Such hematological tumors include, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia.

[0076] As used herein, the term "autoimmune disease" is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate or excessive response to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, inflammation and rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, leukoplakia, myxedema, pernicious anemia, and ulcerative colitis.

[0077] As used herein, the term "therapeutic agent" refers to a drug or derivative thereof that can interact with hyperproliferative cells, such as cancer cells or immune cells, thereby reducing the proliferative state of the cells and / or killing the cells. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, cytotoxic drugs, platinum-based drugs (e.g., cisplatin, carboplatin, oxaliplatin), taxanes (e.g., taxol), etoposide, alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate (MTX)), 5-fluorouracil, gemcitabine, or derivatives thereof, antitumor antibiotics (e.g., mitomycin, doxorubicin), and plant-derived antitumor agents (e.g., vincristine, vindesine, taxol). Such agents further include, but are not limited to, the anticancer agents trimetrexate, temozolomide, methotrexate, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benziguanidine (6-BG), bis-chloronitrosourea (BCNU), and camptothecin, or any therapeutic derivatives thereof. Additional examples of therapeutic agents that may be suitable for use with the methods of the present disclosure include, but are not limited to, antirestenotic agents, pro- or antiproliferative agents, anti-inflammatory agents, antineoplastic agents, antimitotic agents, antiplatelet agents, anticoagulants, antifibrinogens, antithrombin agents, cytostatic agents, antibiotics and other anti-infective agents, antienzymes, antimetabolites, angiogenic agents, cytoprotective agents, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, and / or cardioprotective agents. "Therapeutic agent" also refers to salt, acid, and free base forms of the above agents.

[0078] As used herein, the term "chemotherapeutic agent," when used in the context of cancer therapy, means any agent that causes the death of cancer cells or inhibits the growth or spread of cancer cells. Examples of such chemotherapeutic agents include alkylating agents, antibiotics, antimetabolites, plant-derived 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.

[0079] As used herein, the term "anti-metabolite" refers to a therapeutic agent that inhibits the utilization of a metabolite or its prodrug. Examples of anti-metabolites include methotrexate, methotrexate, 5-fluorouracil, 5-fluorouracil prodrugs such as capecitabine, 5-fluorodeoxyuridine monophosphate, cytarabine, cytarabine prodrugs such as nelarabine, 5-azacytidine, gemcitabine, mercaptopurine, thioguanine, azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, and cladribine. Anti-metabolites useful for practicing the disclosed methods include nucleoside analogs, including purine or pyrimidine analogs. In some embodiments, the gamma polyglutamated methotrexate composition is used in combination with an antimetabolite selected from the group consisting of fluoropyrimidine, 5-fluorouracil, 5-fluoro-2'-deoxycytidine, cytarabine, gemcitabine, troxacitabine, decitabine, azacitidine, pseudoisocytidine, zebularine, ancitabine, fazarabine, 6-azacytidine, capecitabine, N4-octadecylcytarabine, elaidic acid cytarabine, fludarabine, cladribine, clofarabine, nelarabine, forodesine, and pentostatin, or derivatives thereof. In one example, the nucleoside analog is a substrate of a nucleoside deaminase that is an adenosine deaminase or a cytidine deaminase. In some examples, the nucleoside analog is selected from fludarabine, cytarabine, gemcitabine, decitabine, and azacitidine or derivatives thereof. In certain embodiments, the antimetabolite is 5-fluorouracil.

[0080] As used herein, a "taxane" is an anti-cancer drug that interferes with or disrupts microtubule stability, formation, and / or function. Taxanes include paclitaxel and docetaxel and their derivatives, which function on microtubules with the same mode of action as the taxanes from which they are derived. In certain embodiments, the taxane is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the taxane is paclitaxel (Taxol®), docetaxel (Taxotere®), albumin-bound paclitaxel (nab-paclitaxel; Abraxane®), DHA-paclitaxel, or PG-paclitaxel.

[0081] The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, carriers, excipients, stabilizers, diluents, or preservatives. Pharmaceutically acceptable carriers include, for example, one or more compatible solid or liquid fillers, diluents, or encapsulating substances that are suitable for administration to humans or other subjects.

[0082] The present disclosure relates generally to gamma polyglutamated methotrexate (MTX) compositions and methods of making and using the compositions to treat diseases, including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0083] In some embodiments, the present disclosure provides: [1] A composition comprising gamma polyglutamated methotrexate; [2] The composition according to item [1], wherein the gamma polyglutamylated methotrexate contains 1 to 10 glutamyl groups having gamma carboxyl group bonds; [3] The composition according to item [1] or [2], wherein the gamma polyglutamated methotrexate contains 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups with gamma carboxyl group bonds; [4] The composition according to any one of items [1] to [3], wherein the gamma polyglutamated methotrexate is gamma tetraglutamated methotrexate; [5] The composition according to any one of items [1] to [3], wherein the gamma polyglutamated methotrexate is gamma pentaglutamated methotrexate; [6] The composition according to any one of items [1] to [3], wherein the gamma polyglutamated methotrexate is gamma hexaglutamated methotrexate; [7] The composition according to any one of items [1] to [6], wherein the composition is: (a) gamma polyglutamated methotrexate contains an L-glutamyl group with two or more gamma carboxyl group bonds; (b) gamma polyglutamylated methotrexate, wherein each glutamyl group is in the L-form and has a gamma carboxyl group bond; (c) at least one glutamyl group of the gamma polyglutamated methotrexate is in the D-form and has a gamma carboxyl group bond; (d) each glutamyl group of the gamma polyglutamylated methotrexate other than the glutamyl group of methotrexate is in the D-form and has a gamma carboxyl group bond; or (e) the gamma polyglutamated methotrexate contains two or more L-glutamyl groups and at least one D-glutamyl group with a gamma carboxyl linkage; [8] The composition according to item [4], wherein (a) each glutamyl group is in the L-configuration and has a gamma carboxyl linkage, or (b) each glutamyl group other than the glutamyl group of methotrexate is in the D-configuration and each glutamyl group has a gamma carboxyl linkage; [9] The composition according to item [5], wherein (a) each glutamyl group is in the L-configuration and has a gamma carboxyl linkage, or (b) each glutamyl group other than the glutamyl group of methotrexate is in the D-configuration and each glutamyl group has a gamma carboxyl linkage;

[10] The composition according to item [6], wherein (a) each glutamyl group is in the L-configuration and has a gamma carboxyl linkage, or (b) each glutamyl group other than the glutamyl group of methotrexate is in the D-configuration and each glutamyl group has a gamma carboxyl linkage;

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

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

[12] A liposome composition (Lp-γPMTX) containing gamma polyglutamated methotrexate according to any one of items [1] to

[11] ;

[13] The Lp-γPMTX composition according to item

[12] , wherein the gamma polyglutamylated methotrexate contains two or more L-glutamyl groups;

[14] The Lp-γPMTX composition according to item

[12] or

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

[15] The Lp-γPMTX composition according to item

[12] or

[13] , wherein at least one glutamyl group of the gamma polyglutamylated methotrexate is in the D-form;

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

[12] to

[15] , wherein the liposome contains gamma polyglutamated methotrexate containing 1 to 10 glutamyl groups having gamma carboxyl group bonds;

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

[12] to

[16] , wherein the liposome contains gamma polyglutamated methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups;

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

[12] to

[17] , wherein the liposome contains gamma-tetraglutamated methotrexate;

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

[12] to

[17] , wherein the liposome contains gamma-pentaglutamated methotrexate;

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

[12] to

[17] , wherein the liposome contains gamma hexaglutamated methotrexate;

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

[12] to

[20] , wherein the liposome is not PEGylated (PγLp-γPMTX);

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

[12] to

[20] , wherein the liposome is PEGylated (PγLp-γPMTX);

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

[12] to

[22] , wherein the liposome contains at least 1% by weight (w / w) of gamma-polyglutamated methotrexate, or at least 1% of the gamma-polyglutamated MTX starting material is encapsulated (enclosed) in the Lp-γPMTX during the process of producing the Lp-γPMTX;

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

[12] to

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

[25] The Lp-γPMTX 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;

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

[12] to

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

[28] The Lp-γPMTX composition according to item

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

[29] The Lp-γPMTX composition according to item

[27] or

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

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

[27] to

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

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

[27] to

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

[32] The Lp-γPMTX composition according to item

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

[33] The Lp-γPMTX composition according to item

[32] , wherein the steric stabilizer is PEG, and the PEG has a number-average molecular weight (Mn) of 200 to 5,000 daltons;

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

[33] , wherein the liposome is cationic;

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

[12] to

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

[40] The Lp-γPMTX composition according to item

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

[41] The Lp-γPMTX composition according to item

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

[42] The Lp-γPMTX composition according to item

[41] , wherein the pharmaceutically acceptable carrier comprises 1% to 20% trehalose;

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

[39] to

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

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

[39] to

[43] , comprising 5% dextrose suspended in a HEPES buffer solution in the inner space of the liposome;

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

[39] to

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

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

[39] to

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

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

[12] to

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

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

[12] to

[47] , wherein the liposome contains less than 500,000 or less than 200,000 gamma polyglutamated methotrexate molecules;

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

[12] to

[48] , wherein the liposome contains 10 to 100,000 gamma polyglutamated methotrexate molecules or any range therebetween;

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

[50] or

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

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

[50] to

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

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

[39] to

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

[59] The Lp-γPMTX 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-γPMTX 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, resolvin (e.g., D n-6DPA Or D n-3DPAresolvin D, resolvin E, or T-series resolvins such as OXPAC, PGPC, and toll-like receptor (TLR) modulators such as erythropoietin lipids (e.g., E5564);

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

[58] to

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

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

[58] to

[61] , further comprising a hapten;

[63] The Lp-γPMTX composition according to item

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

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

[12] to

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

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

[64] ;

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

[49] ;

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

[12] to

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

[68] A pharmaceutical composition comprising the liposomal gamma polyglutamated methotrexate composition according to any one of items

[12] to

[67] ;

[69] A pharmaceutical composition comprising the gamma polyglutamated methotrexate 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 treating 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 thereof, the method comprising administering to the subject the composition according to any one of items [1] to

[70] ;

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

[12] to

[69] ;

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

[69] ;

[75] A method for killing hyperproliferative cells, comprising contacting the hyperproliferative cells with the liposomal gamma polyglutamated methotrexate composition according to any one of items

[12] to

[69] ;

[76] The method according to item

[74] or

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

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

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

[78] A method for treating cancer, comprising administering an effective amount of the liposomal gamma polyglutamated methotrexate 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 selected from the group consisting of non-hematological tumors, including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors, such as leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias;

[80] The method according to item

[77] or

[78] , wherein the cancer is a member selected from the group consisting of lung cancer, breast cancer, colon cancer, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, ovarian cancer and cervical cancer;

[81] The method according to item

[77] or

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

[82] The method according to any one of items

[77] to

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

[83] A method for treating cancer, comprising administering an effective amount of the Lp-γPMTX 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 thereof;

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

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

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

[12] to

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

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

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

[88] Treatment of: (a) A method for treating 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 the infectious disease; (b) A method for treating an infectious disease, a cardiovascular disease, a metabolic 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, wherein the inflammation is optionally 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, wherein optionally the skin disease is psoriasis;

[89] A method for treating an infectious disease, comprising administering an effective amount of the liposomal gamma polyglutamated methotrexate composition according to any one of items

[12] to

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

[90] A method for delivering gamma polyglutamated methotrexate to a tumor expressing a folate receptor on its surface, comprising administering to a subject having a tumor the Lp-γPMTX composition described in any one of items [1] to

[69] in an amount that delivers a therapeutically effective amount of gamma polyglutamated methotrexate to the tumor;

[91] A method for preparing a gamma polyglutamated methotrexate composition comprising the liposomal gamma polyglutamated methotrexate composition according to any one of items

[12] to

[69] , the method comprising the steps of: forming a mixture in a solution containing liposome components and a gamma polyglutamated antifolate; homogenizing the mixture in the solution to form liposomes; and treating the mixture to form liposomes containing gamma polyglutamated methotrexate;

[92] A method for preparing a gamma polyglutamated methotrexate composition comprising the liposomal gamma polyglutamated methotrexate composition according to any one of items

[12] to

[69] , the method comprising the steps of forming a mixture containing liposome components and gamma polyglutamated methotrexate in a solution; and treating the mixture to form liposomes containing gamma polyglutamated methotrexate;

[93] The method according to item

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

[94] A method for making the composition according to any one of items

[50] to

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

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

[50] to

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

[96] The method according to item

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

[97] The method according to item

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

[98] The method according to any one of items

[95] to

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

[99] The method according to any one of items

[91] to

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

[0084] II. Gamma Polyglutamated Methotrexate (γPMTX) Generally, the present disclosure relates to gamma polyglutamated methotrexate (γPMTX) compositions. γPMTX compositions contain at least one glutamyl group with a gamma carboxyl linkage. These are structurally distinct from L-gamma polyglutamated methotrexate (Lγ1PMTX), which is produced in cells by the enzyme folylpolygamma glutamate synthase (FPGS) during methotrexate therapy.

[0085] In some embodiments, the γPMTX composition comprises 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl groups of methotrexate). In some embodiments, each glutamyl group in the γPMTX other than the glutamyl groups of methotrexate has a gamma linkage. In some embodiments, two or more glutamyl groups in the γPMTX have a gamma linkage. In some embodiments, each glutamyl group in the γPMTX is in the L-form. In some embodiments, each glutamyl group in the γPMTX other than the glutamyl groups of methotrexate is in the D-form. In some embodiments, the γPMTX comprises two or more glutamyl groups in the L-form and one or more glutamyl groups in the D-form.

[0086] In some embodiments, the gamma polyglutamated methotrexate is diglutamated. That is, the gamma polyglutamated methotrexate contains one γ-glutamyl group in addition to the glutamyl groups of methotrexate (γMTX-PG1). In some embodiments, each glutamyl group of the gamma diglutamated methotrexate is in the L-form. In other embodiments, the gamma diglutamated MTX contains a glutamyl group in the D-form.

[0087] In some embodiments, the gamma polyglutamated methotrexate is triglutamylated. That is, the gamma polyglutamated methotrexate contains two γ-glutamyl groups in addition to the glutamyl group of methotrexate (γMTX-PG2). In some embodiments, each glutamyl group of the gamma triglutamated methotrexate is in the L-form. In other embodiments, the gamma triglutamated MTX contains a glutamyl group in the D-form. In a further embodiment, each glutamyl group of the gamma triglutamated methotrexate, other than the glutamyl group of methotrexate, is in the D-form. In a further embodiment, the gamma triglutamated MTX contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

[0088] In some embodiments, gamma polyglutamated methotrexate is tetraglutamylated and thus contains three γ-glutamyl groups in addition to the γ-glutamyl groups in methotrexate (γMTX-PG3). In some embodiments, gamma tetraglutamated MTX contains two or more L-type γ-glutamyl groups. In further embodiments, each γ-glutamyl group in gamma tetraglutamated methotrexate is in the L-type. In other embodiments, gamma tetraglutamated MTX contains a D-type γ-glutamyl group. In some embodiments, gamma tetraglutamated MTX contains two D-type γ-glutamyl groups. In some embodiments, each glutamyl group in gamma tetraglutamated methotrexate, other than the glutamyl group in methotrexate, is in the D-type. In further embodiments, tetraglutamated MTX contains a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0089] In some embodiments, gamma polyglutamated methotrexate is pentaglutamated (γMTX-PG4) and contains a chain of four γ-glutamyl groups linked to the glutamyl groups of methotrexate. In some embodiments, gamma pentaglutamated MTX contains two or more L-glutamyl groups. In further embodiments, each glutamyl group of gamma pentaglutamated methotrexate is in the L-form. In other embodiments, gamma pentaglutamated MTX contains a D-glutamyl group. In some embodiments, gamma tetraglutamated MTX contains two or three D-glutamyl groups. In further embodiments, each γ-glutamyl group of gamma pentaglutamated methotrexate, other than the glutamyl group of methotrexate, is in the D-form. In a further embodiment, pentaglutamated MTX contains a D-glutamyl group and two or more L-glutamyl groups.

[0090] In some embodiments, gamma polyglutamated methotrexate is hexaglutamated (γMTX-PG5) and contains a chain of five γ-glutamyl groups bound to the glutamyl groups of methotrexate. In some embodiments, gamma hexaglutamated MTX contains two or more γ-glutamyl groups in the L form. In further embodiments, each glutamyl group of gamma hexaglutamated methotrexate is in the L form. In other embodiments, gamma hexaglutamated MTX contains a γ-glutamyl group in the D form. In some embodiments, gamma tetraglutamated MTX contains two, three, four, or five γ-glutamyl groups in the D form. In further embodiments, each glutamyl group of gamma hexaglutamated methotrexate other than the glutamyl group of methotrexate is in the D form. In a further embodiment, the hexaglutamylated MTX contains a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0091] In some embodiments, gamma polyglutamated methotrexate is heptaglutamated (γMTX-PG6) and contains a chain of six γ-glutamyl groups linked to the glutamyl groups of methotrexate. In some embodiments, gamma heptaglutamated MTX contains two or more L-type γ-glutamyl groups. In further embodiments, each γ-glutamyl group of gamma heptaglutamated methotrexate is L-type. In other embodiments, gamma heptaglutamated MTX contains D-type γ-glutamyl groups. In some embodiments, gamma tetraglutamated MTX contains 2, 3, 4, 5, or 6 D-type γ-glutamyl groups. In further embodiments, each γ-glutamyl group of gamma heptaglutamated methotrexate, other than the glutamyl groups of methotrexate, is D-type. In a further embodiment, the heptaglutamylated MTX contains a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0092] In some embodiments, gamma polyglutamated methotrexate is octaglutamated (γMTX-PG7), thus comprising a chain of seven γ-glutamyl groups linked to the glutamyl groups of methotrexate. In some embodiments, gamma octaglutamated MTX comprises two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma octaglutamated methotrexate is in the L-form. In other embodiments, gamma octaglutamated MTX comprises a glutamyl group in the D-form. In some embodiments, gamma octaglutamated MTX comprises 2, 3, 4, 5, 6, or 7 γ-glutamyl groups in the D-form. In further embodiments, each glutamyl group of gamma octaglutamated methotrexate, other than the glutamyl group of methotrexate, is in the D-form. In a further embodiment, the octaglutamylated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0093] In some embodiments, gamma polyglutamated methotrexate is nonaglutamated (γMTX-PG8) and contains a chain of eight γ-glutamyl groups bound to the glutamyl groups of methotrexate. In some embodiments, gamma nonaglutamated MTX contains two or more L-glutamyl groups. In further embodiments, each glutamyl group of gamma nonaglutamated methotrexate is in the L-form. In other embodiments, gamma nonaglutamated MTX contains D-glutamyl groups. In a further embodiment, each glutamyl group of gamma nonaglutamated methotrexate, other than the glutamyl group of methotrexate, is in the D-form. In a further embodiment, nonaglutamated MTX contains D-glutamyl groups and two or more L-glutamyl groups.

[0094] In some embodiments, gamma polyglutamated methotrexate is deca-glutamated (γMTX-PG9) and contains a chain of nine γ-glutamyl groups bound to the glutamyl groups of methotrexate. In some embodiments, gamma deca-glutamated MTX contains two or more L-glutamyl groups. In further embodiments, each glutamyl group of gamma deca-glutamated methotrexate is in the L-form. In other embodiments, gamma deca-glutamated MTX contains D-glutamyl groups. In a further embodiment, each glutamyl group of gamma deca-glutamated methotrexate, other than the glutamyl group of methotrexate, is in the D-form. In a further embodiment, deca-glutamated MTX contains a D-glutamyl group and two or more L-glutamyl groups.

[0095] In some embodiments, gamma polyglutamated methotrexate is undecaglutamated (γMTX-PG 10), and comprises a chain of 10 γ-glutamyl groups bound to the glutamyl group of methotrexate. In some embodiments, gamma-undeca-glutamylated MTX comprises two or more L-glutamyl groups. In a further embodiment, each glutamyl group of gamma-undeca-glutamylated methotrexate is in the L-form. In other embodiments, gamma-undeca-glutamylated MTX comprises a D-glutamyl group. In a further embodiment, each glutamyl group of gamma-undeca-glutamylated methotrexate other than the glutamyl group of methotrexate is in the D-form. In a further embodiment, undecaglutamylated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0096] In some embodiments, gamma polyglutamated methotrexate is dodecaglutamated (γMTX-PG 11 ), and comprises a chain of 11 γ-glutamyl groups bound to the glutamyl group of methotrexate. In some embodiments, gamma-dodeca-glutamylated MTX comprises two or more L-glutamyl groups. In a further embodiment, each glutamyl group of gamma-dodeca-glutamylated methotrexate is in the L-form. In other embodiments, gamma-dodeca-glutamylated MTX comprises a D-glutamyl group. In a further embodiment, each glutamyl group of gamma-dodeca-glutamylated methotrexate other than the glutamyl group of methotrexate is in the D-form. In a further embodiment, dodecaglutamylated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0097] In some embodiments, gamma polyglutamated methotrexate is triskite decaglutamated (γMTX-PG 12), and comprises a chain of 12 γ-glutamyl groups bound to the glutamyl group of methotrexate. In some embodiments, gamma-triskai decaglutamated MTX comprises two or more glutamyl groups in the L form. In a further embodiment, each glutamyl group of gamma-triskai decaglutamated methotrexate is in the L form. In other embodiments, gamma-triskai decaglutamated MTX comprises a glutamyl group in the D form. In a further embodiment, each glutamyl group of gamma-triskai decaglutamated methotrexate other than the glutamyl group of methotrexate is in the D form. In a further embodiment, triskai decaglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form.

[0098] In some embodiments, gamma polyglutamated methotrexate is tetradecaglutamated (γMTX-PG 13 ), which comprises a chain of 13 γ-glutamyl groups bound to the glutamyl group of methotrexate. In some embodiments, gamma-tetradecaglutamated MTX comprises two or more L-glutamyl groups. In a further embodiment, each glutamyl group of gamma-tetradecaglutamated methotrexate is in the L-form. In other embodiments, gamma-tetradecaglutamated MTX comprises a D-glutamyl group. In a further embodiment, each glutamyl group of gamma-tetradecaglutamated methotrexate, other than the glutamyl group of methotrexate, is in the D-form. In a further embodiment, tetradecaglutamated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0099] In some embodiments, gamma polyglutamated methotrexate is pentadecaglutamated (γMTX-PG 14), and comprises a chain of 14 γ-glutamyl groups bound to the glutamyl group of methotrexate. In some embodiments, gamma pentadeca-glutamylated MTX comprises two or more L-glutamyl groups. In a further embodiment, each glutamyl group of gamma pentadeca-glutamylated methotrexate is in the L-form. In other embodiments, gamma pentadeca-glutamylated MTX comprises a D-glutamyl group. In a further embodiment, each glutamyl group of gamma pentadeca-glutamylated methotrexate, other than the glutamyl group of methotrexate, is in the D-form. In a further embodiment, pentadeca-glutamylated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0100] In some embodiments, gamma polyglutamated methotrexate is hexadecaglutamated (γMTX-PG 15 ), and comprises a chain of 15 γ-glutamyl groups bound to the glutamyl group of methotrexate. In some embodiments, gamma hexadecaglutamylated MTX comprises two or more L-glutamyl groups. In a further embodiment, each glutamyl group of gamma hexadecaglutamylated methotrexate is in the L-form. In other embodiments, gamma hexadecaglutamylated MTX comprises a D-glutamyl group. In a further embodiment, each glutamyl group of gamma hexadecaglutamylated methotrexate other than the glutamyl group of methotrexate is in the D-form. In a further embodiment, hexadecaglutamylated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0101] In another embodiment, gamma polyglutamated methotrexate is heptadecaglutamated (γMTX-PG 16), which comprises a chain of 16 γ-glutamyl groups bound to the glutamyl group of methotrexate. In some embodiments, gamma heptadeca-glutamylated MTX comprises two or more L-glutamyl groups. In a further embodiment, each glutamyl group of gamma heptadeca-glutamylated methotrexate is in the L-form. In other embodiments, gamma heptadeca-glutamylated MTX comprises a D-glutamyl group. In a further embodiment, each glutamyl group of gamma heptadeca-glutamylated methotrexate, other than the glutamyl group of methotrexate, is in the D-form. In a further embodiment, heptadeca-glutamylated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0102] In some embodiments, gamma polyglutamated methotrexate is octadeca-glutamated (γMTX-PG 17 ), and comprises a chain of 17 γ-glutamyl groups bound to the glutamyl group of methotrexate. In some embodiments, gamma-octadeca-glutamylated MTX comprises two or more L-glutamyl groups. In a further embodiment, each glutamyl group of gamma-octadeca-glutamylated methotrexate is in the L-form. In other embodiments, gamma-octadeca-glutamylated MTX comprises a D-glutamyl group. In a further embodiment, each glutamyl group of gamma-octadeca-glutamylated methotrexate, other than the glutamyl group of methotrexate, is in the D-form. In a further embodiment, octadeca-glutamylated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0103] In some embodiments, gamma polyglutamated methotrexate is eniadecaglutamated (γMTX-PG 18), which comprises a chain of 18 γ-glutamyl groups bound to the glutamyl group of methotrexate. In some embodiments, gamma eniadecaglutamated MTX comprises two or more glutamyl groups in the L form. In further embodiments, each glutamyl group of gamma eniadecaglutamated methotrexate is in the L form. In other embodiments, gamma eniadecaglutamated MTX comprises a glutamyl group in the D form. In a further embodiment, each glutamyl group of gamma eniadecaglutamated methotrexate, other than the glutamyl group of methotrexate, is in the D form. In a further embodiment, eniadecaglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form.

[0104] In some embodiments, gamma polyglutamated methotrexate is eicosiglutamated (γMTX-PG 19 ), and comprises a chain of 19 γ-glutamyl groups bound to the glutamyl group of methotrexate. In some embodiments, gamma mycosiglutamated MTX comprises two or more L-glutamyl groups. In further embodiments, each glutamyl group of gamma mycosiglutamated methotrexate is in the L-form. In other embodiments, gamma mycosiglutamated MTX comprises a D-glutamyl group. In a further embodiment, each glutamyl group of gamma mycosiglutamated methotrexate other than the glutamyl group of methotrexate is in the D-form. In a further embodiment, gamma mycosiglutamated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0105] In some embodiments, gamma polyglutamated methotrexate is eikoshikaihenaglutamated (γMTX-PG 20), and comprises a chain of 20 γ-glutamyl groups bound to the glutamyl group of methotrexate. In some embodiments, the gamma mycoplasma glutamated MTX comprises two or more L-glutamyl groups. In a further embodiment, each glutamyl group of the gamma mycoplasma glutamated methotrexate is in the L-form. In other embodiments, the gamma mycoplasma glutamated MTX comprises a D-glutamyl group. In a further embodiment, each glutamyl group of the gamma mycoplasma glutamated methotrexate, other than the glutamyl group of methotrexate, is in the D-form. In a further embodiment, the gamma mycoplasma glutamated MTX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0106] In some embodiments, gamma polyglutamated methotrexate comprises 4 to 7 glutamyl groups linked to methotrexate (i.e., γMTX-PGn, n=4 to 7), each of the 4 to 7 linked glutamyl groups having a gamma linkage. In some embodiments, each of the 4 to 7 linked glutamyl groups is in the L-form. In other embodiments, each of the 4 to 7 linked glutamyl groups is in the D-form. In other embodiments, the 4 to 7 linked glutamyl groups are in both the L- and D-forms.

[0107] In one embodiment, the gamma polyglutamated methotrexate is tetraglutamated, and each of the three glutamyl groups in the polyglutamate chain linked to methotrexate contains a gamma linkage. In some embodiments, each of the four glutamyl groups is in the L-form. In some embodiments, each glutamyl group in the gamma tetraglutamated methotrexate, except for the glutamyl group in methotrexate, is in the D-form. In other embodiments, at least two of the glutamyl groups in the gamma tetraglutamate methotrexate are in the L-form, and at least one glutamyl group is in the D-form.

[0108] In one embodiment, the gamma polyglutamated methotrexate is pentaglutamated, and each of the four glutamyl groups in the polyglutamate chain linked to methotrexate contains a gamma linkage. In some embodiments, each of the four glutamyl groups is in the L-form. In some embodiments, each glutamyl group in the gamma pentaglutamated methotrexate, other than the glutamyl group in methotrexate, is in the D-form. In other embodiments, at least two glutamyl groups in the gamma pentaglutamated methotrexate are in the L-form and at least one glutamyl group is in the D-form.

[0109] In one embodiment, the gamma polyglutamated methotrexate is hexaglutamated. In some embodiments, each of the five glutamyl groups is in the L form. In some embodiments, each glutamyl group of the gamma hexaglutamated methotrexate, other than the glutamyl group of methotrexate, is in the D form. In other embodiments, at least two glutamyl groups in the gamma hexaglutamated methotrexate are in the L form and at least one glutamyl group is in the D form.

[0110] In another embodiment, the gamma polyglutamated methotrexate is heptaglutamated. In some embodiments, each of the six glutamyl groups is in the L form. In some embodiments, each glutamyl group in the gamma heptaglutamated methotrexate, other than the glutamyl group in methotrexate, is in the D form. In other embodiments, at least two glutamyl groups in the gamma heptaglutamated methotrexate are in the L form and at least one glutamyl group is in the D form.

[0111] In some embodiments, gamma polyglutamated methotrexate (γPMTX) contains a total of 1 to 15, 1 to 10, 2 to 15, 2 to 10, 3 to 15, 3 to 10, 3 to 6, 3 to 5, 4 to 10, 4 to 7, or 4 to 6 glutamyl groups, including the glutamyl groups of methotrexate, or any range therebetween. In some embodiments, each glutamyl group in γPMTX other than the glutamyl groups of methotrexate has a gamma linkage. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in γPMTX have a gamma linkage. In some embodiments, γPMTX contains L- and D-type γ-glutamyl groups. In some embodiments, each glutamyl group in the polyglutamate structure of polyglutamated methotrexate is in the L-type. In some embodiments, each glutamyl group in γPMTX other than the glutamyl groups of methotrexate is in the D form. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the glutamyl groups in γPMTX are in the L form. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in γPMTX are in the D form.

[0112] In further embodiments, the gamma polyglutamylated methotrexate contains 20-100, 20-75, 20-50, 20-40, 20-30, 20-25, or more than 100 gamma glutamyl groups, or any range therebetween. In some embodiments, each glutamyl group of the gamma polyglutamylated methotrexate is in the L-form. In other embodiments, each glutamyl group of the gamma polyglutamylated methotrexate other than the glutamyl group of methotrexate is in the D-form. In alternative embodiments, at least two of the glutamyl groups in the gamma polyglutamylated methotrexate are in the L-form and at least one of the glutamyl groups in the gamma polyglutamylated methotrexate is in the D-form.

[0113] In further embodiments, provided compositions comprise gamma polyglutamated methotrexate comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups with gamma linkages. In some embodiments, the gamma polyglutamated methotrexate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups in the L-form. In some embodiments, the gamma polyglutamated methotrexate comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups in the D-form. In some embodiments, the gamma polyglutamated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 L-glutamyl groups and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 D-glutamyl groups.

[0114] In some embodiments, the gamma polyglutamated methotrexate compositions provided herein can accept one or more additional glutamyl groups, i.e., the compositions can serve as substrates for FPGS (folylpolyglutamate synthetase). Reagents and assays to measure the ability of gamma polyglutamated methotrexate compositions to act as substrates for FPGS (e.g., human FPGS, or rat liver FPGS) are readily available and can be performed routinely.

[0115] In some embodiments, the rate of hepatocyte uptake of a naked gamma-PMTX composition disclosed herein (e.g., gamma-PMTX not conjugated to a delivery vehicle) is significantly reduced compared to the rate of methotrexate uptake under the same physiological conditions. In some embodiments, the rate of hepatocyte uptake of a naked gamma-PMTX composition is less than 30%, 20%, 15%, or 10% of the rate of methotrexate uptake. In further embodiments, the rate of efflux (transport) of a gamma-PMTX composition disclosed herein from hepatocytes is significantly reduced (less than 30%, 20%, 15%, or 10%) compared to methotrexate (MTX).

[0116] In some embodiments, the gamma polyglutamated methotrexate compositions provided herein have greater cytotoxicity against hyperproliferative cells than methotrexate. 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 polyglutamated methotrexate is hexaglutamated methotrexate.

[0117] In some embodiments, the gamma polyglutamated methotrexate compositions provided herein have fewer toxic side effects than methotrexate. In some embodiments, the gamma polyglutamated methotrexate compositions provided herein are less toxic to non-hyperproliferative cells than methotrexate. In some embodiments, the gamma polyglutamated methotrexate compositions provided herein are less toxic to neutrophils, liver cells, or colon epithelial cells than methotrexate. In some embodiments, the neutrophils are human neutrophils, differentiated human neutrophils, or neutrophils differentiated from CD34+ cells. In some embodiments, the liver cells are AML12 liver cells. In some embodiments, the colon epithelial cells are CCD841 colon epithelial cells. In some embodiments, toxicity is measured by an in vitro assay. In some embodiments, the gamma polyglutamated methotrexate is hexaglutamated methotrexate.

[0118] In some embodiments, the gamma polyglutamated methotrexate compositions provided herein have fewer toxic side effects than methotrexate. In some embodiments, the gamma polyglutamated methotrexate compositions provided herein result in fewer or less severe toxic side effects than methotrexate in in vivo assays. In some embodiments, the in vivo assay is performed in an in vivo mouse model. In some embodiments, the gamma polyglutamated methotrexate compositions provided herein result in fewer or less severe hematologic or liver toxic side effects than methotrexate. In some embodiments, hematologic side effects are assessed by mean neutrophil, mean white blood cell, or mean platelet counts. In some embodiments, liver toxic side effects are assessed by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the in vivo assay involves administering 40 mg / kg or 80 mg / kg of a gamma polyglutamated methotrexate composition once a week for four weeks. In some embodiments, the gamma polyglutamated methotrexate is hexaglutamated methotrexate.

[0119] In some embodiments, treatment with the gamma polyglutamated methotrexate compositions provided herein does not induce significant hematologic or liver toxic side effects in an in vivo mouse model. In some embodiments, hematologic side effects are assessed by mean neutrophil, mean white blood cell, or mean platelet counts. In some embodiments, liver toxic side effects are assessed by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the gamma polyglutamated methotrexate compositions provided herein do not significantly reduce mean neutrophil, mean white blood cell, or mean platelet counts. In some embodiments, the gamma polyglutamated methotrexate compositions provided herein do not significantly increase serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. In some embodiments, the gamma polyglutamated methotrexate compositions provided herein do not significantly reduce serum albumin levels. In some embodiments, the in vivo assay involves administering 40 mg / kg or 80 mg / kg of a gamma polyglutamated methotrexate composition once a week for four weeks. In some embodiments, the gamma polyglutamated methotrexate is hexaglutamated methotrexate.

[0120] In some embodiments, the gamma polyglutamated methotrexate composition does not contain fluorine atoms. In some embodiments, the gamma polyglutamated methotrexate composition does not contain 4-fluoroglutamyl groups.

[0121] Gamma polyglutamated methotrexate (γPMTX) compositions and their uses are further described in U.S. Patent Application Nos. 62 / 374,458, 15 / 675,695, 15 / 675,701, and 62 / 583,432, and International Application Nos. PCT / US2017 / 046666 and PCT / US2017 / 046667, the disclosures of each of which are incorporated herein by reference in their entirety.

[0122] A. Polyglutamated Methotrexate Analogs and Derivatives The present disclosure also encompasses gamma polyglutamated methotrexate derivatives and analogs. The compositions and methods disclosed herein are contemplated for application to any and all known polyglutamated methotrexate derivatives or analogs. In some embodiments, polyglutamated methotrexate analog or derivative compositions made and used in accordance with the disclosed compositions and methods are shown in Figures 1I and 1J. In some embodiments, the analog corresponds to a modified form of methotrexate, in which the glutamyl group of methotrexate is not linked to the remainder of the methotrexate molecule via a gamma peptide bond. In some embodiments, the analog is a variant of methotrexate, in which the glutamyl group in methotrexate is in the D-form. In some embodiments, the polyglutamated form of methotrexate or the polyglutamated methotrexate analog or derivative is non-fluorinated.

[0123] In some embodiments, polyglutamated methotrexate analogs or derivatives encompassed by the present disclosure are indoline ring and modified ornithine or glutamic acid containing methotrexate derivatives. In some embodiments, the polyglutamated methotrexate analog or derivative encompassed by the present disclosure is a member selected from the group consisting of indoline moiety-containing methotrexate derivatives, lipophilic amide methotrexate derivatives, alkyl-substituted benzene ring C-containing methotrexate derivatives, polymeric platinol methotrexate derivatives, N-(L-α-aminoacyl) methotrexate derivatives, halogenated methotrexate derivatives, 7-methyl methotrexate derivatives, N-(ac-aminoacyl) methotrexate derivatives, biotin methotrexate derivatives, dichloromethocetate, lipophilic methotrexate derivatives, benzoxazine or benzothiazine moiety-containing methotrexate derivatives, and N delta-acyl-N α-(4-amino-4-deoxypteroyl)-L-ornithine derivatives.

[0124] In some embodiments, polyglutamated methotrexate analogs or derivatives encompassed by the present disclosure include deoxyuridylate methotrexate, 10-deazaaminopterin analogs, 5-deazaaminopterin or 10-deazaaminopterin (10-EDAM) analogs, 5,10-dideazaaminopterin methotrexate analogs, 8-alkyl-7,8-dihydro analogs, L-threo-(2S,4S)-4-fluoroglutamic acid or DL-3,3-difluoroglutamic acid-containing methotrexate analogs, methotrexate tetrahydrofuran ... Doloquinazoline analogs, D-glutamic acid, D-erythro, threo-4-fluoroglutamic acid methotrexate analogs, βγ-methanomethotrexate analogs, γ-tetrazole methotrexate analogs, ortho isomers of aminopterin, hydroxymethyl methotrexate, γ-fluoromethotrexate, gem diphosphonate methotrexate analogs, α- or / and γ-substituted methotrexate analogs, 5-methyl-5-deazamethotrexate analogs, 8-deazamethotrexate analogs, acivicin methotrexate analogs, phosphono Glutamic acid analogs, poly(L-lysine) methotrexate conjugates, disilysine or trilysine methotrexate derivatives, methotrexate-γ-dimyristoylphophatidylethanolamine, iodoacetyllysine methotrexate analogs, 2,ω-diaminoalkaloid acid-containing methotrexate analogs, -methyl-5-deaza analogs, quinazoline methotrexate analogs, pyrazine methotrexate analogs, cysteic or homocysteic acid methotrexate analogs, γ-tert-butyl methotrexate ester, methotrexate fluoride The compound is a member selected from the group consisting of methotrexate analogs, folate methotrexate analogs, 7-hydroxymethotrexate, poly-γ-glutamyl methotrexate analogs, 3′,5′-dichloromethotrexate, diazoketone and chloromethylketone methotrexate analogs, 10-propargyl aminopterin or alkyl methotrexate homologs, lectin derivatives of methotrexate, 3′,5′-dichloromethotrexate, deazamethopterin analogs, cysteic and homocysteic acid methotrexate analogs, and MX068.

[0125] In further embodiments, the gamma polyglutamated methotrexate derivative or analog has a variant polyglutamate chain. In some embodiments, the polyglutamate chain contains one or more natural or synthetic residues other than glutamate. In some embodiments, the polyglutamate chain contains one or more glutamyl groups that do not contain an amide bond. In other embodiments, one or more glutamyl groups of the polyglutamate chain are derivatized.

[0126] B.MTX-PG synthesis The methotrexate polyglutamate compositions provided herein can be obtained by the following synthetic methods known in the art: Procedures for synthesizing methotrexate (including different pharmaceutically acceptable salts or acids (e.g., methotrexate disodium) and crystalline and amorphous forms) and intermediates for synthesizing methotrexate include, but are not limited to, those described in U.S. Patent Nos. 2,512,572; 3,892,801; 3,989,703; 4,057,548; 4,067,867; and 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).

[0127] The addition of glutamyl residues to the glutamyl residues of methotrexate can be carried out using synthetic methods known in the art. In some embodiments, glutamyl residues are added sequentially to the glutamyl residues of methotrexate. In further embodiments, polyglutamates are added to the glutamyl residues of methotrexate using "click chemistry" methods or other bioconjugate chemistries known to those skilled in the art. Alternatively, a peptide of the desired length of glutamyl residues can be generated and added to a precursor that does not contain the glutamyl residues of methotrexate. Peptides can be made using methods known in the art. In some embodiments, an initial glutamyl residue is coupled to wangregin, and additional glutamyl residues are added sequentially by solid-phase peptide synthesis using F-moc chemistry. After the final glutamyl residue is added, the methotrexate precursor is coupled to the peptide, and the molecule is cleaved from the resin.

[0128] C. Methotrexate-PG complex Surprisingly, the inventors have discovered that polyglutamated antifolates, such as methotrexate (γPMTX), can be complexed with other compositions that include therapeutic agents, including cytotoxic compounds, such as platinum-based compounds. Thus, in some embodiments, the present disclosure provides complexes of γPMTX (e.g., the γPMTX disclosed herein) with a therapeutic agent, or a salt or acid thereof.

[0129] In some embodiments, the γPMTX / complex comprises γPMTX and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound such as a chemotherapeutic agent. In further embodiments, the γPMTX / complex comprises a platinum-based drug such as a platinum-based chemotherapeutic agent (e.g., cisplatin, carboplatin, and oxaliplatin). In other embodiments, the γPMTX / complex comprises a taxane-based chemotherapeutic agent (e.g., paclitaxel and docetaxel). In other embodiments, the γPMTX / complex comprises a cyclodextrin. In further embodiments, the γPMTX / complex is encapsulated in a liposome.

[0130] In some embodiments, the present disclosure provides compositions comprising a complex of γPMTX and a therapeutic agent, or a salt or acid thereof. In further embodiments, the γPMTX / therapeutic agent complex comprises one or more γPMTXs comprising 2 to 150, 2 to 100, 2 to 75, 2 to 50, 2 to 24, 2 to 30, 2 to 20, 2 to 19, 2 to 15, 2 to 10, or 2 to 5 glutamyl groups. In some embodiments, the γPMTX / therapeutic agent complex comprises one or more γPMTXs comprising 3 to 10, 3 to 9, 3 to 8, or 3 to 7 glutamyl groups, or any range therebetween. In other embodiments, the γPMTX / therapeutic agent complex comprises one or more γPMTXs comprising 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 one particular embodiment, the complex comprises one or more γPMTXs comprising 3 to 10 glutamyl groups. In a further embodiment, the γPMTX / therapeutic agent complex comprises one or more γPMTXs comprising 3 to 7 glutamyl groups. In another embodiment, the γPMTX / therapeutic agent complex comprises one or more γPMTXs comprising 5 glutamyl groups. In another embodiment, the γPMTX / therapeutic agent complex comprises one or more γPMTXs comprising 6 glutamyl groups. In some embodiments, the therapeutic agent is a cytotoxic compound, or a salt or acid thereof. In a further embodiment, the therapeutic agent is a chemotherapeutic agent, or a salt or acid thereof. In another embodiment, the chemotherapeutic agent is a platinum-based drug. In another embodiment, the chemotherapeutic agent is a taxane-based drug. In a further embodiment, the molar ratio of γPMTX to therapeutic agent in the complex is in the range of 1 to 10:1. In some embodiments, the molar ratio of γPMTX to therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX to cyclodextrin in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween.In some embodiments, the molar ratio of γPMTX / therapeutic agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the γPMTX / therapeutic agent complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0131] In alternative embodiments, the γPMTX complex comprises γPMTX and cyclodextrin. In some embodiments, the molar ratio of γPMTX (e.g., a γPMTX salt) to cyclodextrin in the complex is in the range of 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX to cyclodextrin in the complex is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX to cyclodextrin in the complex is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cyclodextrin in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / cyclodextrin in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cyclodextrin is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the γPMTX / cyclodextrin complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0132] In some embodiments, the present disclosure provides a composition comprising a γPMTX / platinum-based chemotherapeutic agent conjugate. In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or a salt or acid thereof. In other embodiments, the γPMTX / platinum-based chemotherapeutic agent conjugate comprises an analog of cisplatin, carboplatin, or oxaliplatin, or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / platinum-based agent in the conjugate is in the range of 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / platinum-based agent in the conjugate is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / platinum-based agent in the conjugate is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX to platinum-based drug in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX to platinum-based drug in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / platinum-based drug is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPMTX / platinum-based drug complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0133] In further embodiments, the γPMTX / platinum-based chemotherapeutic agent conjugate comprises an analog of cisplatin, carboplatin, oxaliplatin, or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / platinum-based analog in the conjugate is in the range of 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / platinum-based analog in the conjugate is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / platinum-based agent in the conjugate is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX to platinum-based analog in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX to platinum-based drug in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / platinum-based analog is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPMTX / platinum-based analog complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0134] In further embodiments, the present disclosure provides a complex comprising γPMTX and cisplatin, or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / cisplatin (or a cisplatin salt or acid) in the complex is in the range of 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cisplatin (or a cisplatin salt or acid) in the complex is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / cisplatin (or a cisplatin salt or acid) in the complex is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cisplatin (or cisplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / cisplatin (or cisplatin salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cisplatin (or a cisplatin salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPMTX / cisplatin (or a cisplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0135] In another embodiment, the present disclosure provides a complex comprising γPMTX and carboplatin, or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / carboplatin (or a carboplatin salt or acid) in the complex is in the range of 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / carboplatin (or a carboplatin salt or acid) in the complex is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / carboplatin (or a carboplatin salt or acid) in the complex is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / carboplatin (or carboplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / carboplatin (or carboplatin salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / carboplatin (or a carboplatin salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPMTX / carboplatin (or a carboplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0136] In another embodiment, the present disclosure provides a complex comprising γPMTX and oxaliplatin, or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / oxaliplatin (or an oxaliplatin salt or acid) in the complex is in the range of 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / oxaliplatin (or an oxaliplatin salt or acid) in the complex is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / oxaliplatin (or an oxaliplatin salt or acid) in the complex is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / oxaliplatin (or oxaliplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / oxaliplatin (or oxaliplatin salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / oxaliplatin (or an oxaliplatin salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPMTX / oxaliplatin (or an oxaliplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0137] In a further embodiment, the present disclosure provides a conjugate comprising γPMTX and a platinum-based chemotherapeutic agent ("platin") selected from the group consisting of nedaplatin, heptaplatin, lobaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexorumaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamines, satraplatin, enloplatin, JM216, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or a salt or acid thereof. In other embodiments, the γPMTX / platinum-based chemotherapeutic agent conjugate comprises nedaplatin, heptaplatin, lobaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexorumaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enloplatin, JM216, NK121, CI973, DWA2114R, NDDP, or an analog of nedaplatin, or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / platinum (or platinum salt or acid) in the conjugate is in the range of 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / platinum (or platinum salt or acid) in the conjugate is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / platinum (or platinum salt or acid) in the complex is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / platinum (or platinum salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / platinum (or platinum salt or acid) in the complex is in the range of 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween.In some embodiments, the molar ratio of γPMTX / platinum (or a platinum salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPMTX / platinum (or a salt or acid or analog thereof) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0138] In some embodiments, the present disclosure provides a composition comprising a γPMTX / taxane chemotherapeutic agent (taxane) conjugate. In some embodiments, the taxane chemotherapeutic agent is selected from the group consisting of paclitaxel (PTX), docetaxel (DTX), larotaxel (LTX), and cabazitaxel (CTX), or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / taxane in the conjugate is in the range of 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / taxane (or taxane salt or acid) in the conjugate is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / taxane (or taxane salt or acid) in the conjugate is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX to taxane (or taxane salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX to taxane (or taxane salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPMTX to taxane (or taxane salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPMTX / taxane drug complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0139] In further embodiments, the present disclosure provides a complex comprising γPMTX and paclitaxel (PTX) or a salt or acid thereof. In other embodiments, the γPMTX / taxane chemotherapeutic agent complex comprises a paclitaxel (PTX) analog, or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / paclitaxel (or a paclitaxel salt or acid) in the complex is in the range of 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / paclitaxel (or a paclitaxel salt or acid) in the complex is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / paclitaxel (or a paclitaxel salt or acid) in the complex is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / paclitaxel (or paclitaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / paclitaxel (or paclitaxel salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / paclitaxel (or paclitaxel salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPMTX / paclitaxel (or paclitaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0140] In further embodiments, the present disclosure provides a complex comprising γPMTX and docetaxel (DTX) or a salt or acid thereof. In other embodiments, the γPMTX / taxane chemotherapeutic agent complex comprises a docetaxel (DTX) analog, or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / docetaxel (or a docetaxel salt or acid) in the complex is in the range of 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / docetaxel (or a docetaxel salt or acid) in the complex is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / docetaxel (or a docetaxel salt or acid) in the complex is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / docetaxel (or docetaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / docetaxel (or docetaxel salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / docetaxel (or a salt or acid of docetaxel) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPMTX / docetaxel (or a salt or acid of docetaxel) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0141] In further embodiments, the present disclosure provides a complex comprising γPMTX and larotaxel (LTX) or a salt or acid thereof. In other embodiments, the γPMTX / taxane chemotherapeutic agent complex comprises an analog of larotaxel (LTX), or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / larotaxel (or larotaxel salt or acid) in the complex is in the range of 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / larotaxel (or larotaxel salt or acid) in the complex is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / larotaxel (or larotaxel salt or acid) in the complex is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / larotaxel (or larotaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / larotaxel (or larotaxel salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / larotaxel (or larotaxel salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPMTX / larotaxel (or larotaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0142] In further embodiments, the present disclosure provides a complex comprising γPMTX and cabazitaxel (CTX) or a salt or acid thereof. In other embodiments, the γPMTX / taxane chemotherapeutic agent complex comprises a cabazitaxel (CTX) analog, or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / cabazitaxel (or a cabazitaxel salt or acid) in the complex is in the range of 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / cabazitaxel (or a cabazitaxel salt or acid) in the complex is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / cabazitaxel (or a cabazitaxel salt or acid) in the complex is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cabazitaxel (or cabazitaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / cabazitaxel (or cabazitaxel salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cabazitaxel (or cabazitaxel salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPMTX / cabazitaxel (or cabazitaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0143] In further embodiments, the present disclosure provides a complex comprising γPMTX and another antimetabolite, or a salt or acid thereof. An antimetabolite is a chemical compound that is similar to a metabolite required for normal biochemical reactions but differs sufficiently in structure to interfere with one or more normal cell functions, such as cell division. In some embodiments, the present disclosure provides a complex comprising γPMTX and methotrexate (MTX), or a salt or acid thereof. In some embodiments, the present disclosure provides a complex comprising γPMTX and an antimetabolite selected from the group consisting of gemcitabine, fluorouracil, capecitabine, antifolate (e.g., methotrexate), tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, and pharmaceutically acceptable salts or acids or derivatives of any of these. In some embodiments, the molar ratio of γPMTX to the antimetabolite (or antimetabolite salt or acid) in the complex is in the range of 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX to the antimetabolite (or antimetabolite salt or acid) in the complex is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX to the antimetabolite (or antimetabolite salt or acid) in the complex is in the range of 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX to the antimetabolite (or antimetabolite salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX to the antimetabolite (or antimetabolite salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween.In some embodiments, the molar ratio of γPMTX / antimetabolite (or antimetabolite salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPMTX / antimetabolite (or antimetabolite salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0144] In a further embodiment, the present disclosure provides a complex of γPMTX (e.g., the γPMTX disclosed herein) and cyclodextrin. Cyclodextrins (CDs) are a group of cyclic oligosaccharides that have been shown to improve the physicochemical properties of many drugs through complexation. CDs are cyclic oligosaccharides composed of several D-glucose units linked by α-(1,4) bonds. This cyclic structure provides a hydrophobic interior cavity and gives CDs a shortened pyramidal shape. Many hydroxyl groups are located on the ends of the ring, making CDs both lipophilic and water-soluble. As a result, CDs can form complexes with a wide variety of hydrophobic drugs, thereby changing the physicochemical properties of these complexed drugs.

[0145] The terms "cyclodextrin" or "CD," unless otherwise specified, generally refer to parent or derivatized cyclic oligosaccharides capable of complexing with methotrexate-PG, containing a variable number of (α-1,4)-linked D-glucopyranoside units. Each cyclodextrin glucopyranoside subunit has secondary hydroxyl groups at the 2 and 3 positions and a primary hydroxyl group at the 6 position. The terms "parent," "underivatized," or "inactive" cyclodextrin refer to cyclodextrins of the basic formula CH, containing D-glucopyranoside units. 12This refers to a cyclodextrin with an O6 and glucose structure and no additional chemical substituents (e.g., α-cyclodextrin, consisting of six D-glucopyranoside units; β-cyclodextrin, consisting of seven D-glucopyranoside units; and γ-cyclodextrin, consisting of eight D-glucopyranoside units). The physical and chemical properties of the parent cyclodextrin can be modified by derivatizing the hydroxyl groups with other functional groups. Any substance located in the cyclodextrin internal phase is said to be "complexed" with the cyclodextrin or to form a complex (inclusion complex) with the cyclodextrin.

[0146] As used herein, there are no particular limitations on the cyclodextrin component of a γPMTX / cyclodextrin complex, so long as the cyclodextrin can form a complex with γPMTX. In certain embodiments, the cyclodextrin has been derivatized to have ionizable (e.g., weakly basic and / or weakly acidic) functional groups to facilitate complexation with γPMTX and / or liposome encapsulation.

[0147] Modification of cyclodextrin hydroxyl groups, such as those facing away from the cyclodextrin internal phase, with ionizable chemical groups is known to facilitate the addition of cyclodextrin and therapeutic agents complexed with the cyclodextrin. In some embodiments, the cyclodextrin in the γPMTX / cyclodextrin complex has at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 hydroxyl groups substituted with ionizable chemical groups. The term "charged cyclodextrin" refers to a cyclodextrin having hydroxyl groups substituted with one or more of its charged moieties. Such moieties may themselves be charged groups or may include organic moieties (e.g., C1-C6 alkyl or C1-C6 alkyl ether moieties) substituted with one or more charged moieties.

[0148] In some embodiments, the "ionizable" or "charged" moiety of a CD derivative is weakly ionizable. A weakly ionizable moiety is a weakly basic or weakly acidic moiety. A weakly basic functional group (W) has a pKa with CH3-W of about 6.0-9.0, 6.5-8.5, 7.0-8.0, 7.5-8.0, and any range therebetween (endpoints included). Similarly, a weakly acidic functional group (X) has a logarithmic dissociation constant (pKa) with CH3-X of about 3.0-7.0, 4.0-6.5, 4.5-6.5, 5.0-6.0, 5.0-5.5, and any range therebetween (endpoints included). Representative anionic moieties include, but are not limited to, carboxylate, carboxymethyl, succinyl, sulfonyl, phosphate, sulfoalkyl ether, sulfate carbonate, thiocarbonate, dithiocarbonate, phosphate, phosphonate, sulfonate, nitrate, and borate groups. Representative cationic moieties include, but are not limited to, amino, guanidine, and quaternary ammonium groups.

[0149] In another embodiment, the derivatized cyclodextrin is a "polyanion" or a "polycation." A polyanion is a derivatized cyclodextrin with two or more negatively charged groups, resulting in a net negative ionic charge of three or more units. A polycation is a derivatized cyclodextrin with two or more positively charged groups, resulting in a net positive ionic charge of three or more units.

[0150] 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. A chargeable amphiphile can therefore be a weak acid or base. "Amphoteric," as used herein, refers to a derivatized cyclodextrin having ionizable groups of both anionic and cationic character, where (a) at least one, and optionally both, cationic and anionic amphiphiles are chargeable and have at least one charged group with a pK between 4 and 8-8.5, (b) the cationic charge predominates at pH 4, and (c) the anionic charge predominates at pH 8-8.5.

[0151] In some embodiments, the "ionizable" or "charged" derivatized cyclodextrins, whether polyionic, amphiphilic, or otherwise, are generally weakly ionizable (e.g., having a pKai of about 4.0-8.5, 4.5-8.0, 5.0-7.5, 5.5-7.0, 6.0-6.5, and any range therebetween, inclusive).

[0152] Any one, some, or all of the hydroxyl groups of any one, some, or all of the α-D-glucopyranoside units of a cyclodextrin can be modified with an ionizable chemical group as described herein. Because each cyclodextrin hydroxyl group differs in chemical reactivity, reaction with the modifying moiety can produce an amorphous mixture of regio- and optical isomers. Alternatively, the pre-modified α-D-glucopyranoside units can be reacted to form a homogeneous product, depending on the specific chemistry.

[0153] The aggregate substitution occurring in a mixture of cyclodextrin derivatives is described by a term called the degree of substitution. For example, 6-ethylenediamino-β-cyclodextrin having a degree of substitution of 7 will be composed of a distribution of 6-ethylenediamino-β-cyclodextrin isomers in which the number of ethylenediamino groups per 6-ethylenediamino-β-cyclodextrin molecule is 7. The degree of substitution of a mixture of cyclodextrin derivatives can be routinely determined using mass spectrometry or nuclear magnetic resonance spectroscopy.

[0154] In one embodiment, at least one hydroxyl moiety facing away from the interior of the cyclodextrin is substituted with an ionizable chemical group. For example, at least one γ-D-glucopyranoside unit among all three of the C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and C2-C3-C6 hydroxyls is substituted with an ionizable chemical group. Any such hydroxyl combination can be combined with any of the degrees of substitution described herein, as well as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and up to all alpha-D-glucopyranoside units in the modified cyclodextrin. One such derivative is sulfoalkyl ether cyclodextrin (SAE-CD). The sulfobutyl ether derivative of beta cyclodextrin (SBE-β-CD) has been shown to have significantly improved aqueous solubility compared to the parent cyclodextrin.

[0155] Additional cyclodextrin derivatives that can be complexed with therapeutic agents in the disclosed liposomal compositions include sugammadex or Org-25969, in which the 6-hydroxy group on γ-CD has been replaced with a carboxythioacetate ether linkage, and hydroxybutenyl-β-CD. Alternative forms of cyclodextrin include 2,6-di-O-methyl-β-CD (DIMEB), 2-hydroxypropyl-3-cyclodextrin (HP-β-CD), randommethylated-β-cyclodextrin (RAMEB), sulfobutyl ether Examples of suitable cyclodextrins include beta-cyclodextrin (SBE-β-CD), sulfobutylether-γ-cyclodextrin (SBEγCD), sulfobutylated beta-cyclodextrin sodium salt, (2-hydroxypropyl)-gamma-cyclodextrin, (2-hydroxypropyl)-beta-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, 2,6-di-O-methyl)-beta-cyclodextrin (DIMEB-50 heptakis), 2,3,6-tri-O-methyl)-beta-cyclodextrin (TRIMEB heptakis), methyl-beta-cyclodextrin, octakis(6-deoxy-6-iodo)-γ-cyclodextrin, and octakis(6-deoxy-6-bromo)-γ-cyclodextrin.

[0156] In some embodiments, the cyclodextrin has high solubility in water to facilitate entrapment of a larger amount of cyclodextrin in the liposome internal phase. In some embodiments, the solubility of the cyclodextrin in water is at least 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, or more. In some embodiments, the water solubility of the cyclodextrin is within the range of 10-150 mg / mL, 20-100 mg / mL, 20-75 mg / mL, and any range therebetween (inclusive).

[0157] In some embodiments, a large binding constant between the cyclodextrin and γPMTX and / or other therapeutic agent complexed with the cyclodextrin is preferred and can be obtained by selecting the number of glucose units in the cyclodextrin based on the size of the therapeutic agent (e.g., Albers et al., Crit. Rev. Therap. Drug Carrier Syst. 12:311-337 (1995); Stella et al. al., Toxicol. Pathol. 36:30-42 (2008). If the binding constant is pH dependent, a cyclodextrin can be selected that has a high binding constant at the pH of the liposome internal phase. As a result, the solubility (nominal solubility) of the therapeutic agent in the presence of the cyclodextrin can be further improved. In some embodiments, the binding constant between the cyclodextrin and the therapeutic agent is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or greater. In some embodiments, the binding constant between the cyclodextrin and the therapeutic agent is 100-1,200, 200-1,000, 300-750, and any range therebetween.

[0158] In some embodiments, the cyclodextrin of the γPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is underivatized.

[0159] In some embodiments, the cyclodextrin of the γPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is derivatized. In further embodiments, the cyclodextrin derivative of the complex has Formula I: [ka] wherein n is 4, 5, or 6; R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently -H, a linear or branched C1-C8 alkylene group, or an optionally substituted linear or branched C1-C6 group, and at least one of R1, R2, R3, R4, R5, R6, R7, R8, and R9 is a linear or branched C1-C8 alkylene (e.g., C1-C8-(alkylene)-SO3 - (base).

[0160] In some embodiments, the cyclodextrin derivative of the γPMTX / cyclodextrin complex and / or cyclodextrin / therapeutic agent complex has Formula II: [ka] wherein n is 4, 5, or 6; R1, R2, R3, R4, R5, R6, R7, R8, and R9 each independently represent -O- or -O-(C2-C6 alkylene)-SO3 - group; and at least one of R1 and R2 is independently -O-(C2-C6 alkylene)-SO3 - groups; and S1, S2, S3, S4, S5, S6, S7, S8, and S9 are each independently a pharmaceutically acceptable cation. In a further embodiment, the pharmaceutically acceptable cation is Li + , Na + , or K + Alkali metals such as Ca 2+ , or Mg 2+ and ammonium ions and amine cations such as cations of (C-C)-alkylamines, piperidines, pyrazines, (C-C)-alkanolamines, and (C-C)-cycloalkanolamines. In some embodiments, at least one of R and R is independently selected from -O-(CH). man SO3- group, -O-(C2-C6 alkylene)-SO3- group, where m is 2 to 6, preferably 2 to 4 (e.g., -O-CH2CH2CH2SO3- or -O-CH2CH2CH2CH2SO3-); and S1, S2, S3, S4, S5, S6, S7, S8, and S9 are each independently H or a pharmaceutically acceptable cation, including, for example, an alkali metal (e.g., Li + , Na + , K. + ), alkaline earth metals (e.g., Ca 2+ , Mg 2+ ), ammonium ions and amine cations such as the cations of (C1-C6)-alkylamines, piperidines, pyrazines, (C1-C6)-alkanolamines and (C4-C8)-cycloalkanolamines.

[0161] In some embodiments, the cyclodextrin derivative of the γPMTX / cyclodextrin complex and / or cyclodextrin / therapeutic agent complex is a cyclodextrin disclosed in U.S. Patent Nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, and 6,869,939; and WO 02005 / 117911, the contents of each of which are expressly incorporated herein by reference.

[0162] In some embodiments, the cyclodextrin derivative of the γPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is a sulfoalkyl ether cyclodextrin. In some embodiments, the cyclodextrin derivative of the complex is sulfobutyl ether-3-cyclodextrin, such as CAPTISOL® (CyDex Pharma. Inc., Lenexa, Kansas). Methods for making sulfobutyl ether-3-cyclodextrin and other sulfoalkyl ether cyclodextrins are known in the art.

[0163] In some embodiments, the cyclodextrin derivative of the γPMTX / cyclodextrin complex and / or cyclodextrin / therapeutic agent complex has formula III: [ka] wherein R is (a)(H) 21-X or (-(CH2)4-SO3Na) X , and x = 1.0–10.0, 1.0–5.0, 6.0–7.0, or 8.0–10.0; (b)(H) 21-X or (-(CH2CH(OH)CH3) X , and x = 1.0–10.0, 1.0–5.0, 6.0–7.0, or 8.0–10.0; (c)(H) 21-X or (sulfoalkyl ether) X , and x = 1.0–10.0, 1.0–5.0, 6.0–7.0, or 8.0–10.0; or (d)(H) 21-X or (-(CH2)4-SO3Na) X , and x = 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0.

[0164] In a further embodiment, the γPMTX / cyclodextrin complex and / or cyclodextrin / therapeutic agent complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0165] III. γPMTX Delivery Carrier In alternative embodiments, the present disclosure provides γPMTX delivery systems and their use for delivering a payload of γPMTX to a cell or cells in vitro or in vivo. In some embodiments, γPMTX is complexed with or incorporated into a delivery vehicle. Such delivery vehicles are known in the art and include, but are not limited to, liposomes, lipospheres, polymers, peptides, proteins, antibodies (e.g., ADCs such as antibody-γPMTX conjugates), cellular components, cyclic oligosaccharides (e.g., cyclodextrins), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), lipoprotein particles, and combinations thereof. In certain embodiments, the delivery vehicle is a liposome. In other specific embodiments, the delivery vehicle is an antibody or an antigen-binding antibody fragment.

[0166] A. Liposomes In some embodiments, the present disclosure provides a liposome composition comprising liposomes encapsulating (i.e., loaded with) gamma polyglutamated methotrexate (e.g., γPMTX as disclosed herein). In some embodiments, the liposomes in the liposome composition comprise γPMTX containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups (including the glutamyl groups of methotrexate). In some embodiments, the gamma polyglutamated methotrexate in Lp-γPMTX contains two or more L-glutamyl groups. In other embodiments, the gamma polyglutamated methotrexate in Lp-γPMTX contains D-glutamyl groups. In further embodiments, the gamma polyglutamated methotrexate in Lp-γPMTX contains D-glutamyl groups and two or more L-glutamyl groups. In further embodiments, the gamma polyglutamated methotrexate in Lp-γPMTX contains two or more glutamyl groups with gamma carboxyl bonds. In some embodiments, the liposome composition comprises liposomes containing γ-pentaglutamated MTX. In further embodiments, the liposomes comprise L-γ-pentaglutamated MTX, D-γ-pentaglutamated MTX, or L- and D-γ-pentaglutamated MTX. In some embodiments, the liposome composition comprises liposomes containing γ-hexaglutamated MTX (Lp-γPMTX). In further embodiments, the liposomes comprise L-γ-hexaglutamated MTX, D-γ-hexaglutamated MTX, or L- and D-γ-hexaglutamated MTX. In some embodiments, the liposome composition comprises anionic or neutral liposomes. In some embodiments, the liposome composition comprises cationic liposomes. In some embodiments, the Lp-γPMTX composition is not PEGylated. In some embodiments, the Lp-γPMTX composition is non-targeted (NTLp-γPMTX). In other embodiments, the Lp-γPMTX composition is targeted (TLp-γPMTX). In some embodiments, the liposome composition comprises liposomes having a diameter of 20 nm to 500 nm, or any range therebetween.In some embodiments, the liposome composition comprises liposomes having a diameter of 20 nm to 400 nm, or any range therebetween. In some embodiments, the liposome composition comprises liposomes having a diameter of 20 nm to 300 nm, or any range therebetween. In some embodiments, the liposome composition comprises liposomes having a diameter of 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposome composition comprises liposomes having a diameter of 20 nm to 150 nm, or any range therebetween. In further embodiments, the liposome composition comprises liposomes having a diameter of 80 nm to 120 nm, or any range therebetween. In further embodiments, 30 to 70%, 30 to 60%, or 30 to 50% w / w of gamma polyglutamated methotrexate, or any range therebetween, is encapsulated (entrapped) in Lp-γPMTX during the liposome preparation process. In some embodiments, the Lp-αPMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of gamma polyglutamated MTX. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% gamma polyglutamated methotrexate is encapsulated in the Lp-γPMTX during the liposome preparation process.

[0167] In some embodiments, provided liposomes further comprise an immunostimulatory agent, a detectable marker, or both, disposed on the exterior surface of the liposome. The immunostimulatory agent or detectable marker can be ionically or covalently bound to the exterior surface of the liposome, optionally including, for example, binding to a steric stabilizing component of the liposome.

[0168] The term "immunostimulatory agent," also known as "immunostimulant" and "immunostimulator," refers to a substance that stimulates immunity (including a pre-existing immune response) by inducing activation or increased activity of any component of the immune system. These immunostimulatory agents include one or more of haptens, adjuvants, protein immunostimulators, nucleic acid immunostimulators, and chemical immunostimulators. Many adjuvants include substances designed to stimulate the immune response, such as lipid A, proteins derived from Bordetella pertussis, or Mycobacterium tuberculosis. Certain adjuvants are commercially available, for example, Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron, or zinc; insoluble suspensions of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A; IFN-alpha, IFN-gamma, FLT3 ligand; and immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3). Cytokines such as GM-CSF, interleukins 2, 7, 12, and 15, and other similar growth factors can also be used as adjuvants. In a preferred embodiment, the immunostimulant may be at least one selected from the group consisting of fluorescein, DNP, beta-glucan, beta-1,3-glucan, and beta-1,6-glucan. In a further preferred embodiment, the immunostimulant is a toll-like receptor (TLR) modulator. In a further embodiment, the toll-like receptor (TLR) modulator is one or more of oxidized low-density lipoprotein (e.g., OXPAC, PGPC), erythrolamid lipid (e.g., E5564), and resolvin.In some embodiments, the liposomes contain fluorescein isothiocyanate (FITC), which, based on our experiments, surprisingly functions as both an immunostimulant and a detectable marker.

[0169] In some embodiments, the liposome comprises a detectable marker, which may include, for example, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, an enzyme, a dye, an ink, a magnetic compound, a biocatalyst, or a pigment that is detectable by any suitable means known in the art, including, for example, at least, magnetic resonance imaging (MRI), optical imaging, fluorescence / luminescence imaging, and / or nuclear imaging techniques.

[0170] In some embodiments, the immunostimulatory agent and / or detectable marker is bound to the outer surface by co-incubation with the liposome. For example, the immunostimulatory agent and / or detectable marker can be bound to the liposome membrane by hydrophobic interactions or ionic bonds, such as avidin / biotin bonds or metal chelate bonds (e.g., Ni-NTA). Alternatively, the immunostimulatory agent or detectable marker can be covalently bound to the outer surface of the liposome, for example, by covalently binding to a liposome component or a steric stabilizer such as PEG.

[0171] In some embodiments, the liposome further comprises an agent that increases uptake of the liposome into a desired intracellular compartment, including the cytosol.

[0172] In some embodiments, the liposomes comprise a mitochondrial targeting agent. In some embodiments, the liposomes comprise triphenylphosphonium (TPP). Methods and mechanisms for surface functionalization of liposomes with TPP are known in the art (e.g., attaching TPP to a lipid anchor via a PEG spacer group and modifying the TPP with a stearyl group (stearyltriphenylphosphonium (STPP))). In some embodiments, the liposomes comprise high-density octaarginine. In some embodiments, the liposomes comprise sphingomyelin and / or sphingomyelin metabolites. Sphingomyelin metabolites used to formulate the liposomes of the present invention can include, for example, ceramide, sphingosine, or sphingosine 1-phosphate. In some embodiments, the liposomes comprise rhodamine 123. In some embodiments, the liposomes comprise a mitochondrial penetrating peptide. In some embodiments, the liposomes contain a mitofusin peptide, a mitochondrial targeting signal peptide, and an antennapedia helix III homeodomain cell membrane penetrating peptide (ANT) (e.g., RQIKIWFQNRRMKWKKRKKRRQRRR (SEQ ID NO: 1), RKKRRXRRRGC, where X is any natural or unnatural amino acid (SEQ ID NO: 2), CCGCCAAGAAGCG (SEQ ID NO: 3), GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGA GCTGAGCGGCGTGGCGCGGGGGCGTCAT (SEQ ID NO: 4), ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTCATCCGTCAGCTC (SEQ ID NO: 5), or ACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTG (SEQ ID NO: 6)), or a mitochondrial permeability fragment thereof.

[0173] In some embodiments, the liposomes in the provided liposome compositions comprise a mitochondrial penetrant selected from a guanidine-rich peptoid, a tetraguanidinium, a triguanidinium, a diguanidinium, a monoguanidinium, a guanidine-rich polycarbamate, a beta-oligoarginine, a proline-rich dendrimer, and a phosphonium salt (e.g., methyltriphenylphosphonium and / or tetraphenylphosphonium).

[0174] In some embodiments, the liposomes in the provided liposome compositions comprise sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposomes comprise sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposomes comprise DOPE, sphingomyelin, stearyl octaarginine sphingomyelin, and stearyl octaarginine. In some embodiments, the liposomes comprise DOPE, sphingomyelin, stearyl octaarginine sphingomyelin, and stearyl octaarginine in a molar ratio of 9:2:1. In some embodiments, the liposomes comprise the MITO Porter® system or a variant thereof.

[0175] In some embodiments, the liposomes in the provided liposome compositions contain an agent, such as a membrane permeabilizing agent, that facilitates delivery of the liposomes across a cell membrane and provides the liposomes with the ability to bypass the endocytic pathway and the harsh environment of the lysosome. Membrane permeabilizing agents are known in the art and are routinely used and applicable to the manufacture and use of the provided liposome compositions. In some embodiments, the membrane permeabilizing agent / lysosomal bypassing agent is chloroquine. In some embodiments, the membrane permeabilizing agent is a cell-penetrating peptide.In some embodiments, the liposomes in the provided liposome compositions comprise a membrane permeabilizer selected from the group consisting of RKKRRQRRR (SEQ ID NO: 7), GRKKRRQRRRTPQ (SEQ ID NO: 8), YGRKKRRQRRR (SEQ ID NO: 9), AAVALLPAVLLALLA (SEQ ID NO: 10), MGLGLHLLVLAAALQ (SEQ ID NO: 11), GALFLGFLGAAGS™ (SEQ ID NO: 12), AGYLLGKINLKALAALAKKIL (SEQ ID NO: 13), RVIRVWFQNKRCKDKK (SEQ ID NO: 14). , RQIKIWFQNRRMKWKK (SEQ ID NO: 15), GLFEAIAGFIENGWEGMIDG (SEQ ID NO: 16), GWTLNSAGYLLGKIN (SEQ ID NO: 17), RSQSRSRYYRQRQRS (SEQ ID NO: 18), LAIPEQEY (SEQ ID NO: 19), LGIAEQEY (SEQ ID NO: 20), LGIPAQEY (SEQ ID NO: 21), LGIPEAEY (SEQ ID NO: 22), LGIPEQAY (SEQ ID NO: 23), LGIAEAEY (SEQ ID NO: 24), LGIPEAAY (SEQ ID NO: 25), LGIAEQAY (SEQ ID NO: 26). 26), LGIAEAAY (SEQ ID NO: 27), LLIILRRRIRKQAHAHSK (SEQ ID NO: 28), LKALAALAKKIL (SEQ ID NO: 29), KLALKLALKALKAALKLA (SEQ ID NO: 30), KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 31), DHQLNPAF (SEQ ID NO: 32), DPKGDPKG (SEQ ID NO: 33), VTVTVTVTVTGKGDPKPD (SEQ ID NO: 34), RQIKIWFQNRRMKWKK (SEQ ID NO: 35), GRKKRRQRRRPPQ (SEQ ID NO: 36), G WTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 37), GRKKRRQRRR (SEQ ID NO: 38), RRRRRRR (SEQ ID NO: 39), RRRRRRRR (SEQ ID NO: 40), RRRRRRRR (SEQ ID NO: 41), RRRRRRRRRR (SEQ ID NO: 42), RRRRRRRRRRR (SEQ ID NO: 43), and YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG(R)n (wherein n=2-15R in the L- and / or D-form) (SEQ ID NO: 44), or a cell-permeable fragment thereof.

[0176] As discussed above, liposomes may contain a steric stabilizer that can extend their lifespan in the circulation. For those embodiments incorporating a steric stabilizer, the steric stabilizer may be at least one member selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidylpolyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymers; oligoglycerin, polyethylene glycol and polypropylene oxide-containing copolymers, poloxamer 188, and polyvinyl alcohol. In some embodiments, the steric stabilizer or stabilizers is PEG. In one embodiment, the steric stabilizer is PEG. In a further embodiment, the PEG has a number-average molecular weight (Mn) of 200 to 5,000 daltons. These PEGs can be of any structure, such as linear, branched, star or comb structures, and are commercially available.

[0177] In some embodiments, the liposome composition comprises PEGylated liposomes (PLp-γPMTX). In some embodiments, the PEGylated liposomes in the liposome composition comprise γPMTX containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the gamma polyglutamated methotrexate in Lp-γPMTX contains two or more L-glutamyl groups. In other embodiments, the gamma polyglutamated methotrexate in Lp-γPMTX contains D-glutamyl groups. In further embodiments, the gamma polyglutamated methotrexate in Lp-γPMTX contains D-glutamyl groups and two or more L-glutamyl groups. In some embodiments, the liposome composition comprises PEGylated liposomes containing γ-pentaglutamated MTX. In further embodiments, the liposomes comprise L-γ-pentaglutamated MTX, D-γ-pentaglutamated MTX, or L- and D-γ-pentaglutamated MTX. In some embodiments, the liposome composition comprises PEGylated liposomes comprising γ-hexaglutamated MTX. In further embodiments, the liposomes comprise L-γ-hexaglutamated MTX, D-γ-hexaglutamated MTX, or L- and D-γ-hexaglutamated MTX. In some embodiments, the liposome composition comprises PEGylated liposomes that are anionic or neutral. In some embodiments, the liposome composition comprises PEGylated liposomes that are cationic. In some embodiments, the PLp-γPMTX composition is non-targeted (NTPLp-γPMTX). In other embodiments, the PLp-γPMTX composition is targeted (TPLp-γPMTX). In some embodiments, the liposome composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w gamma polyglutamated methotrexate.In some embodiments, liposome compositions comprising PEGylated liposomes comprising at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% gamma polyglutamated methotrexate are encapsulated in PLp-γPMTX during the liposome fabrication process. In some embodiments, the liposome compositions comprise PEGylated liposomes having a diameter in the range of 20 nm to 500 nm. In some embodiments, the liposome compositions comprise PEGylated liposomes having a diameter in the range of 20 nm to 400 nm. In some embodiments, the liposome compositions comprise PEGylated liposomes having a diameter in the range of 20 nm to 300 nm. In some embodiments, the liposome compositions comprise PEGylated liposomes having a diameter in the range of 20 nm to 200 nm. In a further embodiment, the liposome composition comprises PEGylated liposomes having diameters ranging from 80 nm to 120 nm.

[0178] In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated methotrexate in provided liposome compositions is pentaglutamated. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated methotrexate in provided liposome compositions is hexaglutamated. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated methotrexate in the composition has 4 to 10, 4 to 6, or more than 5 γ-glutamyl groups.

[0179] In some embodiments, the gamma polyglutamated methotrexate composition (e.g., a delivery vehicle such as polyglutamates and liposomes containing polyglutamates) is in an aqueous solution. In some embodiments, the γPMTX composition is delivered as a liposome composition in an amount of 1 square meter (m 2 In a further embodiment, the γPMTX composition is administered at a dose of about 0.005 to about 5000 mg per square meter (m) of body surface area, or any range therebetween. In a further embodiment, the γPMTX composition is administered at a dose of about 0.005 to about 5000 mg per square meter (m) of body surface area, or any range therebetween.2 The dose is about 0.1 to about 1000 mg of γPMTX per 1000 mg of body surface area, or any range therebetween.

[0180] (1) Liposome composition Lipids and other components of liposomes contained in the liposome composition can be any lipid, combination of lipids and ratios, or combination of lipids and other liposome components and their respective ratios known in the art. However, those skilled in the art will understand that liposomal encapsulation of any particular drug, such as, but not limited to, gamma-polyglutamated MTX discussed herein, can involve substantially routine experimentation to obtain a useful and functional liposome formulation. Generally, the provided liposomes can have any liposome structure, such as a structure with an internal space isolated from the external medium by one or more lipid bilayers, or any microcapsule structure with a semipermeable membrane with a lipophilic core that separates the interior. The lipid bilayer can be any structure of amphiphilic molecules characterized by hydrophilic and hydrophobic portions. Typically, the amphiphilic molecules in the bilayer are arranged in a two-dimensional sheet, with the hydrophobic portions facing the inside of the sheet and the hydrophilic portions facing the outside. The amphiphilic molecules forming the provided liposomes can be any known or yet to be discovered amphiphilic molecules (e.g., synthetic or naturally occurring lipids or biocompatible lipids). Liposomes can be formed from amphiphilic polymers and surfactants, such as polymersomes and niosomes. In the present disclosure, these liposome-forming materials are also referred to as "lipids," although this disclosure is not limited thereto.

[0181] The liposome composition formulations provided herein can be in a liquid or dry form, such as a dry powder or dry cake. The dry powder or dry cake can undergo primary drying, for example, under lyophilization conditions, or can undergo primary drying only or both primary and secondary drying. In the dry form, the powder or cake can have, for example, 1% to 6% moisture, e.g., 2% to 5% moisture, or 2% to 4% moisture. One example of a drying method is lyophilization (also called freeze-drying or cryodesication). Any of the disclosed compositions and methods can include liposomes, lyophilized liposomes, or liposomes reconstituted from lyophilized liposomes. In some embodiments, the disclosed compositions and methods include one or more lyoprotectants or cryoprotectants. These protectants are typically sugars (mono-, di-, and polysaccharides), polyhydric alcohols and their derivatives, glycerol or polyhydroxy compounds such as polyethylene glycol, trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol, or aminoglycosides. In further embodiments, the lyoprotectant or cryoprotectant comprises up to 10% or up to 20% of the solution outside the liposome, inside the liposome, or both outside and inside the liposome.

[0182] In some embodiments, liposomes contain steric stabilizers that extend their lifespan in the circulation. One or more steric stabilizers, such as hydrophilic polymers (polyethylene glycol (PEG)), glycolipids (monosialoganglioside (GM1)), or others, occupy the space immediately adjacent to the liposome surface, excluding other macromolecules from this space. This prevents plasma opsonins from accessing and binding to the liposome surface, thereby inhibiting macrophage interaction with such liposomes or any other clearance mechanism, and extending the lifespan of liposomes in the circulation. In some embodiments, the steric stabilizer or stabilizers is PEG or a combination comprising PEG. In further embodiments, the steric stabilizer is PEG or a combination comprising PEG with a number-average molecular weight (Mn) of 200 to 5,000 daltons. These PEGs can be of any architecture, including linear, branched, star-shaped, or comb-shaped, and are commercially available.

[0183] The diameter of the disclosed liposomes is not particularly limited. In some embodiments, the liposomes have a diameter in the range of, for example, 30 nm to 150 nm (nanometers). In other embodiments, the liposomes have a diameter in the range of 40 nm to 70 nm.

[0184] The properties of liposomes are influenced by the nature of the lipids used to prepare the liposomes. A wide variety of lipids have been used to prepare liposomes. These include cationic, anionic, and neutral lipids. In some embodiments, liposomes containing gamma polyglutamated methotrexate are anionic or neutral. In other embodiments, provided liposomes are cationic. The charge (e.g., anionic, neutral, or cationic) can be routinely determined by measuring the zeta potential of the liposomes. The zeta potential of the liposomes can be positive, zero, or negative. In some embodiments, the zeta potential of the liposomes is less than or equal to zero. In some embodiments, the zeta potential of the liposomes is in the range of 0 to -150 mV. In other embodiments, the zeta potential of the liposomes is in the range of -30 to -50 mV.

[0185] In some embodiments, cationic lipids are used to create cationic liposomes, which are often used as gene transfer agents. The positive charges on the cationic liposomes allow them to interact with the negative charges on the cell surface. After the cationic liposomes bind to cells, the liposomes are transported into the interior of the cells by endocytosis.

[0186] In some preferred embodiments, neutral to anionic liposomes are used. In preferred embodiments, anionic liposomes are used. For example, the use of a mixture of neutral lipids such as HSPC and anionic lipids such as PEG-DSPE forms anionic liposomes, which are less likely to nonspecifically bind to normal cells. Specific binding to tumor cells can be achieved using tumor-targeting antibodies, such as folate receptor antibodies, including folate receptor alpha antibodies, folate receptor beta antibodies, and / or folate receptor delta antibodies.

[0187] As an example, at least one (or some) lipid is an amphipathic lipid, defined as having a hydrophilic and a hydrophobic portion (usually a hydrophilic head and a hydrophobic tail). The hydrophobic portion usually faces the hydrophobic phase (e.g., within the bilayer), while the hydrophilic portion usually faces the aqueous phase (e.g., outside the bilayer). The hydrophilic portion can include polar or charged groups such as carbohydrates, phosphate, carboxylic acid, sulfato, amino, sulfhydryl, nitro, hydroxy, and other similar groups. The hydrophobic portion can include nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0188] Typically, for example, the lipid is a phospholipid, including, but not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, etc. It should be understood that other lipid membrane components, such as cholesterol, sphingomyelin, and cardiolipin, can also be used.

[0189] The lipids comprising the liposomes provided herein can be anionic and neutral (including zwitterionic and polar) lipids, including anionic and neutral phospholipids. Neutral lipids exist in uncharged or neutral zwitterionic form at selected pHs. At physiological pH, such lipids include, for example, dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol. Examples of zwitterionic lipids include, but are not limited to, dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS). Anionic lipids are negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and anionic modifying groups attached to neutral lipids.

[0190] In this specification, anionic and neutral lipids are collectively referred to as non-cationic lipids. Such lipids may contain phosphorus, but they are not so limited. Examples of non-cationic lipids include lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), lecithin, ... 1-Ethanolamine (DSPE), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans These include PE, palmitoyloleoylphosphatidylethanolamine (POPE), 1-stearoyl-2-oleoylphosphatidiethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, and cholesterol.

[0191] Liposomes can be constructed using any liposome assembly method using liposomal components (also referred to as liposome components) known in the art. Liposome components include, for example, lipids such as DSPE, HSPC, cholesterol, and derivatives of these components. Other suitable lipids are commercially available, for example, from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). A partial list of available negatively or neutrally charged lipids suitable for making anionic liposomes can be, for example, at least one of the following: DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DOPE, DMPA·Na, DPPA·Na, DOPA·Na, DMPG·Na, DPPG·Na, DOPG·Na, DMPS·Na, DPPS·Na, DOPS·Na, DOPE-glutaryl·(Na)2, tetramyristoylcardiolipin·(Na)2, DSPE-mPEG-2000·Na, DSPE-mPEG-5000·Na, and DSPE-maleimidePEG-2000·Na.

[0192] In some embodiments, the γPMTX compositions provided herein are formulated in liposomes containing cationic lipids. In one embodiment, the cationic lipids are, but are not limited to, those described in WO 2012 / 040184, WO 2011 / 153120, WO 2011 / 149733, WO 2011 / 090965, WO 2011 / 043913, WO 2011 / 022460, WO 2012 / 061259, WO 2012 / 054365, WO 2012 The cationic lipids are selected from those described in US Patent Nos. 2004 / 044638, 2010 / 080724, 2010 / 21865 and 2008 / 103276, U.S. Patent Nos. 7,893,302, 7,404,969, 8,283,333, U.S. Patent Application Publication Nos. 20100036115, and 20120202871. Each of these patents is incorporated herein by reference in its entirety. In another embodiment, the cationic lipid may be selected from, but is not limited to, Formula A as described in WO 2012 / 040184, WO 2011 / 153120, WO 201 / 1149733, WO 2011 / 090965, WO 2011 / 043913, WO 2011 / 022460, WO 2012 / 061259, WO 2012 / 054365, and WO 2012 / 044638, each of which is incorporated herein by reference in its entirety. In yet another embodiment, the cationic lipid may be selected from, but is not limited to, formulas CLI-CLXXIX of WO2008103276, formulas CLI-CLXXIX of U.S. Patent No. 7,893,302, formulas CLI-CLXXXXII of U.S. Patent No. 7,404,969, and formulas I-VI of U.S. Patent Application Publication No. 20100036115, each of which is incorporated herein by reference in its entirety. As non-limiting examples, the cationic lipid may be selected from the following: (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethyl-hexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N-Dimethylheneicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-Dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N-Dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-Dimethylheptacosa-18,21-dien-10-amine, (15Z,18Z)-N,N-Dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-Dimethyl-tricosa-14,17-dien-4-amine, (19Z,22Z)-N,N- Dimethyloctacosa-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-8-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyl-triaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacosa-19,22-dien-9-amine N,N-dimethylhexacos-18-en-10-amine, (17Z)-N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosane-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethyl-heptacos-20-en-10-amine, (15Z)-N,N-dimethylheptacosane Cos-15-en-10-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylhentriacont-22-en-10-amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21-[R1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N, N-Dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-Dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-Dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine, N,N-Dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyl octadecan-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethyl-pentadecan-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, R-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)prop-n-2-amine, S-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z-)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-Octadeca-9,12-dien-1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)-N,N- Dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine oxy]-N,N-dimethyl-1-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethyl-propan-2-amine, (2S)-1-[(13Z)-docosa-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(1 3Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-dien-1-yloxylpropan-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]-methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{[-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,2-trien-10-amine or pharmaceutically acceptable salts or acids or stereoisomers thereof.

[0193] In one embodiment, the lipid may be a cleavable lipid such as those described in WO 2012 / 170889, which is incorporated herein by reference in its entirety.

[0194] Cationic lipids can be routinely synthesized using methods known in the art and / or as described in WO 2012 / 040184, WO 2011 / 153120, WO 2011 / 149733, WO 2011 / 090965, WO 201 / 1043913, WO 2011 / 022460, WO 2012 / 061259, WO 2012 / 054365, WO 2012 / 044638, WO 2010 / 080724, and WO 2010 / 21865, which are incorporated herein by reference in their entireties.

[0195] Lipid derivatives can include, for example, at least one or more steric stabilizers and / or functional groups attached (preferably covalently) to the liposome component (after which the steric stabilizer and / or functional group would be considered part of the liposome component). The functional group includes a group that can be used to attach the liposome component to another moiety, such as a protein. Such functional groups include at least maleimide. These steric stabilizers include at least one member selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidylpolyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymers; and polyvinyl alcohol.

[0196] In some embodiments, the γPMTX composition is formulated in a lipid-polycation complex. Formation of the lipid-polycation complex can be achieved using methods known in the art and / or as described in U.S. Patent Application Publication No. 20120178702, which is incorporated herein by reference in its entirety. Non-limiting examples of polycations include cationic peptides or polypeptides such as polylysine, polyornithine, and / or polyarginine, and cationic peptides described in International Publication No. 2012 / 013326, which is incorporated herein by reference in its entirety. In another embodiment, the γPMTX composition is formulated in a lipid-polycation complex, which further includes a neutral lipid such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0197] The liposome components can include any molecule (e.g., chemical / reagent / protein) attached thereto, and in some embodiments, the liposome components provided include at least a member selected from the group DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide. In some embodiments, the liposome components provided include DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide. In preferred embodiments, the liposome components comprising the liposome include DSPE; DSPE-FITC; DSPE-maleimide; cholesterol; and HSPC.

[0198] In further embodiments, the liposomes of the liposome compositions provided herein comprise an oxidized phospholipid. In some embodiments, the liposomes comprise an oxidized phospholipid that is a member selected from the group consisting of phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, and 1-palmitoyl-2-arachidonoyl-sn-glycero-2-phosphate. In some embodiments, the phospholipid has an unsaturated bond. In some embodiments, the phospholipid is an arachidonic acid containing phospholipid. In further embodiments, the phospholipid is sn-2-oxygenated. In further embodiments, the phospholipid is unfragmented.

[0199] In some embodiments, liposomes of the disclosed liposome compositions contain oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-folylcholine (OxPAPC). As used herein, the term "oxPAPC" refers to lipids produced by oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-folylcholine (PAPC), resulting in a mixture of oxidized phospholipids containing fragmented or full-length oxygenated sn-2 residues. Well-characterized oxidative fragment species contain 5-carbon sn-2 residues with omega aldehyde or omega carboxyl groups. Oxidation of arachidonic acid residues also produces phospholipids containing esterified isoprostanes. OxPAPC includes HOdiA-PC, KOdiA-PC, HOOA-PC, and KOOA-PC species, among many other oxidation products present in oxPAPC. In further embodiments, oxPAPC is an epoxyisoprostane-containing phospholipid. In further embodiments, the oxPAPC is 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2-(epoxycyclopentenone)-sn-glycero-3-folylcholine (PECPC), and / or 1-palmitoyl-2-(epoxy-isoprostane E2)-sn-glycero-4-phosphocholine (PEIPC). In some embodiments, the phospholipid has an unsaturated bond. In some embodiments, the phospholipid is an arachidonic acid containing phospholipid. In further embodiments, the phospholipid is sn-2-oxygenated. In further embodiments, the phospholipid is unfragmented.

[0200] In some embodiments, the liposomal gamma polyglutamated methotrexate composition is PEGylated (i.e., PEGylated liposomal gamma polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate (PLp-γPMTX or TPLp-γPMTX)). In some embodiments, the PLp-γPMTX or TPLp-γPMTX is water-soluble. That is, the PLp-γPMTX or TPLp-γPMTX is in the form of an aqueous solution.

[0201] In some embodiments, the liposomes of the disclosed liposome compositions comprise a lipid selected from the following: 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC); 1-palmitoyl-2-(9'oxo-nonanoyl)-sn-glycero-3-phosphocholine; 1-palmitoyl-2-arachinodoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and 1-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine. In further embodiments, the liposomes comprise PGPC.

[0202] In some embodiments, the pH of the solution containing the liposome composition is pH 5-8, or pH 2-6, or pH 2-8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is pH 5-8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is pH 6-7, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is pH 6-7.5, 6.5-7.5, 6.7-7.5, or 6.3-7.0, or any range therebetween.

[0203] In some embodiments, at least one component of the liposomal lipid bilayer is functionalized (or reactive). As used herein, a functionalized component is a component that contains a reactive group that can be used to crosslink reagents and moieties to the lipid. Once a lipid is functionalized, any liposomes it forms are also functionalized. In some embodiments, the reactive group is one that reacts with a crosslinker (or other moiety) to form a crosslink. The reactive group in the liposomal lipid bilayer is located anywhere on the lipid that can contact the crosslinker and allow crosslinking with another moiety (e.g., a steric stabilizer or targeting moiety). In some embodiments, the reactive group is in the head group of a lipid, such as a phospholipid. In some embodiments, the reactive group is a maleimide group. Maleimide groups can crosslink with each other in the presence of a dithiol crosslinker, such as, but not limited to, dithiothreitol (DTT).

[0204] It should be understood that the use of other functionalized lipids, other reactive groups, and other cross-linking agents beyond those described above is also contemplated. In addition to maleimide groups, other examples of contemplated reactive groups include, but are not limited to, other thiol-reactive groups, amino groups such as primary or secondary amines, carboxyl groups, hydroxyl groups, aldehyde groups, alkyne groups, azide groups, carbonyl groups, haloacetyl (e.g., iodoacetyl) groups, imidoester groups, N-hydroxysuccinimide esters, sulfhydryl groups, and pyridyl disulfide groups.

[0205] Functionalized and non-functionalized lipids are available from a number of commercial sources, such as Avanti Polar Lipids (Alabaster, AL) and Lipoid LLC (Newark, NJ).

[0206] (2) Liposome internal space In a further non-limiting embodiment, a provided liposome comprises an interior space. In some embodiments, the interior space comprises, but is not limited to, an aqueous solution. In some embodiments, the interior space comprises gamma polyglutamated methotrexate provided herein. In further embodiments, the interior space of the liposome comprises a tonicity agent. In some embodiments: In some embodiments, the concentration (wt%) of the tonicity agent is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 1-50%, or any range therebetween. In some embodiments, the interior space of the liposome comprises a sugar (e.g., trehalose, maltose, sucrose, lactose, mannose, mannitol, glycerol, dextrose, fructose, etc.). In further embodiments, the concentration (wt%) of the sugar is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 1-50%, or any range therebetween. In some embodiments, the pH of the interior space of the liposome is pH 2-8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is between 5 and 8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is between 6 and 7, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is between 6 and 7.5, 6.5 and 7.5, 6.7 and 7.5, or 6.3 and 7.0, or any range therebetween. In some embodiments, the interior space contains a buffer. In further embodiments, the buffer is a buffer selected from HEPES, citrate, or sodium phosphate (e.g., monobasic sodium phosphate and / or dibasic sodium phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., monobasic sodium phosphate and / or dibasic sodium phosphate). In some embodiments, the buffer is at a concentration of 15 to 200 mM, or any range therebetween. In still further embodiments, the buffer is at a concentration of 5-200 mM, 15-200 mM, 5-100 mM, 15-100 mM, 5-50 mM, 15-50 mM, 5-25 mM, 5-20 mM, 5-15 mM, or any range therebetween.In some embodiments, the buffer is HEPES at a concentration of 15-200 mM, or any range therebetween. In some embodiments, the buffer is citrate at a concentration of 15-200 mM, or any range therebetween. In some embodiments, the buffer is sodium phosphate at a concentration of 15-200 mM, or any range therebetween. In some embodiments, the interior space of the liposome contains a combined concentration of sodium acetate and calcium acetate of 5 mM-500 mM, or 50 mM-500 mM, or any range therebetween.

[0207] In some embodiments, the interior space of the liposome contains trehalose. In further embodiments, the concentration (wt%) of trehalose is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 5-20%, or any range therebetween. In still further embodiments, the concentration (wt%) of trehalose is 1-15%, or any range therebetween. In additional embodiments, the trehalose is present at about 5%-20% (wt%) trehalose, or any combination of one or more lyoprotectants or cryoprotectants at a total concentration of 5%-20%. In some embodiments, the pH of the solution containing the liposome composition is 6-7.5, 6.5-7.5, 6.7-7.5, or 6.3-7.0, or any range therebetween. In some embodiments, the interior space contains a buffer solution. In some embodiments, the buffer is selected from HEPES, citrate, or sodium phosphate (e.g., monobasic sodium phosphate and / or dibasic sodium phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., monobasic sodium phosphate and / or dibasic sodium phosphate). In some embodiments, the buffer is at a concentration of 15-200 mM, or any range therebetween. In still further embodiments, the buffer is at a concentration of 5-200 mM, 15-200 mM, 5-100 mM, 15-100 mM, 5-50 mM, 15-50 mM, 5-25 mM, 5-20 mM, 5-15 mM, or any range therebetween. In some embodiments, the buffer is HEPES at a concentration of 15-200 mM, or any range therebetween. In some embodiments, the buffer is citrate at a concentration of 15-200 mM, or any range therebetween. In some embodiments, the buffer is sodium phosphate at a concentration of 15-200 mM, or any range therebetween. In further embodiments, the interior space of the liposome contains sodium acetate and / or calcium acetate.In some embodiments, the interior space of the liposome comprises a total concentration of sodium acetate and calcium acetate of 5 mM to 500 mM, or 50 mM to 500 mM, or any range therebetween.

[0208] In some embodiments, the interior space of the liposome contains dextrose. In further embodiments, the concentration (wt%) of dextrose is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 5-20%, or any range therebetween. In still further embodiments, the concentration (wt%) of dextrose is 1-15%, or any range therebetween. In additional embodiments, dextrose is present at a concentration of about 5%-20% (wt%) dextrose, or any combination of one or more lyoprotectants or cryoprotectants is present at a total concentration of 5%-20%. In some embodiments, the pH of the solution containing the liposome composition is 6-7.5, 6.5-7.5, 6.7-7.5, or 6.3-7.0, or any range therebetween. In some embodiments, the interior space contains a buffer. In some embodiments, the buffer is selected from HEPES, citrate, or sodium phosphate (e.g., monobasic sodium phosphate and / or dibasic sodium phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., monobasic sodium phosphate and / or dibasic sodium phosphate). In some embodiments, the buffer is at a concentration of 15-200 mM, or any range therebetween. In still further embodiments, the buffer is at a concentration of 5-200 mM, 15-200 mM, 5-100 mM, 15-100 mM, 5-50 mM, 15-50 mM, 5-25 mM, 5-20 mM, 5-15 mM, or any range therebetween. In some embodiments, the buffer is HEPES at a concentration of 15-200 mM, or any range therebetween. In some embodiments, the buffer is citrate at a concentration of 15-200 mM, or any range therebetween. In some embodiments, the buffer is sodium phosphate at a concentration of 15-200 mM, or any range therebetween. In further embodiments, the interior space of the liposome contains sodium acetate and / or calcium acetate.In some embodiments, the interior space of the liposome comprises a total concentration of sodium acetate and calcium acetate of 5 mM to 500 mM, or 50 mM to 500 mM, or any range therebetween.

[0209] In further embodiments, the present disclosure provides liposome compositions comprising liposomes encapsulating (loaded with) gamma polyglutamated methotrexate (e.g., γPMTX as disclosed herein). In some embodiments, the liposomes in the liposome composition comprise γPMTX containing 4, 5, 2 to 10, 4 to 6, or more than 5 γ-glutamyl groups (including the glutamyl groups of methotrexate). In some embodiments, the gamma polyglutamated methotrexate in Lp-γPMTX contains two or more L-glutamyl groups. In other embodiments, the gamma polyglutamated methotrexate in Lp-γPMTX contains D-glutamyl groups. In further embodiments, the gamma polyglutamated methotrexate in Lp-γPMTX contains a D-glutamyl group and two or more L-glutamyl groups. In further embodiments, the gamma polyglutamated methotrexate in Lp-γPMTX contains two or more glutamyl groups with gamma carboxyl bonds. In some embodiments, the liposome composition comprises a liposome containing γ-pentaglutamated MTX. In further embodiments, the liposome comprises L-γ-pentaglutamated MTX, D-γ-pentaglutamated MTX, or L- and D-γ-pentaglutamated MTX. In some embodiments, the liposome composition comprises a liposome containing γ-hexaglutamated MTX (Lp-γPMTX). In further embodiments, the liposome comprises L-γ-hexaglutamated MTX, D-γ-hexaglutamated MTX, or L- and D-γ-hexaglutamated MTX.

[0210] In some embodiments, the targeted PEGylated liposomal gamma polyglutamated (e.g., pentaglutamated or hexaglutamated) methotrexate comprises a vehicle comprising an interior space; aqueous gamma polyglutamated methotrexate disposed within the interior space; and a liposome comprising a targeting moiety comprising a protein having specific affinity for at least one folate receptor, the targeting moiety being disposed on the exterior surface of the liposome. In some embodiments, the vehicle is an aqueous solution. In some embodiments, the interior space, the exterior space (e.g., the vehicle), or both the interior space and the vehicle comprise one or more of the above-listed lyoprotectants or cryoprotectants. In some embodiments, the cryoprotectant is mannitol, trehalose, sorbitol, or sucrose.

[0211] In some embodiments, gamma polyglutamated methotrexate (e.g., Lp-γPMTX, including PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX)-encapsulated liposomes have an interior space containing fewer than 500,000 or fewer than 200,000 gamma polyglutamated methotrexate molecules. In some embodiments, the liposome interior space contains 10 to 100,000 gamma polyglutamated methotrexate molecules, or any range therebetween. In some embodiments, the liposome interior space contains 10,000 to 100,000 gamma polyglutamated methotrexate molecules, or any range therebetween. In some embodiments, the liposomes are not PEGylated and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma polyglutamated methotrexate molecules. In some embodiments, the liposomes are non-PEGylated and the interior space of the liposomes contains 10 to 100,000 gamma polyglutamated methotrexate molecules, or any range therebetween. In further embodiments, the liposomes are non-PEGylated and the interior space of the liposomes contains 10,000 to 100,000 gamma polyglutamated methotrexate molecules, or any range therebetween. In some embodiments, the liposomes are targeted and non-PEGylated (TLp-γPMTX) and have an interior space containing less than 500,000 or less than 200,000 gamma polyglutamated methotrexate molecules. In some embodiments, the liposomes are targeted and non-PEGylated and the interior space of the liposomes contains 10 to 100,000 gamma polyglutamated methotrexate molecules, or any range therebetween. In a further embodiment, the liposome is targeted and non-pegylated, and the interior space of the liposome contains 10,000 to 100,000 gamma polyglutamated methotrexate molecules, or any range therebetween.In some embodiments, the liposomes are non-targeted and non-PEGylated (NTLp-γPMTX) and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma polyglutamated methotrexate molecules. In some embodiments, the liposomes are non-targeted and non-PEGylated and the interior space of the liposomes contains 10 to 100,000 gamma polyglutamated methotrexate molecules, or any range therebetween. In further embodiments, the liposomes are non-targeted and non-PEGylated and the interior space of the liposomes contains 10,000 to 100,000 gamma polyglutamated methotrexate molecules, or any range therebetween.

[0212] In some embodiments, the liposome encapsulates gamma polyglutamated methotrexate containing 2 to 10 glutamyl groups (i.e., Lp-γPMTX, including PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX) and has an interior space containing fewer than 500,000 or fewer than 200,000 gamma polyglutamated methotrexate molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome interior space contains 10 to 100,000 gamma polyglutamated methotrexate molecules containing 2 to 10 glutamyl groups, or any range therebetween. In further embodiments, the liposome interior space contains 10,000 to 100,000 gamma polyglutamated methotrexate molecules containing 2 to 10 glutamyl groups, or any range therebetween. In some embodiments, the liposomes are not PEGylated and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma polyglutamated methotrexate molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposomes are not PEGylated and the interior space of the liposomes contains 10 to 100,000 gamma polyglutamated methotrexate molecules containing 2 to 10 glutamyl groups, or any range therebetween. In further embodiments, the liposomes are not PEGylated and the interior space of the liposomes contains 10,000 to 100,000 gamma polyglutamated methotrexate molecules containing 2 to 10 glutamyl groups, or any range therebetween. In some embodiments, the liposomes are targeted and non-PEGylated (TLp-γPMTX) and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma polyglutamated methotrexate molecules containing 2-10 glutamyl groups. In some embodiments, the liposomes are targeted and non-PEGylated and the interior space of the liposomes contains 10-100,000 or any range therebetween gamma polyglutamated methotrexate molecules containing 2-10 glutamyl groups.In further embodiments, the liposomes are targeted and non-PEGylated, and the interior space of the liposomes contains 10,000 to 100,000, or any range therebetween, gamma polyglutamated methotrexate molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposomes are non-targeted and non-PEGylated (NTLp-γPMTX), and have an interior space containing fewer than 500,000 or fewer than 200,000, gamma polyglutamated methotrexate molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposomes are non-targeted and non-PEGylated, and the interior space of the liposomes contains 10 to 100,000, or any range therebetween, gamma polyglutamated methotrexate molecules containing 2 to 10 glutamyl groups. In a further embodiment, the liposome is non-targeted and non-pegylated, and the interior space of the liposome contains 10,000 to 100,000 gamma polyglutamated methotrexate molecules containing 2 to 10 glutamyl groups, or any range therebetween.

[0213] In some embodiments, gamma polyglutamated methotrexate (i.e., Lp-γPMTX, including PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX)-encapsulated liposomes have an interior space containing fewer than 500,000 or fewer than 200,000 gamma tetraglutamated methotrexate molecules. In some embodiments, the liposome interior space contains 10 to 100,000 gamma tetraglutamated methotrexate molecules, or any range therebetween. In some embodiments, the liposome interior space contains 10,000 to 100,000 gamma tetraglutamated methotrexate molecules, or any range therebetween. In some embodiments, the liposome is not PEGylated and has an interior space containing fewer than 500,000 or fewer than 200,000 gamma tetraglutamated methotrexate molecules. In some embodiments, the liposomes are non-PEGylated and the interior space of the liposomes contains 10 to 100,000 gamma-tetraglutamated methotrexate molecules, or any range therebetween. In further embodiments, the liposomes are non-PEGylated and the interior space of the liposomes contains 10,000 to 100,000 gamma-tetraglutamated methotrexate molecules, or any range therebetween. In some embodiments, the liposomes are targeted and non-PEGylated (TLp-γPMTX) and have an interior space containing less than 500,000 or less than 200,000 gamma-tetraglutamated methotrexate molecules. In some embodiments, the liposomes are targeted and non-PEGylated and the interior space of the liposomes contains 10 to 100,000 gamma-tetraglutamated methotrexate molecules, or any range therebetween. In a further embodiment, the liposome is targeted and non-pegylated, and the interior space of the liposome contains 10,000 to 100,000 gamma tetraglutamated methotrexate molecules, or any range therebetween.In some embodiments, the liposomes are non-targeted and non-PEGylated (NTLp-γPMTX) and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma-tetraglutamated methotrexate molecules. In some embodiments, the liposomes are non-targeted and non-PEGylated and the interior space of the liposomes contains 10 to 100,000 gamma-tetraglutamated methotrexate molecules, or any range therebetween. In further embodiments, the liposomes are non-targeted and non-PEGylated and the interior space of the liposomes contains 10,000 to 100,000 gamma-tetraglutamated methotrexate molecules, or any range therebetween.

[0214] In some embodiments, gamma-polyglutamated methotrexate (i.e., Lp-γPMTX, including PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX)-encapsulated liposomes have an interior space containing fewer than 500,000 or fewer than 200,000 gamma-pentaglutamated methotrexate molecules. In some embodiments, the liposome interior space contains 10 to 100,000 gamma-pentaglutamated methotrexate molecules, or any range therebetween. In some embodiments, the liposome interior space contains 10,000 to 100,000 gamma-pentaglutamated methotrexate molecules, or any range therebetween. In some embodiments, the liposome is not PEGylated and has an interior space containing fewer than 500,000 or fewer than 200,000 gamma-pentaglutamated methotrexate molecules. In some embodiments, the liposomes are non-PEGylated and the interior space of the liposomes contains 10 to 100,000 gamma pentaglutamated methotrexate molecules, or any range therebetween. In further embodiments, the liposomes are non-PEGylated and the interior space of the liposomes contains 10,000 to 100,000 gamma pentaglutamated methotrexate molecules, or any range therebetween. In some embodiments, the liposomes are targeted and non-PEGylated (TLp-γPMTX) and have an interior space containing less than 500,000 or less than 200,000 gamma pentaglutamated methotrexate molecules. In some embodiments, the liposomes are targeted and non-PEGylated and the interior space of the liposomes contains 10 to 100,000 gamma pentaglutamated methotrexate molecules, or any range therebetween. In a further embodiment, the liposome is targeted and non-pegylated, and the interior space of the liposome contains 10,000 to 100,000 gamma pentaglutamated methotrexate molecules, or any range therebetween.In some embodiments, the liposomes are non-targeted and non-PEGylated (NTLp-γPMTX) and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma pentaglutamated methotrexate molecules. In some embodiments, the liposomes are non-targeted and non-PEGylated and the interior space of the liposomes contains 10 to 100,000 gamma pentaglutamated methotrexate molecules, or any range therebetween. In further embodiments, the liposomes are non-targeted and non-PEGylated and the interior space of the liposomes contains 10,000 to 100,000 gamma pentaglutamated methotrexate molecules, or any range therebetween.

[0215] In some embodiments, gamma hexaglutamated methotrexate (i.e., Lp-γPMTX, including PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX)-encapsulated liposomes have an interior space containing fewer than 500,000 or fewer than 200,000 gamma hexaglutamated methotrexate molecules. In some embodiments, the liposome interior space contains 10 to 100,000 gamma hexaglutamated methotrexate molecules, or any range therebetween. In further embodiments, the liposome interior space contains 10,000 to 100,000 gamma hexaglutamated methotrexate molecules, or any range therebetween. In some embodiments, the liposomes are non-PEGylated and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma hexaglutamated methotrexate molecules. In some embodiments, the liposomes are non-PEGylated and have an interior space containing 10 to 100,000 gamma hexaglutamated methotrexate molecules, or any range therebetween. In further embodiments, the liposomes are non-PEGylated and have an interior space containing 10,000 to 100,000 gamma hexaglutamated methotrexate molecules, or any range therebetween. In some embodiments, the liposomes are targeted and are non-PEGylated (TLp-γPMTX) and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma hexaglutamated methotrexate molecules. In some embodiments, the liposomes are targeted and non-PEGylated, and the interior space of the liposomes contains 10 to 100,000 gamma hexaglutamated methotrexate molecules, or any range therebetween. In further embodiments, the liposomes are targeted and non-PEGylated, and the interior space of the liposomes contains 10,000 to 100,000 gamma hexaglutamated methotrexate molecules, or any range therebetween.In some embodiments, the liposomes are non-targeted and non-PEGylated (NTLp-γPMTX) and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma hexaglutamated methotrexate molecules. In some embodiments, the liposomes are non-targeted and non-PEGylated and the interior space of the liposomes contains 10 to 100,000 gamma hexaglutamated methotrexate molecules, or any range therebetween. In further embodiments, the liposomes are non-targeted and non-PEGylated and the interior space of the liposomes contains 10,000 to 100,000 gamma hexaglutamated methotrexate molecules, or any range therebetween.

[0216] In some embodiments, the present disclosure provides liposomal gamma polyglutamated methotrexate compositions, wherein liposomes encapsulate gamma polyglutamated methotrexate, or a salt or acid thereof, and one or more aqueous pharmaceutically acceptable carriers. In some embodiments, the interior space of the liposomes contains trehalose. In some embodiments, the interior space of the liposomes contains 5% to 20% (wt%) trehalose. In some embodiments, the interior space of the liposomes contains HBS at a concentration of 1 to 200 mM and a pH of 2 to 8. In some embodiments, the interior space of the liposomes has a pH of 5 to 8, or any range therebetween. In some embodiments, the interior space of the liposomes has a pH of 6 to 7, or any range therebetween. In some embodiments, the interior space of the liposomes contains a combined concentration of sodium acetate and calcium acetate of 50 mM to 500 mM, or any range therebetween.

[0217] A. Non-polyglutamylated polyglutamylated antifolates In some embodiments, liposomal gamma polyglutamated methotrexate (e.g., Lp-γPMTX, including PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX) compositions comprise gamma polyglutamated methotrexate (e.g., γPMTX disclosed herein) and one or more non-polyglutamylated polyglutamylatable antifolate compositions.

[0218] In some embodiments, Lp-γPMTX (e.g., PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX) comprises gamma polyglutamated methotrexate (e.g., the γPMTX disclosed herein) and methotrexate (MTX). In some embodiments, Lp-γPMTX (i.e., liposomal gamma polyglutamated methotrexate) comprises gamma polyglutamated methotrexate and a polyglutamylatable antifolate selected from the group consisting of methotrexate (MTX), pemetrexed (PMX), lometrexol (LMX), raltitrexed (RTX), pralatrexate, AG2034, GW1843, aminopterin, and LY309887. In some embodiments, Lp-γPMTX comprises gamma polyglutamated methotrexate and lometrexol. In some embodiments, Lp-γPMTX comprises gamma polyglutamated methotrexate and pemetrexed. In some embodiments, Lp-γPMTX comprises gamma polyglutamated methotrexate and leucovorin. In some embodiments, Lp-γPMTX comprises gamma polyglutamated methotrexate and a triazine antifolate derivative (e.g., a sulfonyluride triazine such as NSC127755). In some embodiments, Lp-γPMTX comprises gamma polyglutamated methotrexate and a serine hydroxymethyltransferase (SHMT2) inhibitor. In some embodiments, the SHMT2 inhibitor is an antifolate (e.g., a polyglutamylatable or non-polyglutamylatable antifolate). In some embodiments, the SHMT2 inhibitor is an antifolate.

[0219] B. Non-polyglutamylatable antifolates In some embodiments, Lp-γPMTX (e.g., PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX) comprises gamma polyglutamated methotrexate (e.g., γPMTX disclosed herein) and a so-called "non-polyglutamylatable" antifolate. In some embodiments, the liposome comprises gamma polyglutamated methotrexate and a non-polyglutamylatable antifolate that inhibits one or more enzymes in the folate cycle metabolic pathway. In further embodiments, the non-polyglutamylatable antifolate inhibits one or more enzymes selected from thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide (GAR) transformylase, and aminoimidazolecarboxamide ribonucleotide (AICAR) transformylase. In some embodiments, the liposomes comprise gamma-polyglutamated methotrexate and a non-polyglutamated antifolate that inhibits DHFR. In some embodiments, the liposomes comprise gamma-polyglutamated methotrexate and a non-polyglutamated antifolate that inhibits TS. In some embodiments, the liposomes comprise gamma-polyglutamated methotrexate and a non-polyglutamated antifolate that inhibits GAR or AICAR transformylase. In further embodiments, the non-polyglutamated antifolate is selected from the group consisting of trimetrexate (TMQ), piritrexim (BW301U), and tarotrexin (PT523). In further embodiments, the non-polyglutamylatable antifolate is selected from the group consisting of nolatrexed (AG337), previtrexed (ZD9331, BGC9331), BGC945 (ONX0801).

[0220] C platinum In some embodiments, liposomes comprising gamma polyglutamated methotrexate (e.g., Lp-γPMTX, such as PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX) comprise gamma polyglutamated methotrexate (e.g., γPMTX disclosed herein) and a platinum-based chemotherapeutic agent or a salt or acid thereof. In some embodiments, the liposomes comprise a gamma polyglutamated methotrexate / platinum-based drug conjugate (e.g., as described in Section IIC).

[0221] In some embodiments, the Lp-γPMTX comprises a platinum-based chemotherapeutic agent selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or a salt or acid thereof. In other embodiments, the Lp-γPMTX comprises an analog of a platinum-based chemotherapeutic agent selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or a salt or acid thereof.

[0222] In some embodiments, Lp-γPMTX comprises gamma polyglutamated methotrexate and cisplatin, or a salt or acid thereof. In some embodiments, Lp-γPMTX comprises gamma polyglutamated methotrexate and a cisplatin analog, or a salt or acid thereof.

[0223] In some embodiments, the Lp-γPMTX comprises gamma polyglutamated methotrexate and carboplatin, or a salt or acid thereof. In some embodiments, the liposome comprises gamma polyglutamated methotrexate and a carboplatin analog, or a salt or acid thereof.

[0224] In some embodiments, the Lp-γPMTX comprises gamma polyglutamated methotrexate and oxaliplatin, or a salt or acid thereof. In some embodiments, the liposome comprises gamma polyglutamated methotrexate and an oxaliplatin analog, or a salt or acid thereof.

[0225] In some embodiments, the liposome comprises gamma polyglutamated methotrexate (e.g., γPMTX disclosed herein) and a platinum-based chemotherapeutic agent selected from the group consisting of nedaplatin, heptaplatin, and lobaplatin, or a salt or acid thereof. In some embodiments, Lp-γPMTX comprises gamma polyglutamated methotrexate and an analog of a platinum-based chemotherapeutic agent selected from the group consisting of nedaplatin, heptaplatin, and lobaplatin, or a salt or acid thereof.

[0226] In some embodiments, the Lp-γPMTX comprises gamma polyglutamated methotrexate and a platinum-based chemotherapeutic agent selected from the group consisting of stratoplatin, paraplatin, platinol, cycloplatin, dexorumaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamines, satraplatin, enloplatin, JM216, 254-S, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or a salt or acid thereof. In some embodiments, Lp-γPMTX comprises gamma polyglutamated methotrexate and an analog of a platinum-based chemotherapeutic agent selected from the group consisting of stratoplatin, paraplatin, platinol, cycloplatin, dexorumaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamines, satraplatin, enloplatin, JM216, 254-S, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or a salt or acid thereof.

[0227] In some embodiments, the liposome composition comprises a liposome further comprising one or more of an immunostimulatory agent, a detectable marker, and a maleimide disposed on at least one of the PEG or exterior surface of the liposome.

[0228] D Cyclodextrin In further embodiments, the γPMTX liposome comprises γPMTX (eg, a γPMTX disclosed herein) and a cyclodextrin (eg, a cyclodextrin in Section IIC herein).

[0229] In some embodiments, the γPMTX liposome comprises a complex formed by a cyclodextrin and a therapeutic agent. 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 drug. In further embodiments, the therapeutic agent of the cyclodextrin / therapeutic agent complex is a member selected from the group consisting of gemcitabine, a gemcitabine-based therapeutic agent, doxorubicin, an antifolate, an antifolate chemotherapeutic agent, or a salt or acid, acid form, or free base form thereof. In further embodiments, the molar ratio of cyclodextrin to therapeutic agent in the complex is in the range of 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to therapeutic agent in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to therapeutic agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50.

[0230] In some embodiments, the γPMTX liposome comprises γPMTX and a cyclodextrin / platinum-based chemotherapeutic agent complex. In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the cyclodextrin / platinum-based chemotherapeutic agent complex comprises an analog of cisplatin, carboplatin, oxaliplatin, or salts or acids thereof. In some embodiments, the molar ratio of cyclodextrin to platinum-based agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to platinum-based drug in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to platinum-based drug in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to platinum-based agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50.

[0231] In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the cyclodextrin / platinum-based chemotherapeutic agent complex comprises an analog of cisplatin, carboplatin, oxaliplatin, or salts or acids thereof. In some embodiments, the molar ratio of cyclodextrin to platinum-based agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to platinum-based agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to platinum-based drug in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to platinum-based drug is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the cyclodextrin / platinum-based drug complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0232] In further embodiments, the present disclosure provides a complex comprising a cyclodextrin and cisplatin, or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin to cisplatin (or a cisplatin salt or acid) in the complex is in the range of 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to cisplatin (or a cisplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to cisplatin (or cisplatin salt or acid) in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to cisplatin (or cisplatin salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the cyclodextrin / cisplatin (or cisplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0233] In another embodiment, the present disclosure provides a complex comprising a cyclodextrin and carboplatin, or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin to carboplatin (or a carboplatin salt or acid) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to carboplatin (or a carboplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to carboplatin (or carboplatin salt or acid) in the complex is 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to carboplatin (or carboplatin salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In a further embodiment, the cyclodextrin / carboplatin (or carboplatin salt or acid) complex is encapsulated in a liposome (eg, as described herein or otherwise known in the art).

[0234] In another embodiment, the present disclosure provides a complex comprising a cyclodextrin and oxaliplatin, or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin to oxaliplatin (or oxaliplatin salt or acid) in the complex is in the range of 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to oxaliplatin (or oxaliplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to oxaliplatin (or oxaliplatin salt or acid) in the complex is 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to oxaliplatin (or oxaliplatin salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In a further embodiment, the cyclodextrin / oxaliplatin (or oxaliplatin salt or acid) complex is encapsulated in a liposome (eg, as described herein or otherwise known in the art).

[0235] In a further embodiment, the present disclosure provides a conjugate comprising a cyclodextrin and a platinum-based chemotherapeutic agent selected from the group consisting of nedaplatin, heptaplatin, lobaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexorumaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamines, satraplatin, enloplatin, JM216, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or a salt or acid thereof. In other embodiments, the cyclodextrin / platinum-based chemotherapeutic agent complex comprises nedaplatin, heptaplatin, lobaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexorumaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enloplatin, JM216, NK121, CI973, DWA2114R, NDDP, or nedaplatin, or a salt or acid analog thereof. In some embodiments, the molar ratio of cyclodextrin to oxaliplatin (or oxaliplatin salt or acid) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to platinum-based chemotherapeutic agent (or salt or acid or analog thereof) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to platinum-based chemotherapeutic agent (or salt or acid or analog thereof) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween.In some embodiments, the molar ratio of cyclodextrin / platinum-based chemotherapeutic agent (or salt or acid or analog thereof) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the cyclodextrin / platinum-based chemotherapeutic agent (or salt or acid or analog thereof) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0236] In some embodiments, the present disclosure provides a composition comprising a cyclodextrin / taxane chemotherapeutic agent complex. In some embodiments, the taxane chemotherapeutic agent is selected from the group consisting of paclitaxel (PTX), docetaxel (DTX), larotaxel (LTX), and cabazitaxel (CTX), or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin to taxane in the complex is in the range of 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to 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, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to taxane in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to taxane is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the cyclodextrin / taxane complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0237] In further embodiments, the present disclosure provides a complex comprising a cyclodextrin and paclitaxel (PTX) or a salt or acid thereof. In other embodiments, the cyclodextrin / taxane chemotherapeutic agent complex comprises paclitaxel (PTX), or a salt or acid analog thereof. In some embodiments, the molar ratio of cyclodextrin to paclitaxel (or a salt or acid of paclitaxel) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to paclitaxel (or paclitaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to paclitaxel (or paclitaxel salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / paclitaxel (or paclitaxel salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the cyclodextrin / paclitaxel (or paclitaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0238] In further embodiments, the present disclosure provides a complex comprising a cyclodextrin and docetaxel (DTX) or a salt or acid thereof. In other embodiments, the cyclodextrin / taxane chemotherapeutic agent complex comprises docetaxel (DTX), or a salt or acid analog thereof. In some embodiments, the molar ratio of cyclodextrin to docetaxel (or a salt or acid of docetaxel) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to docetaxel (or docetaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to docetaxel (or docetaxel salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to docetaxel (or docetaxel salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the cyclodextrin / docetaxel (or docetaxel salt or acid) complex is encapsulated in a liposome...

Claims

[Claim 1] 1. A liposome composition comprising a liposome encapsulating gamma polyglutamated methotrexate and one or more non-polyglutamylatable antifolate compositions or non-polyglutamylatable antifolates, wherein the gamma polyglutamated methotrexate comprises 2 to 15 glutamyl groups with gamma carboxyl linkages; (a) at least two of the glutamyl groups of the gamma polyglutamated methotrexate are in the L-configuration; (b) each of the glutamyl groups of the gamma polyglutamated methotrexate is in the L-configuration; (c) at least one of the glutamyl groups of the gamma polyglutamated methotrexate is in the D form; (d) each of the glutamyl groups of the gamma polyglutamylated methotrexate other than the glutamyl groups of methotrexate is in the D form; or (e) at least two of the glutamyl groups of the gamma polyglutamated methotrexate are in the L-configuration and at least one of the glutamyl groups is in the D-configuration; A liposome composition, wherein the liposomes have a diameter of 50 to 150 nm.

Images