Alpha polyglutamated methotrexate and uses thereof

Abstract

To provide alpha polyglutamated methotrexate and formulations to treat hyperproliferative disorders (e.g., cancer) and disorders of the immune system (e.g., autoimmune diseases such as rheumatoid arthritis).SOLUTION: There is provided a liposomal composition comprising alpha polyglutamated methotrexate and a liposome encapsulating one or more non-polyglutamated, polyglutamatable antifolates 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 alpha polyglutamated methotrexate compositions, including delivery vehicles such as liposomes containing the alpha polyglutamated methotrexate compositions, and methods of making and using the compositions to treat diseases, including hyperproliferative diseases such as cancer, immune system disorders including inflammatory and autoimmune diseases 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 polyglutamylatable 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 alpha 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 alpha 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 novel alpha 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.

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

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

[11] The composition according to any one of items [1] to [9], wherein the polyglutamate is branched;

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

[11] ;

[13] The LαPP composition according to item

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

[14] The Lp-αPMTX composition according to item

[12] or

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

[15] The Lp-αPMTX composition according to item

[12] or

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

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

[12] to

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

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

[12] to

[16] , wherein at least one glutamyl group of the alpha polyglutamylated methotrexate has a gamma carboxyl group bond;

[18] The composition according to any one of items

[12] to

[17] , wherein at least one glutamyl group has both an alpha carboxyl group bond and a gamma carboxyl group bond;

[19] The composition according to any one of items

[12] to

[18] , comprising 2, 3, 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups having both alpha carboxyl group bonds and gamma carboxyl group bonds;

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

[22] , wherein the liposome is PEGylated (PαLp-αPMTX);

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

[12] to

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

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

[12] to

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

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

[12] to

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

[28] The Lp-αPMTX composition according to item

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

[29] The Lp-αPMTX 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-αPMTX 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-αPMTX composition according to any one of items

[27] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

[33] , wherein the liposome is cationic;

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

[12] to

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

[40] The Lp-αPMTX composition of 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-αPMTX composition of item

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

[42] The Lp-αPMTX composition according to item

[41] , wherein the pharmaceutically acceptable carrier comprises 5% by weight to 20% by weight of trehalose;

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

[39] to

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

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

[12] to

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

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

[12] to

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

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

[50] or

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

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

[50] to

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

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

[50] to

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

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

[39] to

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

[59] The Lp-αPMTX 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-αPMTX 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), and oxidized low-density lipoproteins (e.g., OXPAC, PGPC), and toll-like receptor (TLR) modulators such as eritran lipids (e.g., E5564);

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

[58] to

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

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

[58] to

[61] , further comprising a hapten;

[63] The Lp-αPMTX composition of item

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

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

[12] to

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

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

[12] to

[67] ;

[69] A pharmaceutical composition comprising the alpha 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, comprising administering to the subject the liposomal alpha 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 alpha 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 alpha 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 breast cancer, advanced 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 (demenoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma;

[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 a sarcoma such as osteosarcoma;

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

[89] A method for treating an infectious disease, comprising administering an effective amount of the liposomal alpha 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 alpha polyglutamated methotrexate to a tumor expressing a folate receptor on its surface, comprising administering to a subject having a tumor the Lp-αPMTX composition described in any one of items [1] to

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

[91] A method for making an alpha polyglutamated methotrexate composition, including the liposomal alpha polyglutamated methotrexate composition according to any one of items

[12] to

[69] , comprising the steps of: forming a mixture in a solution containing liposome components and an alpha polyglutamated antifolate; homogenizing the mixture in the solution to form liposomes; and treating the mixture to form liposomes containing alpha 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 alpha-polyglutamated methotrexate; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes that encapsulate and / or entrap the alpha-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 an alpha polyglutamated methotrexate (αPMTX) composition, wherein at least one glutamyl residue of the alpha polyglutamated methotrexate is linked via its alpha carboxyl group. In some embodiments, the αPMTX contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl group of methotrexate). In some embodiments, the αPMTX contains two or more L-glutamyl groups. In other embodiments, the αPMTX contains a D-glutamyl group. In further embodiments, the αPMTX contains a D-glutamyl group and two or more L-glutamyl groups. In further embodiments, the αPMTX contains two or more glutamyl groups with gamma linkages. In some embodiments, at least one glutamyl group has both alpha and gamma linkages.

[0014] In one embodiment, the αPMTX 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. In a further embodiment, the tetraglutamated MTX comprises two or more glutamyl groups with gamma bonds.

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

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

[0017] In further embodiments, the present disclosure provides compositions comprising delivery vehicles, such as liposomes, loaded (i.e., encapsulated) and / or otherwise bound to alpha polyglutamated methotrexate, as well as methods for making and using αPMTX-loaded / bound delivery vehicle compositions to deliver alpha 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, for example, hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria. αPMTX-loaded / bound delivery vehicle compositions provide selective delivery of a more cytotoxic payload (polyglutamated 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-αPMTX) comprising liposomes encapsulating (loaded with) alpha polyglutamated methotrexate. In some embodiments, the alpha polyglutamated methotrexate in the Lp-αPMTX comprises 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 20 glutamyl groups (including the glutamyl groups of methotrexate). In some embodiments, the alpha polyglutamated methotrexate in the Lp-αPMTX comprises two or more L-glutamyl groups. In other embodiments, the alpha polyglutamated methotrexate in the Lp-αPMTX comprises a D-glutamyl group. In further embodiments, the alpha polyglutamated methotrexate in the Lp-αPMTX comprises a D-glutamyl group and two or more L-glutamyl groups. In further embodiments, the alpha polyglutamated methotrexate in Lp-αPMTX contains two or more glutamyl groups with gamma linkages. In further embodiments, the alpha polyglutamated methotrexate in Lp-αPMTX contains one or more glutamyl groups with both alpha and gamma linkages. In some embodiments, the alpha polyglutamated methotrexate in Lp-αPMTX contains 2 to 10 glutamyl groups with both alpha and gamma linkages, or any range therebetween. In some embodiments, the polyglutamate chain of the alpha polyglutamated methotrexate is linear. In some embodiments, the polyglutamate chain of the alpha polyglutamated methotrexate is branched.

[0019] In one embodiment, the Lp-αPMTX composition comprises alpha polyglutamylated MTX comprising a chain of three glutamyl groups linked to a glutamyl group of methotrexate (i.e., tetraglutamated 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. In a further embodiment, the tetraglutamylated MTX comprises two or more glutamyl groups with gamma linkages. In some embodiments, the polyglutamate chain of the alpha polyglutamylated methotrexate is linear. In some embodiments, the polyglutamate chain of the alpha polyglutamylated methotrexate is branched.

[0020] In one embodiment, the Lp-αPMTX composition comprises alpha polyglutamylated MTX comprising a chain of four glutamyl groups linked to a glutamyl group of methotrexate (i.e., pentaglutamated methotrexate). In some embodiments, the pentaglutamylated MTX comprises two or more L-glutamyl groups. In other embodiments, the pentaglutamylated MTX comprises a D-glutamyl group. In a further embodiment, the pentaglutamylated MTX comprises a D-glutamyl group and two or more L-glutamyl groups. In a further embodiment, the pentaglutamylated MTX comprises two or more glutamyl groups with gamma linkages. In some embodiments, the polyglutamate chain of the alpha polyglutamylated methotrexate is linear. In some embodiments, the polyglutamate chain of the alpha polyglutamylated methotrexate is branched.

[0021] In one embodiment, the Lp-αPMTX composition comprises alpha polyglutamylated MTX comprising a chain of five glutamyl groups linked to a glutamyl group of methotrexate (i.e., hexaglutamated methotrexate). In some embodiments, the hexaglutamylated MTX comprises two or more L-glutamyl groups. In other embodiments, the hexaglutamylated MTX comprises a D-glutamyl group. In a further embodiment, the hexaglutamylated MTX comprises a D-glutamyl group and two or more L-glutamyl groups. In a further embodiment, the hexaglutamylated MTX comprises two or more glutamyl groups with gamma linkages. In some embodiments, the polyglutamate chain of the alpha polyglutamated methotrexate is linear. In some embodiments, the polyglutamate chain of the alpha polyglutamated methotrexate is branched.

[0022] In some embodiments, the Lp-αPMTX composition is cationic. In some embodiments, the Lp-αPMTX 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-αPMTX 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-αPMTX 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 alpha-polyglutamated MTX. In some embodiments, during the process of making Lp-αPMTX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the alpha-polyglutamated MTX starting material is encapsulated in cationic Lp-αPMTX. In further embodiments, the alpha-polyglutamated methotrexate encapsulated by the liposomes is present in a HEPES buffer solution within the liposomes.

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

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

[0025] In some embodiments, the liposomal alpha polyglutamated methotrexate composition is non-targeted (NTLp-αPMTX). That is, the NTLp-αPMTX 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 alpha polyglutamated methotrexate composition is pegylated (NTPLp-αPMTX).

[0026] In other embodiments, the liposomal alpha-polyglutamated methotrexate compositions are targeted (TLp-αPMTX). That is, the TLp-αPMTX compositions include 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-αPMTX or TPLp-αPMTX is not covalently attached to the liposome. In other embodiments, the targeting moiety of the TLp-αPMTX or TPLp-αPMTX is attached to one or both of the PEG and the exterior surface of the liposome. Targeted liposomal alpha-polyglutamated methotrexate compositions (TLp-αPMTX and TPLp-αPMTX) offer further improvements over the efficacy and safety profile of methotrexate by specifically delivering alpha-polyglutamated (e.g., tetraglutamated, pentaglutamated, and hexaglutamated) methotrexate to target cells, such as cancer cells. In a further embodiment, the targeted liposomal alpha polyglutamated methotrexate composition is PEGylated (TPLp-αPMTX). The functions of the targeting moiety of the TLp-αPMTX and / or TPLp-αPMTX composition include, but are not limited to, targeting the liposome to a desired target cell in vivo or in vitro; interacting with a surface antigen to which the targeting moiety has specific affinity; and delivering the liposomal payload (αPMTX) to the cell.

[0027] Suitable targeting moieties are known in the art and include, but are not limited to, antibodies, antigen-binding antibody fragments, scaffold proteins, polypeptides, and peptides. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is a polypeptide comprising at least 3, 5, 10, 15, 20, 30, 40, 50, or 100 amino acid residues. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In further embodiments, the targeting moiety comprises one or more of an antibody, a humanized antibody, an antigen-binding fragment of an antibody, a single-chain antibody, a single-domain antibody, a bispecific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody. In some embodiments, the targeting moiety 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 nucleotide sequence greater than or equal to 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 comprises a polypeptide that specifically binds to a folate receptor. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the folate receptor bound by the targeting moiety is one or more folate receptors selected from the group consisting of folate receptor alpha (FR-α, FOLR1), folate receptor beta (FR-β, FOLR2), and folate receptor delta (FR-δ, FOLR4). In some embodiments, the folate receptor bound by the targeting moiety is folate receptor alpha (FR-α). In some embodiments, the folate receptor bound by the targeting moiety is folate receptor beta (FR-β). In some embodiments, the targeting moiety specifically binds to FR-α and FR-β.

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

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

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

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

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

[0034] In some embodiments, the liposome composition comprises liposomal αPMTX (e.g., Lp-αPMTX, PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, and TPLp-αPMTX). In some embodiments, the liposomal αPMTX is pegylated (e.g., NTPLp-αPMTX and TPLp-αPMTX). In some embodiments, the liposomal αPMTX 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-αPMTX or TPLp-αPMTX). In further embodiments, the liposome composition comprises pegylated liposomal αPMTX 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-αPMTX). In some embodiments, the liposome composition comprises cationic liposomal αPMTX. In other embodiments, the liposome composition comprises anionic or neutral liposomal αPMTX. In further embodiments, the liposome composition comprises liposomal αPMTX having a diameter of 20 nm to 500 nm, 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposomal αPMTX has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

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

[0036] In some embodiments, the pharmaceutical composition comprises liposomal αPMTX (e.g., Lp-αPMTX, PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, and TPLp-αPMTX). In some embodiments, the liposomal αPMTX composition is pegylated (e.g., NTPLp-αPMTX and TPLp-αPMTX). In some embodiments, the liposomal αPMTX 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-αPMTX or TPLp-αPMTX). In further embodiments, the pharmaceutical composition comprises a pegylated liposomal αPMTX 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-αPMTX). In some embodiments, the pharmaceutical composition comprises cationic liposomal αPMTX. In other embodiments, the pharmaceutical composition comprises anionic or neutral liposomal αPMTX. In a further embodiment, the pharmaceutical composition comprises liposomal αPMTX 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 αPMTX 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 for modulating cellular activation, chemokine production, or metabolic activity, the method comprising contacting a cell with a composition comprising an alpha polyglutamated methotrexate (αPMTX) composition. In some embodiments, the contacted cell is a mammalian cell. In further embodiments, the contacted cell is a human cell. In some embodiments, the contacted cell is a hyperproliferative cell. In further embodiments, the cell is an immune cell. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the αPMTX comprises 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the αPMTX composition comprises alpha tetraglutamated methotrexate. In some embodiments, the αPMTX composition comprises alpha pentaglutamated methotrexate. In other embodiments, the αPMTX composition comprises alpha hexaglutamated methotrexate.

[0038] In further embodiments, the present disclosure provides a method for modulating cellular activation, chemokine production, or metabolic activity, the method comprising contacting a cell with a liposome comprising an alpha polyglutamated methotrexate (αPMTX) composition. In some embodiments, the contacted cell is a mammalian cell. In further embodiments, the contacted cell is a human cell. In some embodiments, the contacted cell is a hyperproliferative cell. In further embodiments, the cell is an immune cell. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the αPMTX comprises 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the αPMTX composition comprises alpha tetraglutamated methotrexate. In some embodiments, the αPMTX composition comprises alpha pentaglutamated methotrexate. In other embodiments, the αPMTX composition comprises alpha hexaglutamated methotrexate.

[0039] In further embodiments, the present disclosure provides a method of killing a cell, the method comprising contacting the cell with a composition comprising an alpha polyglutamated methotrexate (αPMTX) composition. In some embodiments, the contacted cell is a mammalian cell. In further embodiments, the contacted cell is a human cell. In some embodiments, the contacted cell is a hyperproliferative cell. In further embodiments, the 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 still further embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from 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, nonleukemic meningeal carcinomatosis, soft tissue sarcoma (dsemoid, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the αPMTX comprises 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the αPMTX composition comprises alpha-tetraglutamated methotrexate. In some embodiments, the αPMTX composition comprises alpha-pentaglutamated methotrexate. In other embodiments, the αPMTX composition comprises alpha hexaglutamated methotrexate.

[0040] In further embodiments, the present disclosure provides a method of killing a cell, the method comprising contacting the cell with a liposome comprising alpha polyglutamylated methotrexate (e.g., Lp-αPMTX, such as PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, or TPLp-αPMTX). 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 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 still further embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from 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 method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the liposome comprises αPMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome comprises alpha-tetraglutamated methotrexate. In some embodiments, the liposome comprises alpha-pentaglutamated methotrexate. In other embodiments, the liposome comprises alpha hexaglutamated methotrexate.

[0041] 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 alpha 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-αPMTX, such as PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, or TPLp-αPMTX). 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 that specifically binds to 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, FZD 6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD2 2, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD 105, CD133, CD138, criptine, 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) ), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR 1, PDGFRα, PDGFRβ, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery vehicle comprises a targeting moiety that specifically binds to a cell surface antigen, such as a neoantigen, identified as being derived from or expressed by a specific target cancer (tumor). In some embodiments, the targeting moiety specifically binds to a cell surface antigen, such as a neoantigen, identified as being derived from or expressed by 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 αPMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises alpha tetraglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises alpha pentaglutamated methotrexate. In other embodiments, the administered delivery vehicle comprises alpha hexaglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises L-alpha polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises D-alpha polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises L- and D-alpha polyglutamated methotrexate. In some embodiments, the cancer is selected from the group consisting of non-blood cancers, 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 blood cancers, such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. In still further embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, and villous adenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (demenoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma.

[0042] In further embodiments, the present disclosure provides methods of treating cancer, the methods comprising administering an effective amount of a liposome comprising alpha polyglutamated methotrexate (e.g., Lp-αPMTX, such as PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, or TPLp-αPMTX) 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 that specifically binds to 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, FZD 6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD2 2, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD 105, CD133, CD138, criptine, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4, EphB6, integrins (e.g., integrin αvβ3, αvβ5, or αvβ6) ), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR 1, PDGFRα, PDGFRβ, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.This also includes the use of cancer stem cell targeting moieties, such as those targeting CD34, CD133 and CD44, CD138, and CD15. In some embodiments, the liposomes contain a targeting moiety that specifically binds to a cell surface antigen determined to be derived 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 αPMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes contain alpha tetraglutamated methotrexate. In some embodiments, the liposomes contain alpha pentaglutamated methotrexate. In other embodiments, the liposomes contain alpha hexaglutamated methotrexate. In some embodiments, the liposomes contain L-alpha polyglutamated methotrexate. In some embodiments, the liposomes contain D-alpha polyglutamated methotrexate. In some embodiments, the liposome comprises L- and D-alpha polyglutamated methotrexate. 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 blood tumors (e.g., leukemia or lymphoma).

[0043] In a further embodiment, the present disclosure provides a method of treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a liposome composition comprising alpha 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 specifically binds to 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, FZD 6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD2 2, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD 105, CD133, CD138, criptine, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4, EphB6, integrins (e.g., integrin αvβ3, αvβ5, or αvβ6) ), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR 1, PDGFRα, PDGFRβ, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the administered liposomes contain a targeting moiety that specifically binds to a cell surface antigen determined to be derived from or expressed on a particular target tumor, such as a neoantigen. In some embodiments, the administered liposome composition contains PEGylated liposomes (e.g., TPLp-αPMTX). In some embodiments, the administered liposome composition contains non-PEGylated liposomes. In some embodiments, the administered liposomes contain αPMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered liposomes contain alpha-tetraglutamated methotrexate. In some embodiments, the administered liposomes contain alpha-pentaglutamated methotrexate. In other embodiments, the administered liposomes contain alpha-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 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, myeloma and other plasma cell dysplasias or cachexia, and leukemia, lymphoma and other B-cell malignancies. In still further embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, and villous adenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (demenoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma.

[0044] In further embodiments, the present disclosure provides a method for treating cancer, comprising administering an effective amount of a liposome composition to a subject having or at risk of having a cancer that expresses a folate receptor on its cell surface, the liposome composition comprising (a) alpha polyglutamated methotrexate (αPMTX) and (b) a liposome comprising a targeting moiety having specific binding affinity for the folate receptor. In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α) and folate receptor beta (FR-β). In some embodiments, the administered liposome composition comprises a pegylated liposome (e.g., TPLp-αPMTX). In some embodiments, the administered liposome composition comprises a non-pegylated liposome. In some embodiments, the liposomes of the administered liposome composition comprise αPMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutamated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutamated methotrexate. In some embodiments, the liposome compositions are administered to treat cancers 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 still further embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, and villous adenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (demenoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma.

[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 alpha polyglutamated methotrexate (Lp-αPMTX) to a subject undergoing or who has undergone cancer therapy. In some embodiments, the administered liposome composition is PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, or TPLp-αPMTX. In some embodiments, the administered liposome composition comprises PEGylated liposomes (e.g., PLp-αPMTX, NTPLp-αPMTX, or TPLp-αPMTX). In some embodiments, the administered liposome composition comprises targeted liposomes (e.g., TLp-αPMTX or TPLp-αPMTX). In some embodiments, the administered liposome composition comprises PEGylated and targeted liposomes (e.g., TPLp-αPMTX). In some embodiments, the liposomes of the administered liposome composition comprise alpha polyglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha pentaglutamated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha 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 a liposome comprising alpha polyglutamated methotrexate (e.g., Lp-αPMTX, PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, or TPLp-αPMTX) 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 further embodiments, the liposome composition is administered to treat rheumatoid arthritis. In some embodiments, the administered liposome composition comprises a pegylated liposome (e.g., PLp-αPMTX, NTPLp-αPMTX, or TPLp-αPMTX). In some embodiments, the administered liposome composition comprises a targeted liposome (e.g., TLp-αPMTX or TPLp-αPMTX) comprising a targeting moiety having specific affinity for a surface antigen on a target cell (e.g., an immune cell) of interest. In further embodiments, the administered liposome composition comprises pegylated and targeted liposomes (e.g., TPLp-αPMTX). In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutamated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutamated methotrexate. In some embodiments, the immune system disorder is selected from inflammation (e.g., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, systemic lupus erythematosus, and Takayasu's disease, and psoriasis.

[0047] In further embodiments, the present disclosure provides a method of treating an autoimmune disease, the method comprising administering to a subject having or at risk of having an inflammatory disease an effective amount of a liposome composition comprising liposomes containing alpha polyglutamated methotrexate (e.g., Lp-αPMTX, PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, or TPLp-αPMTX). In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., PLp-αPMTX, NTPLp-αPMTX, or TPLp-αPMTX). In some embodiments, the administered liposome composition comprises targeted liposomes (e.g., TLp-αPMTX or TPLp-αPMTX) comprising a targeting moiety having specific affinity for a surface antigen on a target cell (e.g., an immune cell) of interest. In further embodiments, the administered liposome composition comprises pegylated and targeted liposomes (e.g., TPLp-αPMTX). In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutamated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutamated methotrexate. In some embodiments, the autoimmune disorder is selected from rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, systemic lupus erythematosus, and psoriasis.

[0048] In further embodiments, the present disclosure provides methods for treating an inflammatory disease, the method comprising administering to a subject having or at risk of having an inflammatory disease an effective amount of a liposome composition comprising liposomes containing alpha polyglutamated methotrexate (e.g., Lp-αPMTX, PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, or TPLp-αPMTX). In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., PLp-αPMTX, NTPLp-αPMTX, or TPLp-αPMTX). In some embodiments, the administered liposome composition comprises targeted liposomes (e.g., TLp-αPMTX or TPLp-αPMTX) comprising a targeting moiety having specific affinity for a surface antigen on a target cell of interest (e.g., an immune cell). In further embodiments, the administered liposome composition comprises pegylated and targeted liposomes (e.g., TPLp-αPMTX). In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutamated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutamated methotrexate. In some embodiments, the inflammatory disease is selected from acute inflammation, chronic inflammation, systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, and systemic lupus erythematosus.

[0049] The present disclosure also provides a method for delivering alpha polyglutamated methotrexate to a site of inflammation in a subject, the method comprising administering to a subject with inflammation a composition comprising alpha polyglutamated methotrexate (L-αPMTX) and a targeting moiety having specific binding affinity for an epitope on a surface antigen of a cell at the site of inflammation or that otherwise affects the inflammation (e.g., via pro-inflammatory cytokine production). 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-αPMTX). In some embodiments, the administered composition comprises alpha polyglutamated methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the administered composition comprises alpha tetraglutamated methotrexate. In some embodiments, the composition administered comprises alpha-pentaglutamated methotrexate, hi other embodiments, the composition administered comprises alpha-hexaglutamated methotrexate.

[0050] The present disclosure also provides a method for delivering alpha polyglutamated methotrexate to tumor or cancer cells, the method comprising administering to a subject having a tumor a composition comprising alpha polyglutamated methotrexate (L-αPMTX) 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-αPMTX). In some embodiments, the administered composition comprises alpha polyglutamated methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the administered composition comprises alpha tetraglutamated methotrexate. In some embodiments, the composition administered comprises alpha-pentaglutamated methotrexate, hi other embodiments, the composition administered comprises alpha-hexaglutamated methotrexate.

[0051] In further embodiments, the present disclosure provides a method for making a liposome composition, including a liposomal alpha polyglutamated methotrexate (αPMTX) composition, comprising: forming a mixture in solution containing liposome components and α polyglutamated methotrexate; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes containing polyglutamate methotrexate. In some embodiments, the alpha polyglutamated methotrexate contains 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the polyglutamated methotrexate composition comprises alpha tetraglutamated methotrexate. In some embodiments, the polyglutamated methotrexate composition comprises alpha pentaglutamated methotrexate. In other embodiments, the polyglutamated methotrexate composition comprises alpha hexaglutamated methotrexate.

[0052] In one embodiment, the present disclosure provides a kit comprising an alpha polyglutamated methotrexate composition and / or an alpha PMTX delivery vehicle, such as a liposome, comprising alpha PMTX and an alpha PMTX immunoconjugate (e.g., an ADC described herein). [Brief explanation of the drawings]

[0053] [Figure 1]1A-1L show the chemical formulas of methotrexate (FIG. 1A), representative alpha methotrexate alpha polyglutamates: methotrexate diglutamate (FIG. 1B), methotrexate triglutamate (FIGS. 1C and 1D), methotrexate tetraglutamate (FIGS. 1E and 1F), methotrexate pentaglutamate (FIGS. 1G and 1H), methotrexate hexaglutamate (FIGS. 1I and 1J), methotrexate heptaglutamate (FIGS. 1K and 1L), methotrexate octaglutamate (FIGS. 1M and 1N), representative alpha methotrexate polyglutamate (FIG. 1O), and representative methotrexate analogs (FIGS. 1P and 1Q). Figures 1R-1U show the structures of representative branched methotrexate polyglutamates, including a branched polyglutamate with a gamma-glutamyl backbone and alpha-glutamyl branching portions (Figure 1S) and a branched polyglutamate with an alpha-glutamyl backbone and gamma-glutamyl branching portions (Figure 1T). [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above. [Figure 1-6] Same as above. [Figure 1-7] Same as above. [Figure 2] Figure 1 shows the relative efficacy of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6) and its enantiomer liposomal alpha-D hexaglutamate (liposomal aDG6) compared to pemetrexed after 48 hours of exposure to the cancer cell lines SW620 (CRC), HT-29 (colon cancer), H1806 (triple-negative breast cancer), OAW28 (ovarian cancer), H292 (NSCLC, adenocarcinoma subtype), and H2342 (NSCLC, adenocarcinoma subtype). [Figure 3]Figure 1 shows an example of the dose-response relationship expressed as the percentage of viable cells after 48 hours of treatment for free pemetrexed L-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha-targeted antibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6-FR1Ab) in NCI H2342 non-small cell lung cancer (NSCLC), adenocarcinoma subtype. Folate receptor alpha-targeted liposomes containing alpha polyglutamated pemetrexed are predicted to successfully target NCI H2342 non-small cell lung cancer cells and reduce their viability. [Figure 4] Figure 1 shows 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-targeted antibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6-FR1Ab) in HT-29 (colon cancer) cells. Folate receptor alpha-targeted liposomes containing alpha-polyglutamated pemetrexed are also predicted to successfully target HT-29 (colon cancer) cells and reduce viability. [Figure 5] 1 shows the therapeutic efficacy of liposomal pemetrexed alpha-L hexaglutamate (Lps Hexa aG6), liposomal pemetrexed alpha-D hexaglutamate (Lps Hexa aDG6), and pemetrexed against HCC1806 triple-negative breast cancer after 48 hours of exposure. [Figure 6] 1 shows the therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (LPS Hexa aG6), liposomal pemetrexed alpha-D hexaglutamate (LPS Hexa aDG6), and pemetrexed on OAW28 ovarian cancer cells after 48 hours of exposure. [Figure 7]The therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (Lps Hexa aG6) and liposomal pemetrexed alpha-D hexaglutamate (Lps Hexa aDG6) on H292 non-small cell lung cancer cells after 48 hours of exposure are shown in comparison to pemetrexed. [Figure 8] This figure shows the therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and pemetrexed at various dose levels ranging from 16 to 128 nM on H292 non-small cell lung cancer cells after 48 hours of exposure. Within each tested dose range, the liposomal pemetrexed aG6 formulation exhibited superior inhibition of H292 non-small cell lung cancer cells compared to pemetrexed. [Figure 9] This figure shows the therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and pemetrexed at various dose levels ranging from 16 to 128 nM on HCC1806 triple-negative breast cancer cells after 48 hours of exposure. Within each tested dose range, the liposomal pemetrexed aG6 formulation was superior to pemetrexed in inhibiting HCC1806 triple-negative breast cancer cells. [Figure 10] This figure shows the therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and pemetrexed at a range of concentrations on OAW28 ovarian cancer cells after 48 hours of exposure. At a dose of 128 nM, pemetrexed appears to be more effective than the liposomal pemetrexed aG6 liposomal formulation, but at doses of 32 nM and 64 nM, the liposomal formulations have superior therapeutic effects to pemetrexed. At 16 nM, the therapeutic effect of liposomal pemetrexed aG6 is similar to that of pemetrexed. [Figure 11]Figure 1 shows the toxicity of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and pemetrexed at 64 nM, 128 nM, and 264 nM toward differentiated human neutrophils. The figure shows that liposomal pemetrexed aG6 is significantly less toxic than pemetrexed toward differentiated human neutrophils. [Figure 12] The effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal alpha-D hexaglutamate (liposomal aDG6), and the corresponding drugs of pemetrexed at various dose levels ranging from 16 to 128 nM on neutrophils (cultured from CD34+ cells) after 48 h of exposure are shown. [Figure 13] This figure shows the effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and 16 nM, 32 nM, 64 nM, and 128 nM of the corresponding pemetrexed on AML12 liver cells after 48 hours of exposure. Remarkably, none of the liposomal drugs tested appeared to be toxic to AML12 liver cells after treatment with liposomal pemetrexed aG6 at any of the dose levels tested. In contrast, pemetrexed treatment resulted in a reduction in AML12 liver cell counts by approximately 40% at all doses examined. [Figure 14]

[0023] Figure 1 shows the effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), 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 resulted 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 15]The structures of the polyglutamate antifolate, cisplatin (CDDP), and two possible aG6-cisplatin complexes are shown. The pH-dependent formation of inter- and / or intrachain coordination between the carboxyl groups of the polyglutamate antifolate and cisplatin may lead to its degradation into separate molecules of aG6 and cisplatin upon encountering the acidic pH of the lysosome (pH 4-5) and in the presence of intracellular chloride ions. [Figure 16] Hematological parameters: Effect of liposomal aG6 treatment in mice at 40 mg / kg and 80 mg / kg once weekly for 4 weeks on white blood cell (WBC) count, neutrophil count, and platelet count. No significant decrease in mean neutrophil, mean white blood cell, or mean platelet count was observed. [Figure 17] 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 18] 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 19] Relative tumor volumes in immunocompromised female Nu / J mice (6-8 weeks old) inoculated with NCI-H292 (non-small cell lung cancer) cells and treated 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 20]Figures 20A-F show the efficacy of liposomal pemetrexed alpha-L triglutamine over 48 hours against H2342 (NSCLC, adenocarcinoma subtype) (Figure 20A), H292 (NSCLC, adenocarcinoma subtype) (Figure 20B), HT-29 (colon cancer) (Figure 20C), HCC1806 (triple-negative breast cancer) (Figure 20D), MCF7 (ER+ breast cancer) (Figure 20E), and OAW28 (ovarian cancer) (Figure 20F). Figure 1 shows the dose-response 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

[0054] Generally, the present disclosure relates to novel alpha polyglutamated methotrexate compositions. The compositions offer an advancement over existing treatments for hyperproliferative diseases such as cancer. Methods for producing, delivering, and using the alpha polyglutamated methotrexate compositions are also provided. The alpha polyglutamated compositions have uses including, but not limited to, the treatment or prevention of hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0055] 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.

[0056] 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, "consisting of" is considered more exclusive, and "consisting essentially of" is considered intermediate).

[0057] 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.

[0058] 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.

[0059] 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. + , N.H. 4+ , 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.

[0060] 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.

[0061] The terms "polyglutamate," "polyglutamated," and variations thereof refer to a composition comprising at least one chain of two or more linked glutamyl groups. The polyglutamate chain can be linear or branched. A linear polyglutamate chain can, for example, contain glutamyl groups with alpha or gamma carboxyl linkages. A branched polyglutamate chain can, for example, contain one or more glutamyl groups with both alpha and gamma carboxyl linkages to other glutamyl groups, thereby providing branching points for the polyglutamate. Representative branched polyglutamates are shown in Figures 1R-1U. The polyglutamate chain contains an N-terminal glutamyl group and one or more C-terminal glutamyl groups. The N-terminal glutamyl group of a polyglutamate chain is not linked to another glutamyl group through its amino group, but is linked to one or more glutamyl groups through its carboxylic acid group. In some embodiments, the N-terminal glutamyl group of the polyglutamated methotrexate is the glutamyl group of methotrexate. The C-terminal glutamyl group(s) of the polyglutamate chain are bonded to another glutamyl group through their amino group, but not through their carboxylic acid group.

[0062] The terms "polyglutamylated methotrexate," "polyglutamylated MTX," "MTX-PG," and "PMTX" are used interchangeably herein and refer to methotrexate compositions that contain at least one glutamyl group in addition to the glutamyl groups in methotrexate (i.e., MTX-PG). n , n≧1). References to the number of glutamyl groups in αPMTX (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 of MTX is referred to herein as hexaglutamated methotrexate or methotrexate hexaglutamate.

[0063] The terms "alpha glutamyl group," "alpha glutamate," and "alpha linkage," when referring to glutamyl group bonds, refer to glutamyl groups containing an alpha carboxyl group bond. In some embodiments, the alpha linkage is an amide bond between the alpha carboxyl group of one glutamyl group and a second glutamyl group. The alpha linkage can be between a glutamyl group and a glutamyl group in methotrexate, or between a glutamyl group and a second glutamyl group not present in methotrexate, such as a glutamyl group in a polyglutamate chain linked to methotrexate.

[0064] The terms "gamma glutamyl group," "gamma glutamate," and "gamma linkage," when referring to the linkage of a glutamyl group, refer to a glutamyl group containing a gamma carboxyl group linkage. As discussed herein, when methotrexate enters cells, it is polyglutamated by the enzyme folylpolygamma glutamate synthase (FPGS), which sequentially adds L-glutamyl groups to the gamma carboxyl groups of glutamates within methotrexate. Thus, alpha polyglutamylated methotrexate compositions are not formed intracellularly during methotrexate therapy. In some embodiments, the gamma linkage is an amide bond between the gamma carboxyl group of one glutamyl group and a second glutamyl group. The gamma bond can be the 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 that is not present in methotrexate but is linked to methotrexate. In some embodiments, the gamma bond refers to the amide bond of the glutamyl group of methotrexate. Reference to a gamma bond includes the gamma bond of the glutamyl group of methotrexate, unless otherwise specified or unless the context clearly indicates otherwise.

[0065] Unless otherwise indicated, the terms "alpha polyglutamated methotrexate," "αPMTX," "alpha MTX-PG," and iterations thereof are used interchangeably herein to refer to polyglutamated methotrexate compositions containing at least one glutamyl group that contains an alpha linkage. For example, a pentaglutamated MTX composition in which the second glutamyl group has an alpha linkage, but each of the other glutamyl groups has a gamma linkage, is considered alpha MTX-PG in this disclosure. In some embodiments, each glutamyl group of MTX-PG other than the glutamyl group of MTX has an alpha linkage (e.g., MTX-PG where n=5 and G1, G2, G3, G4, and G5 each have an alpha linkage). n In some embodiments, the C-terminal glutamyl group(s) or each glutamyl group of MTX-PG other than the glutamyl group of MTX has an alpha linkage (e.g., MTX-PG where n=5 and G1, G2, G3, and alpha G4 each have an alpha linkage). n In some embodiments, each glutamyl group of MTX-PG other than the C-terminal glutamyl group(s) has an alpha linkage (e.g., MTX-PG where n=5 and MTX and G1, G2, G3, and G4 each have an alpha linkage). n ).

[0066] As used herein, the term "isolated" refers to a composition in a form not found in nature. Isolated alpha 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 alpha 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 made (e.g., cell culture). Alpha 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, the alpha polyglutamated composition is typically mixed with a pharmaceutically acceptable carrier or diluent. In some embodiments, isolated alpha polyglutamate compositions (e.g., alpha polyglutamates and delivery vehicles such as liposomes comprising alpha polyglutamates) contain less than 1% or less than 0.1% undesired DNA or protein content. In some embodiments, alpha polyglutamate compositions (e.g., alpha polyglutamates and delivery vehicles such as liposomes comprising alpha polyglutamates) are "isolated."

[0067] 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.

[0068] The terms "specific affinity" or "specifically bind" 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 target epitope. Due to sequence identity between homologous proteins in different species, a particular affinity, in some embodiments, includes binding substances that recognize proteins or targets 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 more than one protein or target. In certain embodiments, it is understood that a targeting moiety that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific affinity" does not necessarily require exclusive binding, e.g., binding to only one target (although it can include such binding). Thus, a targeting moiety may, in certain embodiments, specifically bind to more than one target. In certain embodiments, multiple targets may be bound by the same targeting moiety.

[0069] 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.

[0070] Expressions known in the art, such as "binding affinity to target", "binding to target" and similar expressions, refer to the affinity constant, a property of targeting moiety that can be directly measured by determining, for example, the amount of binding and dissociation of targeting moiety at a given antigen concentration.Other methods can be used to characterize intermolecular interactions, including but not limited to, competitive analysis, equilibrium analysis and microcalorimetry, and real-time interaction analysis based on surface plasmon resonance interaction (for example, using 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).

[0071] The term "delivery vehicle" generally refers to any composition that acts to support, promote, or facilitate the entry of alpha-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.

[0072] "Subject" means a human or vertebrate mammal, including, but not limited to, a dog, a cat, a horse, a goat, and a primate, e.g., a monkey. Thus, the present invention can also be used to treat a disease or condition in a non-human subject. For example, cancer is one of the leading causes of death in companion animals (i.e., 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 highest safe dose according to sound medical judgment.

[0073] 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.

[0074] 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.

[0075] "Cancer," "tumor," or "malignant tumor" are used interchangeably and refer to any of a number of diseases characterized by uncontrolled, abnormal proliferation of cells, spread of infected cells locally or via the bloodstream and lymphatic system to other parts of the body (metastasis), and a number of distinctive structural and / or molecular features. 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 αPMTX 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 leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. Other types of cancers and tumors that can be treated using the αPMTX 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.

[0076] The terms "treating," or "treatment," or "treat," and the like, refer to both (a) therapeutic measures that cure, slow, lessen the symptoms, and / or halt the progression of the 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, 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. Treatment can involve the use of αPMTX compositions alone or in combination with additional therapeutic agents.

[0077] "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 patient is a human.

[0078] 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. 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 tumor), aggressive fibromatosis, bladder cancer, and central nervous system (CNS) lymphoma.

[0079] 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, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis.

[0080] 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, raltitrexed, 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 anti-proliferative agents, anti-inflammatory agents, antineoplastic agents, antimitotic agents, antiplatelet agents, anticoagulants, antifibrinogens, antithrombin agents, cytostatic agents, antibiotics and other anti-infective agents, antienzymes, antimetabolites, angiogenic agents, cytoprotective agents, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, and / or cardioprotective agents. "Therapeutic agent" also refers to salt, acid, and free base forms of the above agents.

[0081] 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.

[0082] 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 alpha polyglutamated methotrexate composition comprises a fluoropyrimidine, 5-fluorouracil, 5-fluoro-2'-deoxycytidine, cytarabine, gemcitabine, troxacitabine, decitabine, azacitidine, pseudoisocytidine, zebularine, ancitabine, fazarabine, 6-azacytidine, capecitabine, N 4It is used in combination with an antimetabolite selected from the group consisting of octadecylcytarabine, elaidic acid cytarabine, fludarabine, cladribine, clofarabine, nelarabine, forodesine, and pentostatin, or derivatives thereof. In one example, the nucleoside analog is a substrate of nucleoside deaminase, which is adenosine deaminase or cytidine deaminase. In some examples, the nucleoside analog is selected from fludarabine, cytarabine, gemcitabine, decitabine, and azacitidine, or derivatives thereof. In certain embodiments, the antimetabolite is 5-fluorouracil.

[0083] 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.

[0084] The terms "pharmaceutically-acceptable carrier" and "pharmaceutically acceptable carrier" refer to an ingredient, other than an active ingredient, in a pharmaceutical formulation 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.

[0085] The present disclosure relates generally to novel alpha 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.

[0086] In some embodiments, the present disclosure provides: [1] A composition comprising alpha polyglutamated methotrexate, wherein at least one glutamyl group has an alpha carboxyl group linkage; [2] The composition of item [1], wherein the alpha polyglutamated methotrexate contains 1 to 10 glutamyl groups with alpha carboxyl group bonds; [3] The composition according to item [1] or [2], wherein the alpha polyglutamated methotrexate contains 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups; [4] The composition according to any one of items [1] to [3], comprising alpha-tetraglutamated methotrexate; [5] The composition according to any one of items [1] to [3], comprising alpha-pentaglutamated methotrexate; [6] The composition according to any one of items [1] to [3], comprising alphahexaglutamated methotrexate; [7] The composition according to any one of items [1] to [6], wherein the composition is: (a) two or more glutamyl groups have an alpha carboxyl group bond; (b) each glutamyl group other than the glutamyl group of methotrexate has an alpha carboxyl linkage; or (c) two or more glutamyl groups have gamma carboxyl group bonds; [8] The composition according to any one of items [1] to [6], which is the following composition: (a) the C-terminal glutamyl group(s) and each glutamyl group other than the glutamyl group of methotrexate have an alpha carboxyl linkage; or (b) a composition wherein each glutamyl group other than the C-terminal glutamyl group(s) has an alpha carboxyl linkage; [9] The composition according to any one of items [1] to [8], wherein at least one glutamyl group has both an alpha carboxyl group bond and a gamma carboxyl group bond;

[10] The composition according to any one of items [1] to [9], which is: (a) at least two glutamyl groups of the alpha polyglutamated methotrexate are in the L-configuration; (b) each glutamyl group of the alpha polyglutamated methotrexate is in the L-configuration; (c) at least one glutamyl group of the alpha polyglutamated methotrexate is in the D-form; (d) each glutamyl group of the alpha polyglutamylated methotrexate other than the glutamyl group of methotrexate is in the D-form; or (e) at least two of the glutamyl groups of the alpha polyglutamated methotrexate are in the L-configuration and at least one of the glutamyl groups is in the D-configuration;

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

[10] , wherein the polyglutamate is linear;

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

[10] , wherein the polyglutamate is branched;

[13] A liposome composition (Lp-αPMTX) containing alpha polyglutamated methotrexate according to any one of items [1] to

[12] ;

[14] The LαPP composition according to item

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

[15] The Lp-αPMTX composition according to item

[13] or

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

[16] The Lp-αPMTX composition according to item

[13] or

[14] , wherein at least one glutamyl group of the alpha polyglutamated methotrexate is in the D-form;

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

[13] to

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

[18] The Lp-αPMTX composition according to items

[13] to

[17] , wherein at least one glutamyl group of the alpha polyglutamylated methotrexate has a gamma carboxyl group bond;

[19] The composition according to any one of items

[13] to

[18] , wherein at least one glutamyl group has both an alpha carboxyl group bond and a gamma carboxyl group bond;

[20] The composition according to any one of items

[13] to

[19] , comprising 2, 3, 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups having both alpha carboxyl group bonds and gamma carboxyl group bonds;

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

[13] to

[20] , wherein the liposome contains alpha-polyglutamated methotrexate, including alpha-tetraglutamated methotrexate, alpha-pentaglutamated methotrexate, or alpha-hexaglutamated methotrexate;

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

[13] to

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

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

[13] to

[22] , wherein the liposome is PEGylated (PαLp-αPMTX);

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

[28] The Lp-αPMTX composition according to item

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

[29] The Lp-αPMTX 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-αPMTX 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-αPMTX composition according to any one of items

[27] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

[40] The Lp-αPMTX composition of 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-αPMTX composition of item

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

[42] The Lp-αPMTX composition according to item

[41] , wherein the pharmaceutically acceptable carrier comprises 5% by weight to 20% by weight of trehalose;

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

[39] to

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

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

[13] 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-αPMTX composition according to any one of items

[13] to

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

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

[13] to

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

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

[13] to

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

[51] The Lp-αPMTX 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-αPMTX composition according to item

[50] or

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

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

[50] to

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

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

[50] to

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

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

[39] to

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

[59] The Lp-αPMTX 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-αPMTX 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-3DPA , resolvin E, or T-series resolvin), and oxidized low-density lipoprotein (e.g., OXPAC, PGPC), and a Toll-like receptor (TLR) modulator, such as an eritran lipid (e.g., E5564);

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

[58] to

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

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

[58] to

[61] , further comprising a hapten;

[63] The Lp-αPMTX composition of item

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

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

[13] to

[63] , further comprising at least one cryoprotectant selected from the group consisting of mannitol, trehalose, sorbitol, and sucrose in the internal space, the external space, or both;

[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-αPMTX composition according to any one of items

[13] to

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

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

[13] to

[67] ;

[69] A pharmaceutical composition comprising the alpha polyglutamated methotrexate composition according to any one of items [1] to [8];

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

[69] for use in treating a disease;

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

[70] in the manufacture of a drug for 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, comprising administering to the subject the liposomal alpha polyglutamated methotrexate composition according to any one of items

[13] 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 alpha polyglutamated methotrexate composition according to any one of items

[13] 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 alpha polyglutamated methotrexate composition according to any one of items

[13] 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 mesothelioma or non-small cell lung cancer (NSCLC);

[82] The method according to item

[77] or

[78] , wherein the cancer is selected from the group consisting of colorectal cancer, breast 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-αPMTX 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 comprising administering an effective amount of the liposomal alpha polyglutamated methotrexate composition according to any one of items

[13] to

[69] to a subject undergoing or who has 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;

[87] A method for treating an immune system disorder, comprising administering an effective amount of the liposomal alpha 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;

[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;

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

[13] to

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

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

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

[91] A method for making an alpha polyglutamated methotrexate composition, including the liposomal alpha polyglutamated methotrexate composition according to any one of items

[13] to

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

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

[13] to

[69] , comprising the steps of: forming a mixture in a solution containing liposome components and alpha-polyglutamated methotrexate; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes that encapsulate and / or entrap the alpha-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 method, dynamic high-pressure microfluidization, microfluidic mixing, double emulsion, freeze-dried double emulsion, 3D printing, membrane contactor method, and stirring;

[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 alpha-polyglutamated methotrexate; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes that encapsulate and / or entrap the alpha-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 alpha-polyglutamated methotrexate in a solution; treating the mixture to form liposomes that entrap and / or encapsulate the alpha-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 of 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 alpha polyglutamated methotrexate starting material is encapsulated or entrapped in liposomes.

[0087] II. Alpha Polyglutamated Methotrexate (αPMTX) Generally, the present disclosure relates to alpha polyglutamated methotrexate (αPMTX) compositions. αPMTX compositions contain at least one glutamyl group with an alpha linkage. These compositions are structurally distinct from L-gamma polyglutamated methotrexate (LαPMTX), which is produced in cells by the enzyme folylpolygamma glutamate synthase (FPGS) during methotrexate therapy.

[0088] In some embodiments, the αPMTX composition contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, 2 to 6, or more than 5 glutamyl groups (including the glutamyl group of methotrexate). In some embodiments, each glutamyl group in αPMTX other than the glutamyl group of methotrexate has an alpha linkage. In some embodiments, each glutamyl group in αPMTX other than the C-terminal glutamyl group(s) and the glutamyl group of methotrexate has an alpha linkage. In some embodiments, each glutamyl group in αPMTX other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, two or more glutamyl groups in αPMTX have gamma linkages. In some embodiments, at least one glutamyl group of alpha polyglutamated methotrexate has an alpha carboxyl group linkage and a gamma carboxyl group linkage. In some embodiments, each glutamyl group in αPMTX is in the L-form. In some embodiments, each glutamyl group in αPMTX other than the glutamyl group of methotrexate is in the D form. In some embodiments, αPMTX contains two or more glutamyl groups in the L form and one or more glutamyl groups in the D form. In some embodiments, the polyglutamate chain of αPMTX is linear (not branched). In some embodiments, the polyglutamate chain of αPMTX is branched.

[0089] In some embodiments, the alpha polyglutamated methotrexate is diglutamated. That is, the alpha polyglutamated methotrexate contains one additional glutamyl group in addition to the glutamyl groups of methotrexate (αMTX-PG1), and the additional glutamyl group is linked to the glutamyl group in methotrexate via an alpha bond. In some embodiments, each glutamyl group of the alpha diglutamated methotrexate is in the L-form. In other embodiments, the alpha diglutamated MTX contains a glutamyl group in the D-form.

[0090] In some embodiments, the alpha polyglutamated methotrexate is triglutamated. That is, the alpha polyglutamated methotrexate contains two additional glutamyl groups in addition to the glutamyl groups of methotrexate (αMTX-PG2). In some embodiments, each of the two additional glutamyl groups has an alpha linkage. In other embodiments, one of the two additional glutamyl groups has an alpha linkage and the other glutamyl group has a gamma linkage. In some embodiments, one of the two additional glutamyl groups has an alpha linkage. In some embodiments, one of the two additional glutamyl groups has a gamma linkage. In some embodiments, two of the three glutamyl groups have alpha linkages. In other embodiments, one of the three glutamyl groups has an alpha linkage and another glutamyl group has a gamma linkage. In some embodiments, one glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, each glutamyl group of alpha triglutamated methotrexate is in the L-form. In other embodiments, alpha triglutamated MTX comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group of alpha triglutamated methotrexate other than the glutamyl group of methotrexate is in the D-form. In further embodiments, triglutamated MTX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0091] In some embodiments, the alpha polyglutamylated methotrexate is tetraglutamylated, and thus contains three additional glutamyl groups in addition to the glutamyl groups in methotrexate (αMTX-PG3). In some embodiments, each of the three glutamyl groups has an alpha linkage. In other embodiments, one or two of the three additional glutamyl groups have an alpha linkage, and the remaining two or one glutamyl groups each have a gamma linkage. In some embodiments, two of the three additional glutamyl groups have an alpha linkage. In other embodiments, one of the three glutamyl groups has an alpha linkage, and another additional glutamyl group has a gamma linkage. In other embodiments, one of the three additional glutamyl groups has an alpha linkage and a gamma linkage. In other embodiments, three of the four glutamyl groups have alpha linkages. In some embodiments, at least one glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, the alpha-tetraglutamated MTX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group in the alpha-tetraglutamated methotrexate is in the L-form. In other embodiments, the alpha-tetraglutamated MTX contains a glutamyl group in the D-form. In further embodiments, each glutamyl group in the alpha-triglutamated methotrexate, other than the glutamyl group in methotrexate, is in the D-form. In further embodiments, the tetraglutamated MTX contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0092] In some embodiments, the alpha polyglutamated methotrexate is pentaglutamated (αMTX-PG4) and comprises a chain of four additional glutamyl groups attached to the glutamyl groups of methotrexate. In some embodiments, each of the four additional glutamyl groups in the chain has an alpha linkage. In some embodiments, each of the four additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha linkage. In other embodiments, one, two, or three of the four additional glutamyl groups have an alpha linkage, and the remaining three, two, or one glutamyl groups, respectively, are attached to the glutamyl groups of the molecule via a gamma linkage. In other embodiments, one or two of the four additional glutamyl groups have an alpha linkage, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl groups of the molecule via a gamma linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the five glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the five glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, the alpha-pentaglutamated MTX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group in the alpha-pentaglutamated methotrexate is in the L-form. In other embodiments, the alpha-pentaglutamated MTX contains a glutamyl group in the D-form. In further embodiments, each glutamyl group in the alpha-pentaglutamated methotrexate other than the glutamyl group in methotrexate is in the D-form. In further embodiments, the pentaglutamated MTX contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0093] In some embodiments, the alpha polyglutamated methotrexate is hexaglutamated (αMTX-PG5) and comprises a chain of five additional glutamyl groups attached to the glutamyl groups of methotrexate. In some embodiments, each of the five additional glutamyl groups in the chain has an alpha linkage. In some embodiments, each of the five additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha linkage. In some embodiments, four of the five additional glutamyl groups in the chain have alpha linkages. In other embodiments, one, two, three, or four of the five additional glutamyl groups are attached to the glutamyl groups of the molecule via alpha linkages, and the remaining four, three, two, or one glutamyl groups, respectively, are attached to the glutamyl groups of the molecule via gamma linkages. In other embodiments, one, two, three, or four of the five additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the six glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the six glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, five of the six glutamyl groups have alpha linkages. In some embodiments, the alpha hexaglutamated MTX contains two or more glutamyl groups in the L form. In a further embodiment, each glutamyl group in the alpha hexaglutamated methotrexate is in the L form. In another embodiment, the alpha hexaglutamated MTX contains a glutamyl group in the D form. In a further embodiment, each glutamyl group in the alpha hexaglutamated methotrexate other than the glutamyl group in methotrexate is in the D form. In further embodiments, the hexaglutamylated MTX contains a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0094] In some embodiments, the alpha polyglutamated methotrexate is heptaglutamated (αMTX-PG6) and comprises a chain of six additional glutamyl groups attached to the glutamyl groups of methotrexate. In some embodiments, each of the six additional glutamyl groups has an alpha linkage. In some embodiments, each of the six additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha linkage. In some embodiments, five of the six additional glutamyl groups in the chain have alpha linkages. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have alpha linkages, and the remaining five, four, three, two, or one glutamyl groups, respectively, have gamma linkages. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the seven glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the seven glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, six of the seven glutamyl groups have alpha linkages. In some embodiments, the alphaheptaglutamated MTX contains two or more glutamyl groups in the L form. In a further embodiment, each glutamyl group in the alphaheptaglutamated methotrexate is in the L form. In another embodiment, the alphaheptaglutamated MTX contains a glutamyl group in the D form. In a further embodiment, each glutamyl group in the alphaheptaglutamated methotrexate other than the glutamyl group in methotrexate is in the D form. In further embodiments, the heptaglutamated MTX contains a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0095] In some embodiments, the alpha polyglutamated methotrexate is octaglutamated (αMTX-PG7) and comprises a chain of seven additional glutamyl groups attached to the glutamyl groups of methotrexate. In some embodiments, each of the seven additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha linkage. In some embodiments, six of the seven additional glutamyl groups in the chain have an alpha linkage. In some embodiments, each of the seven additional glutamyl groups has an alpha linkage. In other embodiments, one, two, three, four, five, or six of the seven additional glutamyl groups have an alpha linkage, and the remaining six, five, four, three, two, or one glutamyl groups, respectively, have a gamma linkage. In other embodiments, one, two, three, four, five, or six of the seven additional glutamyl groups have an alpha linkage, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl groups of the molecule via a gamma linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the eight glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the eight glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, seven of the eight glutamyl groups have alpha linkages. In some embodiments, the alpha-octaglutamated MTX comprises two or more glutamyl groups in the L-form. In a further embodiment, each glutamyl group in the alpha-octaglutamated methotrexate is in the L-form. In other embodiments, the alpha-octaglutamated MTX comprises a glutamyl group in the D-form. In a further embodiment, each glutamyl group in the alpha-octaglutamated methotrexate other than the glutamyl group in methotrexate is in the D-form. In a further embodiment, the octaglutamated MTX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chains are linear. In other embodiments, the polyglutamate chains are branched.

[0096] In some embodiments, the alpha polyglutamated methotrexate is nonaglutamated (αMTX-PG8) and contains a chain of eight additional glutamyl groups attached to the glutamyl groups of methotrexate. In some embodiments, each of the eight additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha linkage. In some embodiments, seven of the eight additional glutamyl groups in the chain have an alpha linkage. In some embodiments, each of the eight additional glutamyl groups has an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, or 7 of the eight additional glutamyl groups have an alpha linkage, and the remaining 7, 6, 5, 4, 3, 2, or 1 glutamyl groups, respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, or 7 of the eight additional glutamyl groups have an alpha linkage, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl groups of the molecule via a gamma linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the nine glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the nine glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, eight of the nine glutamyl groups have an alpha linkage. In some embodiments, the alpha nonaglutamated MTX contains two or more glutamyl groups in the L-form. In a further embodiment, each glutamyl group in the alpha nonaglutamated methotrexate is in the L-form. In other embodiments, the alpha nonaglutamated MTX contains a glutamyl group in the D-form. In a further embodiment, each glutamyl group in the alpha nonaglutamated methotrexate other than the glutamyl group in methotrexate is in the D-form. In a further embodiment, the nonaglutamated MTX contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chains are linear. In other embodiments, the polyglutamate chains are branched.

[0097] In some embodiments, the alpha polyglutamated methotrexate is deca-glutamated (αMTX-PG9) and comprises a chain of nine additional glutamyl groups attached to the glutamyl groups of methotrexate. In some embodiments, each of the nine additional glutamyl groups has an alpha linkage. In some embodiments, each of the nine additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha linkage. In some embodiments, eight of the nine additional glutamyl groups in the chain have an alpha linkage. In other embodiments, one, two, three, four, five, six, seven, or eight of the nine additional glutamyl groups have an alpha linkage, and the remaining eight, seven, six, five, four, three, two, or one glutamyl groups, respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 of the 9 additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 10 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 10 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 9 of the 10 glutamyl groups have alpha linkages. In some embodiments, the alphadecaglutamylated MTX contains two or more glutamyl groups in the L-form. In a further embodiment, each glutamyl group of the alphadecaglutamylated methotrexate is in the L-form. In other embodiments, the alphadecaglutamylated MTX contains a glutamyl group in the D-form. In a further embodiment, each glutamyl group of alpha-decaglutamated methotrexate, other than the glutamyl group of methotrexate, is D-type. In a further embodiment, deca-glutamated MTX contains a glutamyl group of D-type and two or more glutamyl groups of L-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0098] In some embodiments, the alpha polyglutamated methotrexate is undecaglutamated (αMTX-PG 10). In some embodiments, each of the 10 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 10 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 9 of the 10 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an alpha linkage, and the remaining 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an alpha linkage, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via a gamma linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 11 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 11 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 10 of the 11 glutamyl groups have an alpha linkage. In some embodiments, the alpha-undeca-glutamated MTX contains two or more glutamyl groups in the L-form. In a further embodiment, each glutamyl group in the alpha-undeca-glutamated methotrexate is in the L-form. In other embodiments, the alpha-undeca-glutamated MTX contains a glutamyl group in the D-form. In a further embodiment, each glutamyl group in the alpha-undeca-glutamated methotrexate other than the glutamyl group in methotrexate is in the D-form. In a further embodiment, the undecaglutamated MTX contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.

[0099] In some embodiments, the alpha polyglutamated methotrexate is dodecaglutamated (αMTX-PG11 In some embodiments, each of the 11 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 11 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 10 of the 11 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alpha linkage, and the remaining 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alpha linkage, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via a gamma linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 12 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 12 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 11 of the 12 glutamyl groups have an alpha linkage. In some embodiments, the alpha dodecaglutamated MTX contains two or more glutamyl groups in the L form. In a further embodiment, each glutamyl group in the alpha dodecaglutamated methotrexate is in the L form. In other embodiments, the alpha dodecaglutamated MTX contains a glutamyl group in the D form. In a further embodiment, each glutamyl group in the alpha dodecaglutamated methotrexate other than the glutamyl group in methotrexate is in the D form. In a further embodiment, the dodecaglutamated MTX contains a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.

[0100] In some embodiments, the alpha polyglutamated methotrexate is triskite decaglutamated (αMTX-PG 12In some embodiments, each of the 12 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 12 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 11 of the 12 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alpha linkage, and the remaining 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups, respectively, have gamma linkages. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alpha linkage, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 13 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 13 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 12 of the 13 glutamyl groups have an alpha linkage. In some embodiments, alpha-Triskai deca-glutamated MTX contains two or more glutamyl groups in the L-form. In a further embodiment, each glutamyl group in alpha-Triskai deca-glutamated methotrexate is in the L-form. In another embodiment, alpha-Triskai deca-glutamated MTX contains a glutamyl group in the D-form. In a further embodiment, each glutamyl group in alpha-Triskai deca-glutamated methotrexate other than the glutamyl group of methotrexate is in the D-form. In further embodiments, the triskaidecaglutamated MTX contains a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0101] In some embodiments, the alpha polyglutamated methotrexate is tetradecaglutamated (αMTX-PG 13In some embodiments, each of the 13 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 13 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 12 of the 13 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have an alpha linkage, and the remaining 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups, respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have an alpha linkage, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via a gamma linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 14 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 14 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 13 of the 14 glutamyl groups have an alpha linkage. In some embodiments, the alpha-tetradecaglutamated MTX comprises two or more glutamyl groups in the L-form. In a further embodiment, each glutamyl group in the alpha-tetradecaglutamated methotrexate is in the L-form. In another embodiment, the alpha-tetradecaglutamated MTX comprises a glutamyl group in the D-form. In a further embodiment, each glutamyl group in the alpha-tetradecaglutamated methotrexate other than the glutamyl group in methotrexate is in the D-form. In a further embodiment, the tetradecaglutamated MTX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chains are linear. In other embodiments, the polyglutamate chains are branched.

[0102] In some embodiments, the alpha polyglutamated methotrexate is pentadecaglutamated (αMTX-PG 14In some embodiments, each of the 14 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 14 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 13 of the 14 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14 additional glutamyl groups have an alpha linkage, and the remaining 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups, respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14 additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 15 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 15 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 14 of the 15 glutamyl groups have alpha linkages. In some embodiments, the alpha-pentadeca-glutamylated MTX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alpha-pentadeca-glutamylated methotrexate is in the L-form. In other embodiments, the alpha pentadeca-glutamated MTX comprises a glutamyl group in the D-form. In a further embodiment, each glutamyl group in the alpha pentadeca-glutamated methotrexate other than the glutamyl group in methotrexate is in the D-form. In a further embodiment, the pentadeca-glutamated MTX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0103] In some embodiments, the alpha polyglutamated methotrexate is hexadecaglutamated (αMTX-PG 15In some embodiments, each of the 15 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 15 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 14 of the 15 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15 additional glutamyl groups have an alpha linkage, and the remaining 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups, respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15 additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 16 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 16 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 15 of the 16 glutamyl groups have alpha linkages. In some embodiments, the alphahexadecaglutamated MTX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alphahexadecaglutamated methotrexate is in the L-form. In other embodiments, the alphahexadecaglutamated MTX comprises a glutamyl group of D-form. In a further embodiment, each glutamyl group of the alphahexadecaglutamated methotrexate other than the glutamyl group of methotrexate is of D-form. In a further embodiment, the hexadecaglutamated MTX comprises a glutamyl group of D-form and two or more glutamyl groups of L-form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0104] In another embodiment, the alpha polyglutamated methotrexate is heptadecaglutamated (αMTX-PG 16In some embodiments, each of the 16 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 16 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 15 of the 16 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the 16 additional glutamyl groups have an alpha linkage, and the remaining 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups, respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the 16 additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 17 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 17 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 16 of the 17 glutamyl groups have alpha linkages. In some embodiments, the alphaheptadecaglutamated MTX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alphaheptadecaglutamated methotrexate is in the L-form. In other embodiments, the alpha heptadeca-glutamated MTX comprises a glutamyl group of D-form. In a further embodiment, each glutamyl group of the alpha heptadeca-glutamated methotrexate other than the glutamyl group of methotrexate is of D-form. In a further embodiment, the heptadeca-glutamated MTX comprises a glutamyl group of D-form and two or more glutamyl groups of L-form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0105] In some embodiments, the alpha polyglutamated methotrexate is octadeca-glutamated (αMTX-PG 17In some embodiments, each of the 17 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 17 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 16 of the 17 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the 17 additional glutamyl groups have an alpha linkage, and the remaining 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups, respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the 17 additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 18 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 18 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 17 of the 18 glutamyl groups have alpha linkages. In some embodiments, the alpha-octadeca-glutamylated MTX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alpha-octadeca-glutamylated methotrexate is in the L-form. In other embodiments, the alpha-octadeca-glutamated MTX comprises a glutamyl group in the D-form. In a further embodiment, each glutamyl group in the alpha-octadeca-glutamated methotrexate other than the glutamyl group in methotrexate is in the D-form. In a further embodiment, the octadeca-glutamated MTX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0106] In some embodiments, the alpha polyglutamated methotrexate is eniadecaglutamated (αMTX-PG 18In some embodiments, each of the 18 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 18 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 17 of the 18 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have an alpha linkage, and the remaining 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups, respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 19 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 19 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 18 of the 19 glutamyl groups have alpha linkages. In some embodiments, the alphaeniadecaglutamated MTX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alphaeniadecaglutamated methotrexate is in the L-form. In other embodiments, the alpha eniadecaglutamated MTX comprises a glutamyl group in the D form. In a further embodiment, each glutamyl group in the alpha eniadecaglutamated methotrexate other than the glutamyl group in methotrexate is in the D form. In a further embodiment, the eniadecaglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0107] In some embodiments, the alpha polyglutamated methotrexate is eicosiglutamated (αMTX-PG 19In some embodiments, each of the 19 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 19 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 18 of the 19 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups have an alpha linkage, and the remaining 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups, respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 20 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 20 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 19 of the 20 glutamyl groups have alpha linkages. In some embodiments, the alpha-icosiglutamated MTX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alpha-icosiglutamated methotrexate is in the L-form. In other embodiments, the alpha-icosiglutamated MTX comprises a D-glutamyl group. In a further embodiment, each glutamyl group of the alpha-icosiglutamated methotrexate other than the glutamyl group of methotrexate is D-glutamyl. In a further embodiment, the eicosiglutamated MTX comprises a D-glutamyl group and two or more L-glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0108] In some embodiments, the alpha polyglutamated methotrexate is polyglutamated (αMTX-PG 20In some embodiments, each of the 20 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 20 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 19 of the 20 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have an alpha linkage, and the remaining 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups, respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have alpha linkages, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule via gamma linkages. In some embodiments, at least one additional glutamyl group has both alpha and gamma linkages. In some embodiments, at least one of the 21 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 21 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 20 of the 21 glutamyl groups have alpha linkages. In some embodiments, the alpha-coccal glutamylated MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-cosyl-glutamated methotrexate is in the L-form. In another embodiment, alpha-cosyl-glutamated MTX comprises a glutamyl group in the D-form. In a further embodiment, each glutamyl group of alpha-cosyl-glutamated methotrexate other than the glutamyl group of methotrexate is in the D-form. In a further embodiment, alpha-cosyl-glutamated MTX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain is linear.In other embodiments, the polyglutamate chains are branched.

[0109] In some embodiments, the alpha polyglutamated methotrexate comprises 4 to 7 glutamyl groups bound to methotrexate (i.e., αMTX-PGn, n=4-7), and each of the 4 to 7 linked glutamyl groups has an alpha linkage. In some embodiments, the alpha polyglutamated methotrexate comprises 4 to 7 glutamyl groups bound to methotrexate (i.e., αMTX-PGn, n=4-7), and each of the 4 to 7 linked glutamyl groups other than the C-terminal glutamyl group(s) has an alpha 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. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0110] In one embodiment, the alpha polyglutamated methotrexate is tetraglutamated, and each of the three glutamyl groups in the polyglutamate chain attached to methotrexate contains an alpha linkage. In one embodiment, the alpha polyglutamated methotrexate is tetraglutamated, and each of the three glutamyl groups in the polyglutamate chain attached to methotrexate, other than the C-terminal glutamyl group(s), contains an alpha linkage. In some embodiments, each of the four glutamyl groups is L-type. In some embodiments, each glutamyl group in the alpha tetraglutamated methotrexate, other than the glutamyl group in methotrexate, is D-type. In other embodiments, at least two of the glutamyl groups in the alpha tetraglutamate methotrexate are L-type and at least one glutamyl group is D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0111] In one embodiment, the alpha polyglutamated methotrexate is pentaglutamated, and each of the four glutamyl groups in the polyglutamate chain attached to methotrexate comprises an alpha linkage. In one embodiment, the alpha polyglutamated methotrexate is pentaglutamated, and each of the four glutamyl groups in the polyglutamate chain attached to methotrexate, other than the C-terminal glutamyl group(s), comprises an alpha linkage. In some embodiments, each of the four glutamyl groups is L-form. In some embodiments, each glutamyl group in the alpha pentaglutamated methotrexate, other than the glutamyl group in methotrexate, is D-form. In other embodiments, at least two of the glutamyl groups in the alpha pentaglutamated methotrexate are L-form and at least one glutamyl group is D-form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0112] In one embodiment, the alpha polyglutamated methotrexate is hexaglutamated, and each of the five glutamyl groups in the polyglutamate chain attached to methotrexate comprises an alpha linkage. In one embodiment, the alpha polyglutamated methotrexate is hexaglutamated, and each of the five glutamyl groups in the polyglutamate chain attached to methotrexate, other than the C-terminal glutamyl group(s), comprises an alpha linkage. In some embodiments, each of the five glutamyl groups is in the L-form. In some embodiments, each glutamyl group in the alpha hexaglutamated methotrexate, other than the glutamyl group in methotrexate, is in the D-form. In other embodiments, at least two of the glutamyl groups in the alpha hexaglutamated methotrexate are in the L-form and at least one glutamyl group is in the D-form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.

[0113] In another embodiment, the alpha polyglutamated methotrexate is heptaglutamated, and each of the six glutamyl groups in the polyglutamate chain attached to methotrexate contains an alpha linkage. In another embodiment, the alpha polyglutamated methotrexate is heptaglutamated, and each of the six glutamyl groups in the polyglutamate chain attached to methotrexate, other than the C-terminal glutamyl group(s), contains an alpha linkage. In some embodiments, each of the six glutamyl groups is in the L-form. In some embodiments, each glutamyl group in the alpha heptaglutamated methotrexate, other than the glutamyl group in methotrexate, is in the D-form. In other embodiments, at least two of the glutamyl groups in the alpha heptaglutamated methotrexate are in the L-form and at least one glutamyl group is in the D-form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.

[0114] In some embodiments, alpha polyglutamylated methotrexate (αPMTX) 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 αPMTX other than the glutamyl groups of methotrexate has an alpha linkage. In some embodiments, each glutamyl group in αPMTX other than the C-terminal glutamyl group(s) and the glutamyl group of methotrexate has an alpha linkage. In some embodiments, each glutamyl group in αPMTX other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPMTX have alpha linkages. In some embodiments, αPMTX contains L- and D-glutamyl groups. In further embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPMTX have alpha linkages, and 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 glutamyl groups each have gamma linkages. In some embodiments, each glutamyl group in the polyglutamate structure of polyglutamylated methotrexate is in the L-form. In some embodiments, each glutamyl group in αPMTX 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 αPMTX are L-type. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPMTX are D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0115] In some embodiments, alpha polyglutamylated methotrexate (αPMTX) contains a total of 2 to 20, 2 to 15, 2 to 10, 2 to 5, or any range therebetween, including the glutamyl groups of methotrexate. In some embodiments, each glutamyl group in αPMTX other than the glutamyl groups of methotrexate has an alpha linkage. In some embodiments, each glutamyl group in αPMTX other than the C-terminal glutamyl group(s) and the glutamyl group of methotrexate has an alpha linkage. In some embodiments, each glutamyl group in αPMTX other than the C-terminal glutamyl group(s) has an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups have an alpha linkage. In some embodiments, αPMTX contains two or more glutamyl groups with gamma linkages. In further embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups in αPMTX other than the glutamyl groups of methotrexate have alpha linkages, and 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 glutamyl groups each have gamma linkages. In some embodiments, each glutamyl group in αPMTX is in the L-form. In some embodiments, each glutamyl group in αPMTX other than the glutamyl groups of methotrexate is in the D-form. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the glutamyl groups in αPMTX are in the L-form. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the glutamyl groups in αPMTX are in the D-form.

[0116] In some embodiments, the alpha polyglutamated methotrexate contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in addition to the glutamyl groups of methotrexate. In further embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additional glutamyl groups have alpha linkages. In further embodiments, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group in the alpha polyglutamated methotrexate has gamma linkages. In some embodiments, at least one glutamyl group has both alpha and gamma linkages. In some embodiments, the glutamyl groups in methotrexate have alpha linkages. In some embodiments, the glutamyl group in methotrexate has both alpha and gamma linkages.

[0117] In some embodiments, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in the alpha polyglutamated methotrexate are in the L-form, the D-form, or the L- and D-form. In some embodiments, each glutamyl group in the alpha polyglutamated methotrexate is in the L-form. In other embodiments, each glutamyl group in the alpha polyglutamated methotrexate other than the glutamyl group in methotrexate is in the D-form. In alternative embodiments, at least two of the glutamyl groups in the alpha polyglutamated methotrexate are in the L-form and at least one of the glutamyl groups in the alpha polyglutamated methotrexate is in the D-form. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 glutamyl groups in the alpha polyglutamated methotrexate are in the L-form. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 glutamyl groups in the alpha polyglutamated methotrexate are in the D-form.

[0118] In further embodiments, the alpha polyglutamated methotrexate contains 20-100, 20-75, 20-50, 20-40, 20-30, 20-25, or more than 100 glutamyl groups, or any range therebetween. In some embodiments, each glutamyl group of the alpha polyglutamated methotrexate is in the L-form. In other embodiments, each glutamyl group of the alpha polyglutamated 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 alpha polyglutamated methotrexate are in the L-form and at least one of the glutamyl groups in the alpha polyglutamated methotrexate is in the D-form.

[0119] In further embodiments, provided compositions comprise alpha polyglutamated methotrexate comprising glutamyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 alpha linkages. In some embodiments, the alpha polyglutamated methotrexate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 L-glutamyl groups. In some embodiments, the alpha polyglutamated methotrexate comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 D-glutamyl groups. In some embodiments, the alpha polyglutamated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups in the L-form and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups in the D-form. In other embodiments, the alpha polyglutamated methotrexate contains at least one glutamyl group with both alpha and gamma linkages. In some embodiments, the alpha polyglutamated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or more than 10 glutamyl groups with both alpha and gamma linkages.

[0120] In some embodiments, the alpha polyglutamated methotrexate comprises at least one glutamyl group with an alpha linkage and 2, 3, 4, 5, 6, 7, 8, 9, 1-10, 1-20, or more glutamyl groups with gamma linkages. For example, in some embodiments, the alpha polyglutamated methotrexate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10 L-alpha glutamyl group linkages and further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 L-gamma glutamyl group linkages. In some further embodiments, the alpha polyglutamated methotrexate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 L-alpha glutamyl group linkages and further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma glutamyl group linkages. In still further embodiments, the alpha polyglutamated methotrexate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-alpha glutamyl group linkages and further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma glutamyl group linkages. In other further embodiments, the alpha polyglutamated methotrexate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma glutamyl group linkages and further comprises 1, 2, 3, 4, 5, 6, or 1-10 L-gamma glutamyl group linkages. In other embodiments, the alpha polyglutamated methotrexate comprises at least one glutamyl group with both alpha and gamma linkages. In some embodiments, the alpha polyglutamated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or more than 10 glutamyl groups with both alpha and gamma linkages.

[0121] In some embodiments, the alpha 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 alpha 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.

[0122] In some embodiments, the rate of hepatocyte uptake of a naked alpha PMTX composition disclosed herein (e.g., alpha PMTX not conjugated to a delivery vehicle) is significantly reduced compared to the rate of methotrexate uptake under physiological conditions. In some embodiments, the rate of hepatocyte uptake of a naked alpha PMTX composition is less than 30%, 20%, 15%, or 10% of the rate of methotrexate. In further embodiments, the rate of efflux (transport) of an alpha PMTX composition disclosed herein from hepatocytes occurs at a significantly slower rate (less than 30%, 20%, 15%, or 10%) than that of methotrexate.

[0123] In some embodiments, the alpha 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 alpha polyglutamated methotrexate is hexaglutamated methotrexate.

[0124] In some embodiments, the alpha polyglutamated methotrexate compositions provided herein have fewer toxic side effects than methotrexate. In some embodiments, the alpha polyglutamated methotrexate compositions provided herein are less toxic to non-hyperproliferative cells than methotrexate. In some embodiments, the alpha 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 alpha polyglutamated methotrexate is hexaglutamated methotrexate.

[0125] In some embodiments, the alpha polyglutamated methotrexate compositions provided herein have fewer toxic side effects than methotrexate. In some embodiments, the alpha 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 alpha 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 the alpha polyglutamated methotrexate composition once a week for four weeks. In some embodiments, the alpha polyglutamated methotrexate is hexaglutamated methotrexate.

[0126] In some embodiments, treatment with the alpha 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 alpha polyglutamated methotrexate compositions provided herein do not significantly reduce mean neutrophil, mean white blood cell, or mean platelet counts. In some embodiments, the alpha polyglutamated methotrexate compositions provided herein do not significantly increase serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. In some embodiments, the alpha 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 the alpha polyglutamated methotrexate composition once a week for four weeks. In some embodiments, the alpha polyglutamated methotrexate is hexaglutamated methotrexate.

[0127] In some embodiments, the alpha polyglutamated methotrexate composition does not contain fluorine atoms, hi some embodiments, the alpha polyglutamated methotrexate composition does not contain 4-fluoroglutamyl groups.

[0128] Alpha polyglutamated methotrexate (α PMTX) compositions and their uses are further described in U.S. Patent Application No. 62 / 374,458, International Application No. PCT / US2017 / 046666, and International Application No. PCT / US2017 / 046667, the disclosures of each of which are incorporated herein by reference in their entireties.

[0129] A. Polyglutamated Methotrexate Analogs and Derivatives The present disclosure also encompasses alpha 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 according to 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 rest 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 not fluorinated.

[0130] 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.

[0131] 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 analogs, methotrexate analogs, methotrexate analogs, methotrexate analogs, methotrexate analogs, methotrexate analogs, methotrexate analogs, methotrexate analogs, methotrexate analogs, methotrexate analogs, methotrexate analogs, methotrexate analog methotrexate tetrahydroquinazoline 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, phosphonoglutamic acid analogs, poly(L-lysine) methotrexate conjugates, disilysine or trilysine methotrexate derivatives, methotrexate-γ-dimyristoylphosphatidylethanolamine, iodoacetyllysine methotrexate analogs, methotrexate-γ-dimyristoylphosphatidylethanolamine, 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, gamma-tert-butyl methotrexate esters, fluorinated methotrexate analogs, folate methotrexate analogs, 7-hydroxymethotrexate, poly-gamma-glutamyl methotrexate analogs, 3',5'-dichloromethotrexate, diazoketone and chloromethylketone methotrexate analogs, 10-propargyl aminopterin or alkyl methotrexate homologs, lectin derivatives of methotrexate, 3',A member selected from the group consisting of 5'-dichloromethotrexate, deazaamethopterin analogs, cysteic and homocysteic acid methotrexate analogs, and MX068.

[0132] In further embodiments, the alpha 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.

[0133] 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).

[0134] 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.

[0135] C. Methotrexate-PG complex Surprisingly, the inventors have discovered that polyglutamated anti-metabolites, such as polyglutamated methotrexate (αPMTX), can be complexed with other compositions, including therapeutic agents, including cytotoxic compounds, such as platinum-based compounds. Thus, in some embodiments, the present disclosure provides complexes of αPMTX (e.g., the αPMTX disclosed herein) with a therapeutic agent, or a salt or acid thereof.

[0136] In some embodiments, the αPMTX / complex comprises αPMTX and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound such as a chemotherapeutic agent. In further embodiments, the αPMTX / complex comprises a platinum-based drug such as a platinum-based chemotherapeutic agent (e.g., cisplatin, carboplatin, and oxaliplatin). In other embodiments, the αPMTX / complex comprises a taxane-based chemotherapeutic agent (e.g., paclitaxel and docetaxel). In other embodiments, the αPMTX / complex comprises a cyclodextrin. In further embodiments, the αPMTX / complex is encapsulated in a liposome.

[0137] In some embodiments, the present disclosure provides compositions comprising a complex of αPMTX and a therapeutic agent, or a salt or acid thereof. In further embodiments, the αPMTX / therapeutic agent complex comprises one or more αPMTXs containing 2 to 150, 2 to 100, 2 to 75, 2 to 50, 2 to 24, 2 to 30, 2 to 20, 2 to 19, 2 to 15, 2 to 10, or 2 to 5 glutamyl groups. In some embodiments, the αPMTX / therapeutic agent complex comprises one or more αPMTXs containing 3 to 10, 3 to 9, 3 to 8, or 3 to 7 glutamyl groups. In other embodiments, the αPMTX / therapeutic agent complex comprises one or more αPMTXs containing 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 glutamyl groups. In one embodiment, the complex comprises one or more αPMTXs containing 3 to 10 glutamyl groups. In a further embodiment, the αPMTX / therapeutic agent conjugate comprises one or more αPMTXs containing 3 to 7 glutamyl groups. In another embodiment, the αPMTX / therapeutic agent conjugate comprises one or more αPMTXs containing 5 glutamyl groups. In another embodiment, the αPMTX / therapeutic agent conjugate comprises one or more αPMTXs containing 6 glutamyl groups. In some embodiments, the therapeutic agent is a cytotoxic compound, or a salt or acid thereof. In a further embodiment, the therapeutic agent is a chemotherapeutic agent, or a salt or acid thereof. In another embodiment, the 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 αPMTX to therapeutic agent in the conjugate is in the range of 1 to 10:1. In some embodiments, the molar ratio of αPMTX / therapeutic agent in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the molar ratio of αPMTX / therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1.In some embodiments, the αPMTX / therapeutic agent complex is encapsulated in a liposome (eg, as described herein or otherwise known in the art).

[0138] In alternative embodiments, the αPMTX complex comprises αPMTX and cyclodextrin. In some embodiments, the molar ratio of αPMTX (e.g., αPMTX 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 αPMTX 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 αPMTX 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 αPMTX / 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 αPMTX / 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 αPMTX / cyclodextrin in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the αPMTX / cyclodextrin complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0139] In some embodiments, the present disclosure provides a composition comprising an αPMTX / 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 αPMTX / 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 αPMTX / 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 αPMTX / 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 αPMTX / 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 αPMTX to platinum-based chemotherapeutic 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 αPMTX to platinum-based chemotherapeutic 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 αPMTX / platinum-based drug in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPMTX / taxane-based drug complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0140] In further embodiments, the αPMTX / 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 αPMTX / 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 αPMTX / 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 αPMTX / 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 αPMTX / 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 some embodiments, the molar ratio of αPMTX to platinum-based drug in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPMTX / platinum-based analog 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 αPMTX and cisplatin, or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / 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 αPMTX / 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 αPMTX / 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 αPMTX / 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 some embodiments, the molar ratio of αPMTX / cisplatin (or a cisplatin salt or acid) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPMTX / cisplatin (or a cisplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0142] In another embodiment, the present disclosure provides a complex comprising αPMTX and carboplatin, or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / 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 αPMTX / 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 αPMTX / 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 αPMTX / 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 some embodiments, the molar ratio of αPMTX / cyclodextrin in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPMTX / carboplatin (or carboplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0143] In another embodiment, the present disclosure provides a complex comprising αPMTX and carboplatin, or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / 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 αPMTX / 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 αPMTX / 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 αPMTX / 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 some embodiments, the molar ratio of αPMTX / oxaliplatin (or oxaliplatin salt or acid) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPMTX / oxaliplatin (or oxaliplatin 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 conjugate comprising αPMTX 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 αPMTX / 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 αPMTX / 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 αPMTX / 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 αPMTX to 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 αPMTX to platinum (or platinum salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1.In some embodiments, the molar ratio of αPMTX / platinum (or platinum salt or acid) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPMTX / platinum (or salt or acid or analog thereof) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0145] In some embodiments, the present disclosure provides a composition comprising an αPMTX / 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 αPMTX / taxane in the conjugate is in the range of 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of αPMTX / 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 αPMTX / 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 αPMTX / 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 some embodiments, the molar ratio of αPMTX to taxane (or taxane salt or acid) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPMTX / taxane complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0146] In further embodiments, the present disclosure provides a complex comprising αPMTX and paclitaxel (PTX) or a salt or acid thereof. In other embodiments, the αPMTX / taxane chemotherapeutic agent complex comprises a paclitaxel (PTX) analog, or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / 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 αPMTX / 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 αPMTX / 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 αPMTX / 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 some embodiments, the molar ratio of αPMTX / paclitaxel (or paclitaxel salt or acid) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPMTX / paclitaxel (or paclitaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0147] In further embodiments, the present disclosure provides a complex comprising αPMTX and docetaxel (DTX) or a salt or acid thereof. In other embodiments, the αPMTX / taxane chemotherapeutic agent complex comprises a docetaxel (DTX) analog, or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / 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 αPMTX / 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 αPMTX / 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 αPMTX / 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 some embodiments, the molar ratio of αPMTX / docetaxel (or a docetaxel salt or acid) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPMTX / docetaxel (or a docetaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0148] In further embodiments, the present disclosure provides a complex comprising αPMTX and larotaxel (LTX) or a salt or acid thereof. In other embodiments, the αPMTX / taxane chemotherapeutic agent complex comprises an analog of larotaxel (LTX), or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / 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 αPMTX / 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 αPMTX / 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 ααPMTX / 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 some embodiments, the molar ratio of αPMTX / larotaxel (or larotaxel salt or acid) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPMTX / larotaxel (or larotaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0149] In further embodiments, the present disclosure provides a conjugate comprising αPMTX and cabazitaxel (CTX) or a salt or acid thereof. In other embodiments, the αPMTX / taxane chemotherapeutic agent conjugate comprises a cabazitaxel (CTX) analog, or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / cabazitaxel (or cabazitaxel 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 αPMTX / cabazitaxel (or cabazitaxel 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 αPMTX / cabazitaxel (or cabazitaxel 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 αPMTX / 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 some embodiments, the molar ratio of αPMTX / cabazitaxel (or cabazitaxel salt or acid) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPMTX / cabazitaxel (or cabazitaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0150] In further embodiments, the present disclosure provides a complex comprising αPMTX and another antimetabolite, or a salt or acid thereof. An antimetabolite is a chemical compound that is similar in structure to a metabolite required for normal biochemical reactions, but differs sufficiently in structure to interfere with one or more normal cell functions, such as cell division. In some embodiments, the present disclosure provides a complex comprising αPMTX and methotrexate (MTX), or a salt or acid thereof. In some embodiments, the present disclosure provides a complex comprising αPMTX 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 αPMTX 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 αPMTX 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 αPMTX 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 αPMTX / 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 some embodiments, the molar ratio of αPMTX / antimetabolite (or antimetabolite salt or acid) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50.In a further embodiment, the αPMTX / anti-metabolite (or an antimetabolite salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0151] In a further embodiment, the present disclosure provides a complex of αPMTX (e.g., the αPMTX 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 internal 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 altering the physicochemical properties of these complexed drugs.

[0152] 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.

[0153] As used herein, there are no particular limitations on the cyclodextrin component of the αPMTX / cyclodextrin complex, so long as the cyclodextrin can form a complex with αPMTX. 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 αPMTX and / or liposome encapsulation.

[0154] 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 αPMTX / 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] In some embodiments, the "ionizable" or "charged" derivatized cyclodextrins, whether polyionic, amphiphilic, or otherwise, are generally weakly ionizable (i.e., have 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).

[0159] 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.

[0160] 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.

[0161] 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 α-D-glucopyranoside unit among all three of the C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and C2-C3-C6 hydroxyls is substituted with an ionizable chemical group. Any such hydroxyl combination can be combined with any of the degrees of substitution described herein, as well as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and up to all α-D-glucopyranoside units in the modified cyclodextrin. One such derivative is sulfoalkyl ether cyclodextrin (SAE-CD). The sulfobutyl ether derivative of beta cyclodextrin (SBE-β-CD) has been shown to have significantly improved aqueous solubility compared to the parent cyclodextrin.

[0162] Additional cyclodextrin derivatives that can be complexed with therapeutic agents in the disclosed liposomal compositions include sugammadex or Org-25969, in which the 6-hydroxy group on γ-CD has been replaced with a carboxythioacetate ether linkage, and hydroxybutenyl-β-CD. Alternative forms of cyclodextrin include 2,6-di-O-methyl-β-CD (DIMEB), 2-hydroxypropyl-3-cyclodextrin (HP-β-CD), randommethylated-β-cyclodextrin (RAMEB), sulfobutyl ether Examples of suitable cyclodextrins include β-cyclodextrin (SBE-β-CD), sulfobutylether-γ-cyclodextrin (SBEγCD), sulfobutylated-β-cyclodextrin sodium salt, (2-hydroxypropyl)-alpha-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, 2,6-di-O-methyl)-β-cyclodextrin (DIMEB-50 heptakis), 2,3,6-tri-O-methyl)-β-cyclodextrin (TRIMEB heptakis), methyl-β-cyclodextrin, octakis(6-deoxy-6-iodo)-γ-cyclodextrin, and octakis(6-deoxy-6-bromo)-γ-cyclodextrin.

[0163] 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).

[0164] In some embodiments, a large binding constant between the cyclodextrin and αPMTX and / or other therapeutic agents 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.

[0165] In some embodiments, the cyclodextrin of the αPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is underivatized.

[0166] In some embodiments, the cyclodextrin of the αPMTX / 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).

[0167] In some embodiments, the cyclodextrin derivatization of the αPMTX / cyclodextrin complex and / or cyclodextrin / therapeutic agent complex is carried out using a cyclodextrin derivative of 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-CH2CH2CH2S03- or -O-CH2CH2CH2CH2S03-); 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.

[0168] In some embodiments, the cyclodextrin derivatization of the αPMTX / 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.

[0169] In some embodiments, the cyclodextrin derivative of the αPMTX / cyclodextrin complex and / or 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.

[0170] In some embodiments, the cyclodextrin derivative of the αPMTX / 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.

[0171] In a further embodiment, the αPMTX / cyclodextrin complex and / or cyclodextrin / therapeutic agent complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0172] III. αPMTX Delivery Carriers In alternative embodiments, the present disclosure provides αPMTX delivery systems and their use for delivering a payload of αPMTX to a cell or cells in vitro or in vivo. In some embodiments, αPMTX 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-αPMTX 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.

[0173] A. Liposomes In some embodiments, the present disclosure provides a liposome composition comprising liposomes encapsulating (loaded with) alpha polyglutamated methotrexate (e.g., αPMTX as disclosed herein). In some embodiments, the liposomes in the liposome composition comprise αPMTX 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 alpha polyglutamated methotrexate in Lp-αPMTX contains two or more L-glutamyl groups. In other embodiments, the alpha polyglutamated methotrexate in Lp-αPMTX contains D-glutamyl groups. In further embodiments, the alpha polyglutamated methotrexate in Lp-αPMTX contains D-glutamyl groups and two or more L-glutamyl groups. In further embodiments, the alpha polyglutamated methotrexate in Lp-αPMTX contains two or more glutamyl groups with gamma carboxyl linkages. In some embodiments, the alpha polyglutamated methotrexate in Lp-αPMTX contains at least one glutamyl group with both an alpha carboxyl linkage and a gamma carboxyl linkage. In some embodiments, the liposome composition comprises liposomes containing pentaglutamated MTX. In further embodiments, the liposomes comprise L-α pentaglutamated MTX, D-α pentaglutamated MTX, or L- and D-α pentaglutamated MTX. In some embodiments, the liposome composition comprises liposomes containing hexaglutamated MTX (Lp-αPMTX). In further embodiments, the liposomes comprise L-α hexaglutamated MTX, D-α hexaglutamated MTX, or L- and D-α 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-αPMTX composition is not PEGylated. In some embodiments, the Lp-αPMTX composition is non-targeted (NTLp-αPMTX).In other embodiments, the Lp-αPMTX composition is targeted (TLp-αPMTX). 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, 30-70%, 30-60%, or 30-50% w / w of alpha polyglutamated methotrexate, or any range therebetween, is encapsulated (enclosed) in Lp-αPMTX. 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% of the alpha polyglutamated methotrexate is encapsulated in Lp-αPMTX during the liposome preparation process.

[0174] In some embodiments, provided liposomes further comprise an immunostimulatory agent, a detectable marker, or both, disposed on the exterior surface of the liposome. The immunostimulatory agent or detectable marker can be ionically or covalently bound to the exterior surface of the liposome, which may optionally include binding to a steric stabilizing component of the liposome.

[0175] 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 gamma, IFN alpha, 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.

[0176] 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.

[0177] 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.

[0178] In some embodiments, the liposome further comprises an agent that increases uptake of the liposome into a desired intracellular compartment, including the cytosol.

[0179] 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), GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCG AGCTGAGCGGCGTGGCGCGGGGGCGTCAT (SEQ ID NO: 4), ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTCATCCGTCAGCTC (SEQ ID NO: 5), or ACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTG (SEQ ID NO: 6)), or a mitochondrial permeability fragment thereof.

[0180] 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).

[0181] In some embodiments, the liposomes in the provided liposome compositions comprise sphingomyelin and / or stearyloctaarginine. In some embodiments, the liposomes comprise sphingomyelin and / or stearyloctaarginine. In some embodiments, the liposomes comprise DOPE, sphingomyelin, stearyloctaarginine sphingomyelin, and stearyloctaarginine. In some embodiments, the liposomes comprise DOPE, sphingomyelin, stearyloctaarginine sphingomyelin, and stearyloctaarginine in a molar ratio of 9:2:1. In some embodiments, the liposomes comprise a MITO porter system or a variant thereof.

[0182] 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.

[0183] In some embodiments, the liposome comprises a mitochondrial penetrant selected from the group consisting of guanidine-rich peptoids, tetraguanidinium, triguanidinium, diguanidinium, monoguanidinium, guanidine-rich polycarbamates, beta-oligoarginines, proline-rich dendrimers, and phosphonium salts (e.g., methyltriphenylphosphonium and / or tetraphenylphosphonium).

[0184] In some embodiments, the liposome comprises sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposome comprises sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposome comprises DOPE, sphingomyelin, stearyl octaarginine sphingomyelin, and stearyl octaarginine. In some embodiments, the liposome comprises DOPE, sphingomyelin, stearyl octaarginine sphingomyelin, and stearyl octaarginine in a molar ratio of 9:2:1. In some embodiments, the liposome comprises a MITO porter system or a variant thereof.

[0185] In some embodiments, the liposomes contain an agent, such as a membrane permeabilizing agent, that facilitates delivery of the liposomes across the 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 production and use of αPMTX 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 liposome comprises a membrane permeabilizing agent selected from the group consisting of: RQIKIWFQNRRMKWKKRKKRRQRRR (SEQ ID NO: 1), RKKRRXRRRGC (where X is any natural or unnatural amino acid) (SEQ ID NO: 2), CCGCCAAGAAGCG (SEQ ID NO: 3), GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTGAGCGGCGTGGCGCGGGGGCGTCAT (SEQ ID NO: 4), ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTCATCCGTCAGCTC (SEQ ID NO: 5), or ACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTG (SEQ ID NO: 6), or a mitochondrial permeabilizing fragment thereof.

[0186] 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).

[0187] 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.

[0188] 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.

[0189] 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.

[0190] In some embodiments, the liposome composition comprises PEGylated liposomes (PLp-αPMTX). In some embodiments, the PEGylated liposomes in the liposome composition comprise αPMTX containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the alpha polyglutamated methotrexate in Lp-αPMTX contains two or more L-glutamyl groups. In other embodiments, the alpha polyglutamated methotrexate in Lp-αPMTX contains D-glutamyl groups. In further embodiments, the alpha polyglutamated methotrexate in Lp-αPMTX contains D-glutamyl groups and two or more L-glutamyl groups. In further embodiments, the alpha polyglutamated methotrexate in Lp-αPMTX contains two or more glutamyl groups with gamma linkages. In some embodiments, at least one glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, the liposome composition comprises PEGylated liposomes comprising α-pentaglutamated MTX. In further embodiments, the liposomes comprise L-α-pentaglutamated MTX, D-α-pentaglutamated MTX, or L- and D-α-pentaglutamated MTX. In some embodiments, the liposome composition comprises PEGylated liposomes comprising α-hexaglutamated MTX. In further embodiments, the liposomes comprise L-α-hexaglutamated MTX, D-α-hexaglutamated MTX, or L- and D-α-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-αPMTX composition is non-targeted (NTPLp-αPMTX). In other embodiments, the PLp-αPMTX composition is targeted (TPLp-αPMTX). In further embodiments, the liposome composition comprises PEGylated liposomes containing 30-70%, 30-60%, or 30-50% liposome-encapsulated alpha polyglutamated methotrexate, or any range therebetween.In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w / w) of the alpha polyglutamated methotrexate is encapsulated in PLp-αPMTX. In some embodiments, the liposome composition comprises PEGylated liposomes having a diameter ranging from 20 nm to 500 nm. In some embodiments, the liposome composition comprises PEGylated liposomes having a diameter ranging from 20 nm to 200 nm. In further embodiments, the liposome composition comprises PEGylated liposomes having a diameter ranging from 80 nm to 120 nm.

[0191] In some embodiments, greater than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamated methotrexate in the composition has 4 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, greater than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamated methotrexate in the provided liposome compositions is tetraglutamated. In some embodiments, greater than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamated methotrexate in the provided liposome compositions is pentaglutamated. In some embodiments, greater than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamated methotrexate in the provided liposome compositions is hexaglutamated.

[0192] In some embodiments, the alpha polyglutamated methotrexate composition (e.g., a delivery vehicle such as alpha polyglutamates and liposomes containing polyglutamates) is in an aqueous solution. In some embodiments, the αPMTX composition is delivered as a liposome composition in an amount of 1 square meter (m 2In a further embodiment, the αPMTX composition is administered as a liposome composition at a dose of about 0.1 to about 1000 mg of αPMTX per square meter of body surface area, or at any dose range therebetween.

[0193] (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, alpha-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, amphiphilic molecules in the bilayer are arranged in a two-dimensional sheet, with the hydrophobic portions facing the inside of the sheet, while 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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 alpha 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] Anionic and neutral lipids are collectively referred to herein 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), distearoylphosphatidyl-1-ethanolamine (DSPE), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), 16-O-monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, palmitoyloleoylphosphatidylethanolamine (POPE), 1-stearoyl-2-oleoylphosphatidiethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, and cholesterol.

[0204] 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.

[0205] In some embodiments, the αPMTX compositions provided herein are formulated in liposomes comprising a cationic lipid. In one embodiment, the cationic lipid is, but is not limited to, a cationic lipid 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] In some embodiments, the αPMTX 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 αPMTX is formulated in a lipid-polycation complex, which further includes a neutral lipid such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0210] The liposome components can include any molecule (i.e., 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.

[0211] 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.

[0212] 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.

[0213] In some embodiments, the liposomal alpha polyglutamated methotrexate composition is PEGylated (i.e., PEGylated liposomal alpha polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate (PLp-αPMTX or PLp-αPMTX)). In some embodiments, the PLp-αPMTX or PLp-αPMTX is water-soluble. That is, the PLp-αPMTX or PLp-αPMTX is in the form of an aqueous solution.

[0214] 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.

[0215] In some embodiments, the pH of the solution containing the liposome composition is between pH 2 and 8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is between pH 5 and 8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is between pH 6 and 7, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is between pH 6 and 7.5, 6.5 and 7.5, 6.7 and 7.5, or 6.3 and 7.0, or any range therebetween.

[0216] 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).

[0217] 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.

[0218] 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).

[0219] (2) Liposome internal space In further non-limiting embodiments, provided liposomes comprise 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 alpha 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 liposome interior space is between 2 and 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.

[0220] 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%, 5-20%, or 1-50%, 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% 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 15-200 mM, or any range therebetween. In still further embodiments, the HBS citrate buffer is 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 15-200 mM, or any range therebetween. In some embodiments, the buffer is citrate at 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.

[0221] 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%, 5-20%, or 1-50%, 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 dextrose concentration of about 5%-20% (wt%), 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 som...

Claims

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

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