Alpha polyglutamylated pemetrexed and uses thereof

Abstract

To provide alpha polyglutamated pemetrexed compositions to overcome pharmacological challenges related to dose limiting toxicities and treatment resistance associated with pemetrexed therapy.SOLUTION: Disclosed is a liposome composition comprising a liposome encapsulating alpha polyglutamated pemetrexed, where the alpha polyglutamated pemetrexed comprises 2-10 glutamyl groups having an alpha carboxyl group linkage, the liposome is PEGylated, and the liposome composition comprises a targeting moiety that has a specific affinity to a surface antigen on a target cell.SELECTED DRAWING: None

Description

[Background technology]

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

[0002] Pemetrexed disodium is sold under the trade name ALIMTA® (Eli Lilly and Company) and has the chemical name L-glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-, disodium salt, heptahydrate (molecular formula: C 20 H 19 Also known as N5Na2O6·7H2O), it is the active ingredient in antineoplastic drug products approved as a single agent for the treatment of locally advanced or metastatic non-small cell lung cancer and in combination with cisplatin for the treatment of patients with malignant pleural mesothelioma. Pemetrexed has demonstrated activity in clinical trials in a variety of tumor types, including lung cancer, breast cancer, colon cancer, mesothelioma, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, and cervical cancer.

[0003] Folate is an essential cofactor mediating the transfer of one-carbon units involved in nucleotide biosynthesis and DNA repair, homocysteine ​​remethylation (Hcy), and methylation of DNA, proteins, and lipids. The only circulating form of folate in the blood is monoglutamate, and folate monoglutamate is the only form of folate transported across cell membranes, as are monoglutamate-type polyglutamate-compatible antifolate drugs such as pemetrexed. Once inside cells, intracellular folate is converted to polyglutamate by the enzyme folylpolygammaglutamate synthase (FPGS).

[0004] Pemetrexed is a multitarget antifolate drug that acts by disrupting folate-dependent metabolic processes essential for cellular homeostasis and replication. Pemetrexed inhibits three enzymes required for purine and pyrimidine biosynthesis: thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFTase). Inhibition of these enzymes suppresses de novo nucleotide biosynthesis, resulting in disruption of cellular homeostasis and an imbalance of purine and pyrimidine precursors, which prevents accurate DNA replication and ultimately leads to cell death.

[0005] Pemetrexed 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 primary pemetrexed transporter at physiological pH and is widely expressed in normal and diseased cells. Therefore, pemetrexed is often subject to dose-limiting toxicity, a major obstacle in cancer chemotherapy. Once inside cells, pemetrexed is polyglutamylated by FPGS, which can add up to six L-glutamyl groups to the L-gamma carboxyl group bond of pemetrexed. L-gamma polyglutamylation of pemetrexed by FPGS serves at least two major therapeutic purposes: (1) it greatly enhances the affinity and inhibitory activity of pemetrexed for several folate-dependent enzymes, including thymidylate synthase and GARFTase; and (2) it facilitates the accumulation of polyglutamylated pemetrexed, which, unlike pemetrexed (monoglutamate), is not readily transported out of cells by cellular efflux pumps.

[0006] Pemetrexed 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 pemetrexed therapy, limiting its clinical application. Pretreatment with folic acid and B vitamins is currently used to alleviate the most common side effects associated with pemetrexed therapy, including myelosuppression, fatigue, and rash.

[0007] Resistance to pemetrexed therapy is usually associated with one or more of the following: (a) increased cellular efflux pump activity, (b) increased thymidylate synthetase activity, (c) decreased folylpoly-gamma-glutamate synthetase (FPGS) activity, and (d) increased gamma-glutamyl hydrolase (GGH) activity, which cleaves gamma-polyglutamate chains attached to folate and antifolate drugs.

[0008] 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.

[0009] The provided alpha-polyglutamated pemetrexed compositions offer a strategy to overcome the pharmacological challenges associated with dose-limiting toxicity and therapeutic resistance associated with pemetrexed therapy. The provided methods deliver novel alpha-polyglutamated forms of pemetrexed to cancer cells while (1) minimizing / reducing exposure to normal tissue cells, (2) optimizing / improving the cytotoxic effects of pemetrexed-based drugs against cancer cells, and (3) minimizing / reducing the effects of efflux pumps and other resistance mechanisms that limit the therapeutic efficacy of pemetrexed. Summary of the Invention

[0010] The present disclosure relates generally to novel alpha polyglutamated pemetrexed (PMX) 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.

[0011] In some embodiments, the present disclosure provides: [1] A composition comprising alpha polyglutamated pemetrexed, wherein at least one glutamyl group has an alpha carboxyl group bond; [2] The composition according to item [1], wherein the alpha polyglutamylated pemetrexed contains 1 to 10 glutamyl groups having alpha carboxyl group bonds; [3] The composition according to item [1] or [2], wherein the alpha polyglutamated pemetrexed 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 pemetrexed; [5] The composition according to any one of items [1] to [3], comprising alpha-pentaglutamated pemetrexed; [6] The composition according to any one of items [1] to [3], comprising alphahexaglutamated pemetrexed; [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 pemetrexed has an alpha carboxyl linkage; or (c) a composition in which two or more glutamyl groups have a gamma carboxyl group bond; [8] The composition according to any one of items [1] to [6], wherein the composition is: (a) the C-terminal glutamyl group(s) and each glutamyl group other than the glutamyl group of pemetrexed have an alpha carboxyl group bond; 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 pemetrexed are in the L-configuration; (b) each glutamyl group of the alpha polyglutamated pemetrexed is in the L-configuration; (c) at least one glutamyl group of the alpha polyglutamated pemetrexed is in the D-form; (d) each glutamyl group of said alpha polyglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form; or (e) at least two of the glutamyl groups of the alpha polyglutamated pemetrexed 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-αPPMX) containing alpha polyglutamated pemetrexed according to any one of items [1] to

[12] ;

[14] The LαPP composition according to item

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

[15] The Lp-αPPMX composition according to item

[13] or

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

[16] The Lp-αPPMX composition according to item

[13] or

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

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

[13] to

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

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

[13] to

[17] , wherein at least one glutamyl group of the alpha polyglutamylated pemetrexed 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

[18] , comprising 2, 3, 4, 5, 6, 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-αPPMX composition according to any one of items

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

[28] The Lp-αPPMX composition according to item

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

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

[27] to

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

[32] The Lp-αPPMX 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-αPPMX composition according to item

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

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

[13] to

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

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

[13] to

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

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

[13] to

[33] , wherein the liposome is cationic;

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

[13] to

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

[40] The Lp-αPPMX composition according to item

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

[41] The Lp-αPPMX composition according to item

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

[42] The Lp-αPPMX composition according to item

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

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

[39] to

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

[46] The Lp-αPPMX 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-αPPMX 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-αPPMX 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 pemetrexed molecules;

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

[13] to

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

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

[50] , wherein the targeting moiety is attached to one or both of the PEG and the exterior surface of the liposome, and optionally the targeting moiety is covalently attached to one or both of the PEG and the exterior surface of the liposome;

[52] The Lp-αPPMX composition according to item

[50] or

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

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

[50] to

[53] , wherein the targeting moiety is at least 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-αPPMX 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-αPPMX 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-αPPMX 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-αPPMX composition according to

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

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

[39] to

[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-αPPMX 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 D (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-αPPMX composition according to any one of items

[58] to

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

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

[58] to

[61] , further comprising a hapten;

[63] The Lp-αPPMX composition according to item

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

[64] The Lp-αPPMX 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 the internal space and the external space;

[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-αPPMX 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 pemetrexed composition according to any one of items

[13] to

[67] ;

[69] A pharmaceutical composition comprising the alpha polyglutamated pemetrexed 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 pemetrexed 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 pemetrexed 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 pemetrexed 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 a sarcoma such as osteosarcoma;

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

[50] to

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

[84] A maintenance therapy comprising administering an effective amount of the composition according to any one of items [1] to

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

[85] A maintenance therapy comprising administering an effective amount of the liposomal alpha polyglutamated pemetrexed 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 pemetrexed composition according to any one of items [9] to

[69] to a subject having or at risk of having an immune system disorder;

[88] 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;

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

[69] in an amount sufficient to deliver a therapeutically effective amount of alpha polyglutamated pemetrexed to the tumor;

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

[13] to

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

[92] A method for preparing an alpha polyglutamated pemetrexed composition, including the liposomal alpha polyglutamated pemetrexed composition according to any one of items

[13] to

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

[93] The method according to item

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

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

[13] to

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

[96] The method according to item

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

[97] The method according to any one of items

[94] to

[96] , 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;

[98] The method according to any one of items

[94] 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 polyglutamylated PMX starting material is encapsulated or entrapped in Lp-αPPMX.

[0012] In some embodiments, the present disclosure provides alpha polyglutamated pemetrexed (αPPMX) compositions, wherein at least one glutamyl residue of alpha polyglutamated pemetrexed is linked via its alpha carboxyl group. In some embodiments, αPPMX contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl group of pemetrexed). In some embodiments, αPPMX contains two or more glutamyl groups in the L-form. In other embodiments, αPPMX contains a glutamyl group in the D-form. In further embodiments, αPPMX contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In further embodiments, αPPMX contains two or more glutamyl groups with gamma linkages. In some embodiments, at least one glutamyl group has both alpha and gamma linkages.

[0013] In one embodiment, the αPPMX composition contains a chain of three glutamyl groups linked to a glutamyl group in pemetrexed (i.e., tetraglutamylated pemetrexed). In some embodiments, the tetraglutamylated PMX contains two or more L-glutamyl groups. In other embodiments, the tetraglutamylated PMX contains a D-glutamyl group. In a further embodiment, the tetraglutamylated PMX contains a D-glutamyl group and two or more L-glutamyl groups. In a further embodiment, the tetraglutamylated PMX contains two or more glutamyl groups with gamma linkages.

[0014] In one embodiment, the αPPMX composition comprises a chain of four glutamyl groups linked to a glutamyl group in pemetrexed (i.e., pentaglutamated pemetrexed). In some embodiments, the pentaglutamated PMX comprises two or more L-glutamyl groups. In other embodiments, the pentaglutamated PMX comprises a D-glutamyl group. In a further embodiment, the pentaglutamated PMX comprises a D-glutamyl group and two or more L-glutamyl groups. In a further embodiment, the pentaglutamated PMX comprises two or more glutamyl groups with gamma linkages.

[0015] In one embodiment, the αPPMX composition contains a chain of five glutamyl groups linked to a glutamyl group in pemetrexed (i.e., hexaglutamated pemetrexed). In some embodiments, the hexaglutamated PMX contains two or more L-glutamyl groups. In other embodiments, the hexaglutamated PMX contains a D-glutamyl group. In a further embodiment, the hexaglutamated PMX contains a D-glutamyl group and two or more L-glutamyl groups. In a further embodiment, the hexaglutamated PMX contains two or more glutamyl groups with gamma linkages.

[0016] 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 pemetrexed, as well as methods for making and using αPPMX-loaded / bound delivery vehicle compositions to deliver alpha polyglutamated pemetrexed 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. αPPMX-loaded / bound delivery vehicle compositions provide selective delivery of a more cytotoxic payload (e.g., polyglutamated pemetrexed) compared to the cytotoxicity of pemetrexed administered in its monoglutamate state (PMX), thereby improving the efficacy and safety of pemetrexed delivery to cancer cells.

[0017] In further embodiments, the present disclosure provides a composition (Lp-αPPMX) comprising liposomes encapsulating (loaded with) alpha polyglutamated pemetrexed. In some embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 20 glutamyl groups (including the glutamyl group of pemetrexed). In some embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains two or more L-glutamyl groups. In other embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains D-glutamyl groups. In further embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains D-glutamyl groups and two or more L-glutamyl groups. In further embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains two or more glutamyl groups with gamma linkages. In further embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains one or more glutamyl groups with both alpha and gamma linkages. In some embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX 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 pemetrexed is linear. In some embodiments, the polyglutamate chain of the alpha polyglutamated pemetrexed is branched.

[0018] In one embodiment, the Lp-αPPMX composition comprises alpha polyglutamated PMX comprising a chain of three glutamyl groups linked to a glutamyl group of pemetrexed (i.e., tetraglutamated pemetrexed). In some embodiments, the tetraglutamated PMX comprises two or more L-glutamyl groups. In other embodiments, the tetraglutamated PMX comprises a D-glutamyl group. In further embodiments, the tetraglutamated PMX comprises a D-glutamyl group and two or more L-glutamyl groups. In further embodiments, the tetraglutamated PMX comprises two or more glutamyl groups with gamma linkages. In some embodiments, the polyglutamate chain of the alpha polyglutamated pemetrexed is linear. In some embodiments, the polyglutamate chain of the alpha polyglutamated pemetrexed is branched.

[0019] In one embodiment, the Lp-αPPMX composition comprises alpha polyglutamated PMX comprising a chain of four glutamyl groups linked to a glutamyl group of pemetrexed (i.e., pentaglutamated pemetrexed). In some embodiments, the pentaglutamated PMX comprises two or more L-glutamyl groups. In other embodiments, the pentaglutamated PMX comprises a D-glutamyl group. In further embodiments, the pentaglutamated PMX comprises a D-glutamyl group and two or more L-glutamyl groups. In further embodiments, the pentaglutamated PMX comprises two or more glutamyl groups with gamma linkages. In some embodiments, the polyglutamate chain of the alpha polyglutamated pemetrexed is linear. In some embodiments, the polyglutamate chain of the alpha polyglutamated pemetrexed is branched.

[0020] In one embodiment, the Lp-αPPMX composition comprises alpha polyglutamated PMX comprising a chain of five glutamyl groups linked to a glutamyl group of pemetrexed (i.e., hexaglutamated pemetrexed). In some embodiments, the hexaglutamated PMX comprises two or more L-glutamyl groups. In other embodiments, the hexaglutamated PMX comprises a D-glutamyl group. In further embodiments, the hexaglutamated PMX comprises a D-glutamyl group and two or more L-glutamyl groups. In further embodiments, the hexaglutamated PMX comprises two or more glutamyl groups with gamma linkages. In some embodiments, the polyglutamate chain of the alpha polyglutamated pemetrexed is linear. In some embodiments, the polyglutamate chain of the alpha polyglutamated pemetrexed is branched.

[0021] In some embodiments, the Lp-αPPMX composition is cationic. In some embodiments, the Lp-αPPMX liposomes are cationic and have diameters ranging from 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-αPPMX 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-αPPMX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of alpha polyglutamylated PMX. 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 PMX starting material is encapsulated in cationic Lp-αPPMX during the process of making Lp-αPPMX. In further embodiments, the alpha polyglutamated pemetrexed encapsulated by the liposomes is present in a HEPES buffer within the liposomes.

[0022] In other embodiments, the Lp-αPPMX composition is anionic or neutral. In some embodiments, the Lp-αPPMX composition is cationic. In some embodiments, the Lp-αPPMX liposomes are anionic or neutral and have diameters ranging from 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-αPPMX liposomes are anionic or neutral and have diameters ranging from 80 nm to 120 nm, or any range therebetween. In some embodiments, the Lp-αPPMX liposomes are anionic and have diameters ranging from 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-αPPMX 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-αPPMX 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-αPPMX 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, anionic or neutral Lp-αPPMX compositions comprise 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 polyglutamylated PMX. In some embodiments, during the process of making Lp-αPPMX, 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 PMX starting material is encapsulated (encapsulated) in anionic or neutral Lp-αPPMX.In some embodiments, anionic or neutral Lp-αPPMX compositions comprise 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 PMX. In some embodiments, anionic or neutral Lp-αPPMX compositions comprise 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 PMX. In some embodiments, the anionic or neutral Lp-αPPMX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% w / w of alpha hexaglutamated PMX. In further embodiments, the liposomally encapsulated alpha polyglutamated pemetrexed is present in a HEPES buffer solution within the liposomes.

[0023] In a further embodiment, the liposomal alpha polyglutamated pemetrexed composition is PEGylated (PLp-αPPMX).

[0024] In some embodiments, the liposomal alpha polyglutamated pemetrexed composition is non-targeted (NTLp-αPPMX). That is, the NTLp-αPPMX 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 pemetrexed composition is pegylated (NTPLp-αPPMX).

[0025] In other embodiments, the liposomal alpha-polyglutamated pemetrexed compositions are targeted (TLp-αPPMX). That is, the TLp-αPPMX compositions include a targeting moiety that has specific affinity for an epitope (surface antigen) on a target cell of interest. In some embodiments, the TLp-αPPMX or TPLp-αPPMX is not covalently attached to the liposome. In other embodiments, the targeting moiety of the TLp-αPPMX or TPLp-αPPMX is attached to one or both of the PEG and the exterior surface of the liposome. The targeted liposomal alpha-polyglutamated pemetrexed compositions (TLp-αPPMX and TPLp-αPPMX) provide further improvements over the efficacy and safety profile of pemetrexed by specifically delivering alpha-polyglutamated (e.g., tetraglutamated, pentaglutamated, and hexaglutamated) pemetrexed to target cells, such as cancer cells. In a further embodiment, the targeted liposomal alpha polyglutamated pemetrexed composition is PEGylated (TPLp-αPPMX). The functions of the targeting moiety of the TLp-αPPMX and / or TPLp-αPPMX 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 for which the targeting moiety has specific affinity; and delivering the liposomal payload (αPPMX) to the cell.

[0026] 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

[0027] 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 specifically binds to 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.

[0028] In further embodiments, the αPPMX composition comprises one or more of an immunostimulant, a detectable marker, and a maleimide disposed on at least one of the PEG or exterior surface of the liposome. In some embodiments, the liposomal αPPMX composition (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX) is cationic. In other embodiments, the liposomal αPPMX composition (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX) is anionic or neutral. In further embodiments, the liposomes of the liposomal αPPMX composition (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX) have diameters ranging from 20 nm to 500 nm, or any range therebetween. In further embodiments, the liposomes of the liposomal αPPMX composition have diameters ranging from 80 nm to 120 nm, or any range therebetween. In some embodiments, the liposomal αPPMX composition is PEGylated (e.g., PLp-αPPMX, NTPLp-αPPMX, or TPLp-αPPMX). In some embodiments, the liposomal αPPMX composition is targeted (e.g., TLp-αPPMX or TPLp-αPPMX). In further embodiments, the liposomal αPPMX composition is both PEGylated and targeted (e.g., TPLp-αPPMX). In some embodiments, the liposomal αPPMX composition comprises alpha polyglutamated pemetrexed containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomal αPPMX composition comprises alpha tetraglutamated pemetrexed. In some embodiments, the liposomal αPPMX composition comprises alpha pentaglutamated pemetrexed. In other embodiments, the liposomal αPPMX composition comprises alpha hexaglutamated pemetrexed.

[0029] In some embodiments, the liposome composition comprises alpha polyglutamated pemetrexed containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% w / w of alpha polyglutamated PMX. In some embodiments, the Lp-αPPMX composition comprises alpha polyglutamated pemetrexed containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups and 1% to 98.5% w / w of alpha polyglutamated PMX. In some embodiments, the liposomes comprise alpha polyglutamated pemetrexed containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups, and during the process of making Lp-αPPMX, 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 pemetrexed starting material is encapsulated (encapsulated) in Lp-αPPMX.

[0030] In some embodiments, the liposome composition consists of alpha-tetraglutamated pemetrexed 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 PMX. In some embodiments, the Lp-αPPMX composition comprises alpha-tetraglutamated pemetrexed and 1% to 98.5% w / w of alpha-tetraglutamated PMX. In some embodiments, the liposomes comprise alpha-tetraglutamated pemetrexed, and during the process of making Lp-αPPMX, 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 pemetrexed starting material is encapsulated (encapsulated) in Lp-αPPMX.

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

[0032] Also provided are liposome compositions comprising αPPMX-encapsulated liposomes. In some embodiments, the liposome composition comprises a PEGylated αPPMX composition. In some embodiments, the liposome composition comprises an αPPMX composition conjugated to or otherwise incorporated into a targeting moiety. In further embodiments, the liposome composition comprises an αPPMX composition that is PEGylated and conjugated to or otherwise incorporated into a targeting moiety. In some embodiments, the liposome composition comprises αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome composition comprises alpha-tetraglutamated pemetrexed. In some embodiments, the liposome composition comprises alpha-pentaglutamated pemetrexed. In other embodiments, the liposome composition comprises alpha-hexaglutamated pemetrexed.

[0033] In some embodiments, the liposome composition comprises liposomal αPPMX (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, and TPLp-αPPMX). In some embodiments, the liposomal αPPMX is pegylated (e.g., NTPLp-αPPMX and TPLp-αPPMX). In some embodiments, the liposomal αPPMX 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-αPPMX or TPLp-αPPMX). In further embodiments, the liposome composition comprises pegylated liposomal αPPMX 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-αPPMX). In some embodiments, the liposome composition comprises liposomal αPPMX that is cationic. In other embodiments, the liposome composition comprises anionic or neutral liposomal αPPMX. In further embodiments, the liposome composition comprises liposomal αPPMX having a diameter of 20 nm to 500 nm, 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposomal αPPMX has a diameter of 80 nm to 120 nm, or any range therebetween.

[0034] Pharmaceutical compositions comprising alpha polyglutamated pemetrexed (αPPMX) in a delivery vehicle, such as liposomal αPPMX, are also provided. In some embodiments, the pharmaceutical composition comprises a pegylated αPPMX composition. In some embodiments, the pharmaceutical composition comprises an αPPMX composition linked to or otherwise incorporated into a targeting moiety. In further embodiments, the pharmaceutical composition comprises an αPPMX composition that is pegylated and linked to or otherwise incorporated into a targeting moiety. In some embodiments, the pharmaceutical composition comprises αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition comprises alpha tetraglutamated pemetrexed. In some embodiments, the pharmaceutical composition comprises alpha pentaglutamated pemetrexed. In other embodiments, the pharmaceutical composition comprises alpha hexaglutamated pemetrexed.

[0035] In some embodiments, the pharmaceutical composition comprises liposomal αPPMX (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, and TPLp-αPPMX). In some embodiments, the liposomal αPPMX composition is pegylated (e.g., NTPLp-αPPMX and TPLp-αPPMX). In some embodiments, the liposomal αPPMX 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-αPPMX or TPLp-αPPMX). In further embodiments, the pharmaceutical composition comprises a pegylated liposomal αPPMX 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-αPPMX). In some embodiments, the pharmaceutical composition comprises cationic liposomal αPPMX. In other embodiments, the pharmaceutical composition comprises anionic or neutral liposomal αPPMX. In a further embodiment, the pharmaceutical composition comprises liposomal αPPMX 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 αPPMX composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

[0036] 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 polyglutamylated pemetrexed (αPPMX) 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 cells are primary cells or cells from a cell line obtained / derived from a cancer selected from the group consisting of: 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 non-hematological tumors, including melanoma; and hematological tumors, such as leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the αPPMX comprises 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the αPPMX composition comprises alpha-tetraglutamylated pemetrexed. In some embodiments, the αPPMX composition comprises alpha-pentaglutamated pemetrexed. In other embodiments, the αPPMX composition comprises alpha-hexaglutamated pemetrexed.

[0037] 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 pemetrexed (i.e., Lp-αPPMX, such as PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX). 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 cell lines obtained / derived from cancers selected from the group consisting of 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 non-hematological tumors, including melanoma; and hematological tumors, such as leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the liposomes comprise αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes comprise alpha-tetraglutamylated pemetrexed. In some embodiments, the liposomes comprise alpha-pentaglutamated pemetrexed. In other embodiments, the liposomes comprise alpha-hexaglutamated pemetrexed.

[0038] In further embodiments, the present disclosure provides methods for treating cancer, the methods comprising administering an effective amount of a delivery vehicle (e.g., an immunoconjugate or liposome) comprising alpha polyglutamated pemetrexed 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-αPPMX, such as PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX). 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, FZD6 , FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD10 5, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C 242 antigens, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery vehicle comprises a targeting moiety that specifically binds to a cell surface antigen, such as a neoantigen, identified as being derived from or expressed 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 αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises alpha tetraglutamated pemetrexed. In some embodiments, the administered delivery vehicle comprises alpha pentaglutamated pemetrexed. In other embodiments, the administered delivery vehicle comprises alpha hexaglutamated pemetrexed. In some embodiments, the administered delivery vehicle comprises L-alpha polyglutamated pemetrexed. In some embodiments, the administered delivery vehicle comprises D-alpha polyglutamated pemetrexed. In some embodiments, the administered delivery vehicle comprises L- and D-alpha polyglutamated pemetrexed. In some embodiments, 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, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. In some embodiments, the cancer is lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer is breast cancer (e.g., HER2++ or triple-negative breast cancer). In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer cells are ovarian cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is head and neck cancer.In some embodiments, the cancer is osteosarcoma.

[0039] In further embodiments, the present disclosure provides methods of treating cancer, the methods comprising administering an effective amount of liposomes comprising alpha polyglutamylated pemetrexed (e.g., Lp-αPPMX, such as PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX) to a subject having or at risk of having cancer. In some embodiments, the liposomes are PEGylated. In some embodiments, the liposomes are not PEGylated. In further embodiments, the liposomes comprise 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, FZD6 , FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD10 5, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C 242 antigens, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.This also includes the use of cancer stem cell targeting moieties, such as those targeting CD34, CD133 and CD44, CD138, and CD15. In some embodiments, the liposomes contain a targeting moiety that specifically binds to a cell surface antigen, such as a neoantigen, determined to be derived from or expressed on a particular target tumor. In some embodiments, the targeting moiety is an antibody or antigen-binding antibody fragment. In some embodiments, the liposomes contain αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes contain alpha tetraglutamated pemetrexed. In some embodiments, the liposomes contain alpha pentaglutamated pemetrexed. In other embodiments, the liposomes contain alpha hexaglutamated pemetrexed. In some embodiments, the liposomes contain L-alpha polyglutamated pemetrexed. In some embodiments, the liposomes contain D-alpha polyglutamated pemetrexed. In some embodiments, the liposome comprises L- and D-alpha polyglutamated pemetrexed. In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, and hematological tumors (e.g., leukemia or lymphoma). In some embodiments, the cancer is lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer is breast cancer (e.g., HER2++ or triple-negative breast cancer). In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer cells are ovarian cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is osteosarcoma.

[0040] In 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 polyglutamylated pemetrexed and a liposome comprising a targeting moiety having specific affinity for an epitope of a cancer surface antigen.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, FZD6 , FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD10 5, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C 242 antigens, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the 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-αPPMX). In some embodiments, the administered liposome composition contains non-PEGylated liposomes. In some embodiments, the administered liposomes contain αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered liposomes contain alpha-tetraglutamated pemetrexed. In some embodiments, the administered liposomes contain alpha-pentaglutamated pemetrexed. In other embodiments, the administered liposomes contain alpha-hexaglutamated pemetrexed. 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 some embodiments, the liposome composition is administered to treat lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the liposome composition is administered to treat breast cancer (e.g., HER2++ or triple-negative breast cancer). In some embodiments, the liposome composition is administered to treat colorectal cancer. In some embodiments, the liposome composition is administered to treat ovarian cancer. In some embodiments, the liposome composition is administered to treat endometrial cancer. In some embodiments, the liposome composition is administered to treat pancreatic cancer, in some embodiments, the liposome composition is administered to treat liver cancer, in some embodiments, the liposome composition is administered to treat head and neck cancer.In some embodiments, the liposome composition is administered to treat osteosarcoma.

[0041] 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 pemetrexed (αPPMX) 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-αPPMX). In some embodiments, the administered liposome composition comprises a non-pegylated liposome. In some embodiments, the liposomes of the administered liposome composition comprise αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated pemetrexed. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutamated pemetrexed. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutamated pemetrexed. In some embodiments, the liposome composition is administered to treat cancers 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, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. In some embodiments, the liposome composition is administered to treat lung cancer (e.g., NSCLC or mesothelioma).In some embodiments, the liposome composition is administered to treat breast cancer (e.g., HER2++ or triple-negative breast cancer). In some embodiments, the liposome composition is administered to treat colorectal cancer. In some embodiments, the liposome composition is administered to treat ovarian cancer. In some embodiments, the liposome composition is administered to treat endometrial cancer. In some embodiments, the liposome composition is administered to treat pancreatic cancer. In some embodiments, the liposome composition is administered to treat liver cancer. In some embodiments, the liposome composition is administered to treat head and neck cancer. In some embodiments, the liposome composition is administered to treat osteosarcoma.

[0042] In further embodiments, the present disclosure provides a method for maintenance therapy of cancer, the method comprising administering an effective amount of a liposomal composition comprising liposomes containing alpha polyglutamated pemetrexed (Lp-αPPMX) to a subject undergoing or who has undergone cancer therapy. In some embodiments, the administered liposomal composition is PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX. In some embodiments, the administered liposomal composition comprises pegylated liposomes (e.g., PLp-αPPMX, NTPLp-αPPMX, or TPLp-αPPMX). In some embodiments, the administered liposomal composition comprises targeted liposomes (e.g., TLp-αPPMX or TPLp-αPPMX). In some embodiments, the administered liposomal composition comprises pegylated and targeted liposomes (e.g., TPLp-αPPMX). In some embodiments, the liposomes of the administered liposome composition comprise alpha-polyglutamated pemetrexed containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated pemetrexed. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutamated pemetrexed. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutamated pemetrexed.

[0043] In further embodiments, the present disclosure provides methods for treating an immune system disorder, the method comprising administering an effective amount of a liposome composition comprising liposomes containing alpha polyglutamated pemetrexed (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX) 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-αPPMX, NTPLp-αPPMX, or TPLp-αPPMX). In some embodiments, the administered liposome composition comprises a targeted liposome (e.g., TLp-αPPMX or TPLp-αPPMX) 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-αPPMX). In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutamated pemetrexed containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated pemetrexed. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutamated pemetrexed. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutamated pemetrexed.

[0044] The present disclosure also provides a method for delivering alpha polyglutamated pemetrexed to tumor or cancer cells, the method comprising administering to a subject having a tumor a composition comprising alpha polyglutamated pemetrexed (L-αPPMX) 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-αPPMX). In some embodiments, the administered composition comprises alpha polyglutamated pemetrexed containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the administered composition comprises alpha tetraglutamated pemetrexed. In some embodiments, the composition administered comprises alpha-pentaglutamated pemetrexed, hi other embodiments, the composition administered comprises alpha-hexaglutamated pemetrexed.

[0045] In further embodiments, the present disclosure provides a method for making a liposome composition comprising a liposomal alpha polyglutamated pemetrexed (αPPMX) composition, the method comprising: forming a mixture in solution comprising liposome components and α polyglutamated pemetrexed; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes comprising polyglutamated pemetrexed. In some embodiments, the alpha polyglutamated pemetrexed comprises 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the polyglutamated pemetrexed composition comprises alpha tetraglutamated pemetrexed. In some embodiments, the polyglutamated pemetrexed composition comprises alpha pentaglutamated pemetrexed. In other embodiments, the polyglutamated pemetrexed composition comprises alpha hexaglutamated pemetrexed.

[0046] In one embodiment, the present disclosure provides a kit comprising an alpha polyglutamated pemetrexed composition and / or an αPPMX delivery vehicle, such as a liposome, comprising αPPMX and αPPMX immunoconjugates (e.g., ADCs) described herein. [Brief explanation of the drawings]

[0047] [Figure 1]1A-1L show the chemical formulas of pemetrexed (FIG. 1A), representative alpha-pemetrexed alpha polyglutamates pemetrexed diglutamate (FIG. 1B), pemetrexed triglutamate (FIGS. 1C and 1D), pemetrexed tetraglutamate (FIGS. 1E and 1F), pemetrexed pentaglutamate (FIGS. 1G and 1H), pemetrexed hexaglutamate (FIGS. 1I and 1J), pemetrexed heptaglutamate (FIGS. 1K and 1L), pemetrexed octaglutamate (FIGS. 1M and 1N), representative alpha-pemetrexed polyglutamate (FIG. 1O), and representative pemetrexed analogs (FIGS. 1P and 1Q). Figures 1R-1U show the structures of representative branched pemetrexed polyglutamates, including a branched polyglutamate with a gamma-glutamyl backbone and alpha-glutamyl branching moieties (Figure 1S) and a branched polyglutamate with an alpha-glutamyl backbone and gamma-glutamyl branching moieties (Figure 1T). [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] Shown are the effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal alpha-D hexaglutamate (liposomal aDG6), and the corresponding pemetrexed drug on neutrophils (differentiated from CD34+ cells) after 48 hours of exposure at various dose levels ranging from 16 to 128 nM. [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] Figure 1 shows the results of a survival study of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6) treatment in a NSCLC (H292) xenograft model. The survival curves of mice (10) treated with 90 mg / kg liposomal aG6 intravenously once weekly for 4 weeks (90 mg / kg subcutaneously weekly for 6 weeks) are shown as solid circles. The survival curves of mice (10) treated with pemetrexed (167 mg / kg intravenously every 3 weeks for 6 weeks) are shown as solid triangles. Pemetrexed administration of 167 mg / kg in mice is equivalent to a 500 mg / m2 dose in humans. The survival curves of control mice (10) are shown as hollow diamonds. [Figure 21A-F] Liposomal pemetrexed alpha-L triglutamate () for 48 hours against H2342 (NSCLC, adenocarcinoma subtype) (Figure 21A), H292 (NSCLC, adenocarcinoma subtype) (Figure 21B), HT-29 (colon cancer) (Figure 21C), HCC1806 (triple-negative breast cancer) (Figure 21D), MCF7 (ER+ breast cancer) (Figure 21E), and OAW28 (ovarian cancer) (Figure 21F). Figure 1 shows the dose-response relationship for liposomal pemetrexed 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

[0048] Generally, the present disclosure relates to novel alpha polyglutamated pemetrexed compositions. The compositions offer an advancement over existing treatments for hyperproliferative diseases, such as cancer. Methods for producing, delivering, and using the alpha polyglutamated pemetrexed 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.

[0049] 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.

[0050] 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).

[0051] 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.

[0052] 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.

[0053] 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.

[0054] Unless otherwise indicated, the terms "pemetrexed" and "PMX" are used interchangeably and include salt, acid, and / or free base forms of pemetrexed (e.g., pemetrexed disodium). Compositions containing PMX salts may also contain various cations, e.g., Na + , Mg 2+ , K. + , NH4 + , and / or Ca 2+ In certain embodiments, the salt is typically a pharmaceutically acceptable salt. In further particular embodiments, the PMX salt may comprise Na + Pemetrexed, also known as ALIMTA®, LY231514, or MTA, has the chemical name N-[4-2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-1-glutamic acid, or L-glutamic acid, N-[4-2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-c]pyrimidin-5-yl)ethyl]benzoyl]-, disodium salt, heptahydrate. Pemetrexed typically contains one L-gamma glutamyl group and is therefore considered monoglutamated for purposes of this disclosure.

[0055] 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 polyglutamylated pemetrexed is the glutamyl group of pemetrexed. The C-terminal glutamyl group(s) of the polyglutamate chain are bonded to another glutamyl group through their amino groups, but not through their carboxylic acid groups.

[0056] The terms "polyglutamylated pemetrexed," "polyglutamylated PMX," "PMX-PG," "PPMX," and iterations thereof are used interchangeably herein and refer to pemetrexed compositions containing at least one glutamyl group in addition to the glutamyl groups in pemetrexed (i.e., PMX-PG). n , n≧1). References herein to the number of glutamyl groups in αPPMX (PMX-PG) count the glutamyl groups in pemetrexed. For example, a PMX-PG composition containing five glutamyl residues in addition to the glutamyl groups in PMX is referred to herein as hexaglutamated pemetrexed or pemetrexed hexaglutamate.

[0057] The terms "alpha glutamyl group," "alpha glutamate," and "alpha linkage," when referring to a glutamyl group bond, refer to a glutamyl group 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 pemetrexed, or between a glutamyl group and a second glutamyl group not present in pemetrexed, such as a glutamyl group in a polyglutamate chain bound to pemetrexed.

[0058] The terms "gamma glutamyl group," "gamma glutamate," and "gamma linkage," when referring to a glutamyl group bond, refer to a glutamyl group containing a gamma carboxyl group bond. As discussed herein, when pemetrexed enters cells, it is polyglutamylated by the enzyme folylpolygamma glutamate synthase (FPGS), which sequentially adds L-glutamyl groups to the gamma carboxyl groups of glutamates within pemetrexed. Thus, alpha polyglutamylated pemetrexed compositions are not formed intracellularly during pemetrexed 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 a bond between a glutamyl group and a glutamyl group in pemetrexed, or between a glutamyl group and a second glutamyl group, such as a glutamyl group in a polyglutamate chain attached to pemetrexed, that is not present in pemetrexed. In some embodiments, the gamma bond refers to the amide bond of the glutamyl group of pemetrexed. Reference to a gamma bond includes the gamma bond of the glutamyl group of pemetrexed, unless otherwise specified or unless the context clearly indicates otherwise.

[0059] Unless otherwise indicated, the terms "alpha polyglutamated pemetrexed," "αPPMX," "alpha PMX-PG," and iterations thereof are used interchangeably herein to refer to polyglutamated pemetrexed compositions containing at least one glutamyl group that contains an alpha linkage. For example, a pentaglutamated PMX 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 PMX-PG in this disclosure. In some embodiments, each glutamyl group of PMX-PG other than the glutamyl group of PMX has an alpha linkage (e.g., PMX-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 PMX-PG other than the glutamyl group of PMX has an alpha linkage (e.g., PMX-PG where n=5 and G1, G2, G3, and G4 each have an alpha linkage). n In some embodiments, each glutamyl group of PMX-PG other than the C-terminal glutamyl group(s) has an alpha linkage (e.g., PMX-PG where n=5 and each of the glutamyl groups of PMX and G1, G2, G3, and G4 has an alpha linkage). n ).

[0060] 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 antifolates are 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, alpha polyglutamated compositions are 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."

[0061] 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.

[0062] 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.

[0063] 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.

[0064] Expressions known in the art, such as " target binding affinity ", " target binding " and similar expressions, refer to the affinity constant, which can be directly measured by determining the amount of targeting moiety that binds and dissociates at a given antigen concentration.Other methods can be used to characterize intermolecular interactions, including but not limited to, competitive analysis, equilibrium analysis and microcalorimetry, and real-time interaction analysis based on surface plasmon resonance interaction (for example, using BIACORE® device).These methods are well known to those skilled in the art and are described, for example, in Neri et al., Tibtech 14:465-470 (1996), and Jansson et al., J.Biol.Chem.272:8189-8197 (1997).

[0065] The term "delivery vehicle" generally refers to any composition that acts to support, promote, or facilitate the entry of alpha-polyglutamated pemetrexed 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.

[0066] "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.

[0067] 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.

[0068] 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 stenosis or restenosis after angioplasty.

[0069] "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 αPPMX compositions provided herein include, but are not limited to, non-hematological tumors, including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors, such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. Other types of cancers and tumors that can be treated using the αPPMX 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.

[0070] 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 terms "treating" or "treatment" or "treat" refer 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 terms "treating" or "treatment" or "treat" refer 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 terms "treating" or "treatment" or "treat" refer to the reduction or stabilization of size, tumor cell growth or survival, or cancer cell number. Treatment can involve the use of α-PPMX compositions alone or in combination with additional therapeutic agents.

[0071] "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.

[0072] 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.

[0073] 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.

[0074] 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. Further examples of therapeutic agents that may be suitable for use with the methods of the present disclosure include, but are not limited to, antirestenotic agents, pro- or anti-proliferative agents, anti-inflammatory agents, antineoplastic agents, antimitotic agents, antiplatelet agents, anticoagulants, antifibrinogens, antithrombin agents, cytostatic agents, antibiotics and other anti-infective agents, antienzymes, antimetabolites, angiogenic agents, cytoprotective agents, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, and / or cardioprotective agents. "Therapeutic agent" also refers to salt, acid, and free base forms of the above agents.

[0075] 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.

[0076] 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, pemetrexed, 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 pemetrexed composition is used in combination with an antimetabolite selected from the group consisting of fluoropyrimidine, 5-fluorouracil, 5-fluoro-2'-deoxycytidine, cytarabine, gemcitabine, troxacitabine, decitabine, azacitidine, pseudoisocytidine, zebularine, ancitabine, fazarabine, 6-azacytidine, capecitabine, N4-octadecylcytarabine, elaidic acid cytarabine, fludarabine, cladribine, clofarabine, nelarabine, forodesine, and pentostatin, or derivatives thereof. In one example, the nucleoside analog is a substrate of a nucleoside deaminase that is an adenosine deaminase or a cytidine deaminase. In some examples, the nucleoside analog is selected from fludarabine, cytarabine, gemcitabine, decitabine, and azacitidine or derivatives thereof. In certain embodiments, the antimetabolite is 5-fluorouracil.

[0077] 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.

[0078] 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.

[0079] The present disclosure relates generally to novel alpha polyglutamated pemetrexed (PMX) 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.

[0080] In some embodiments, the present disclosure provides: [1] A composition comprising alpha polyglutamated pemetrexed, wherein at least one glutamyl group has an alpha carboxyl group bond; [2] The composition according to item [1], wherein the alpha polyglutamylated pemetrexed contains 1 to 10 glutamyl groups having alpha carboxyl group bonds; [3] The composition according to item [1] or [2], wherein the alpha polyglutamated pemetrexed 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 pemetrexed; [5] The composition according to any one of items [1] to [3], comprising alpha-pentaglutamated pemetrexed; [6] The composition according to any one of items [1] to [3], comprising alphahexaglutamated pemetrexed; [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 pemetrexed has an alpha carboxyl linkage; or (c) a composition in which two or more glutamyl groups have a gamma carboxyl group bond; [8] The composition according to any one of items [1] to [6], wherein the composition is: (a) the C-terminal glutamyl group(s) and each glutamyl group other than the glutamyl group of pemetrexed have an alpha carboxyl group bond; 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 pemetrexed are in the L-configuration; (b) each glutamyl group of the alpha polyglutamated pemetrexed is in the L-configuration; (c) at least one glutamyl group of the alpha polyglutamated pemetrexed is in the D-form; (d) each glutamyl group of said alpha polyglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form; or (e) at least two of the glutamyl groups of the alpha polyglutamated pemetrexed 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-αPPMX) containing alpha polyglutamated pemetrexed according to any one of items [1] to

[12] ;

[14] The LαPP composition according to item

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

[15] The Lp-αPPMX composition according to item

[13] or

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

[16] The Lp-αPPMX composition according to item

[13] or

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

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

[13] to

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

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

[13] to

[17] , wherein at least one glutamyl group of the alpha polyglutamylated pemetrexed 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

[18] , comprising 2, 3, 4, 5, 6, 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-αPPMX composition according to any one of items

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

[28] The Lp-αPPMX composition according to item

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

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

[27] to

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

[32] The Lp-αPPMX 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-αPPMX composition according to item

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

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

[13] to

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

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

[13] to

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

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

[13] to

[33] , wherein the liposome is cationic;

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

[13] to

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

[40] The Lp-αPPMX composition according to item

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

[41] The Lp-αPPMX composition according to item

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

[42] The Lp-αPPMX composition according to item

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

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

[39] to

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

[46] The Lp-αPPMX 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-αPPMX 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-αPPMX 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 pemetrexed molecules;

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

[13] to

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

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

[50] , wherein the targeting moiety is attached to one or both of the PEG and the exterior surface of the liposome, and optionally the targeting moiety is covalently attached to one or both of the PEG and the exterior surface of the liposome;

[52] The Lp-αPPMX composition according to item

[50] or

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

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

[50] to

[53] , wherein the targeting moiety is at least 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-αPPMX 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-αPPMX 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-αPPMX 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-αPPMX composition according to item

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

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

[39] to

[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-αPPMX 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 D (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-αPPMX composition according to any one of items

[58] to

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

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

[58] to

[61] , further comprising a hapten;

[63] The Lp-αPPMX composition according to item

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

[64] The Lp-αPPMX 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 the internal space and the external space;

[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-αPPMX 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 pemetrexed composition according to any one of items

[13] to

[67] ;

[69] A pharmaceutical composition comprising the alpha polyglutamated pemetrexed 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 pemetrexed 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 pemetrexed 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 pemetrexed 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 a sarcoma such as osteosarcoma;

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

[50] to

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

[84] A maintenance therapy comprising administering an effective amount of the composition according to any one of items [1] to

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

[85] A maintenance therapy comprising administering an effective amount of the liposomal alpha polyglutamated pemetrexed 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 pemetrexed composition according to any one of items [9] to

[69] to a subject having or at risk of having an immune system disorder;

[88] 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;

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

[69] in an amount sufficient to deliver a therapeutically effective amount of alpha polyglutamated pemetrexed to the tumor;

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

[13] to

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

[92] A method for preparing an alpha polyglutamated pemetrexed composition, including the liposomal alpha polyglutamated pemetrexed composition according to any one of items

[13] to

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

[93] The method according to item

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

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

[13] to

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

[96] The method according to item

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

[97] The method according to any one of items

[94] to

[96] , 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;

[98] The method according to any one of items

[94] 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 polyglutamylated PMX starting material is encapsulated or entrapped in Lp-αPPMX.

[0081] II. Alpha Polyglutamated Pemetrexed (αPPMX) In general, the present disclosure relates to alpha polyglutamylated pemetrexed (αPPMX) compositions. αPPMX compositions contain at least one glutamyl group with an alpha linkage. These compositions are structurally distinct from L-gamma polyglutamylated pemetrexed (LαPPMX), which is generated in cells by the enzyme folylpolygamma glutamate synthase (FPGS) during pemetrexed therapy.

[0082] In some embodiments, the αPPMX composition contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl group of pemetrexed). In some embodiments, each glutamyl group in αPPMX other than the glutamyl group of pemetrexed has an alpha linkage. In some embodiments, each glutamyl group in αPPMX other than the C-terminal glutamyl group(s) and the glutamyl group of pemetrexed has an alpha linkage. In some embodiments, each glutamyl group in αPPMX other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, two or more glutamyl groups in αPPMX have gamma linkages. In some embodiments, at least one glutamyl group of alpha polyglutamylated pemetrexed has an alpha carboxylic group linkage and a gamma carboxylic group linkage. In some embodiments, each glutamyl group in αPPMX is in the L-configuration. In some embodiments, each glutamyl group in αPPMX other than the glutamyl group of pemetrexed is in the D form. In some embodiments, αPPMX 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 αPPMX is linear (not branched). In some embodiments, the polyglutamate chain of αPPMX is branched.

[0083] In some embodiments, the alpha polyglutamated pemetrexed is diglutamated. That is, the alpha polyglutamated pemetrexed contains one additional glutamyl group in addition to the glutamyl groups of pemetrexed (αPMX-PG1), and the additional glutamyl group is linked to the glutamyl group in pemetrexed via an alpha bond. In some embodiments, each glutamyl group in alpha diglutamated pemetrexed is in the L-form. In other embodiments, the alpha diglutamated PMX contains a glutamyl group in the D-form.

[0084] In some embodiments, the alpha polyglutamated pemetrexed is triglutamated. That is, the alpha polyglutamated pemetrexed contains two additional glutamyl groups in addition to the glutamyl groups of pemetrexed (αPMX-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 pemetrexed is in the L-form. In other embodiments, alpha triglutamated PMX contains a glutamyl group in the D-form. In further embodiments, each glutamyl group of alpha triglutamated pemetrexed, other than the glutamyl group of pemetrexed, is in the D-form. In further embodiments, triglutamated PMX 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.

[0085] In some embodiments, alpha polyglutamated pemetrexed is tetraglutamated and thus contains three additional glutamyl groups in addition to the glutamyl groups in pemetrexed (αPMX-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-tetraglutamylated PMX comprises two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alpha-tetraglutamylated pemetrexed is in the L-form. In other embodiments, the alpha-tetraglutamylated PMX comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group of the alpha-tetraglutamylated pemetrexed, other than the glutamyl group of pemetrexed, is in the D-form. In further embodiments, the tetraglutamylated PMX 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.

[0086] In some embodiments, alpha polyglutamylated pemetrexed is pentaglutamylated (αPMX-PG4) and comprises a chain of four additional glutamyl groups linked to glutamyl groups of pemetrexed. 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 linked to glutamyl groups of the molecule via gamma linkages. 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 linked to 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 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 PMX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group in alpha-pentaglutamated pemetrexed is in the L-form. In other embodiments, the alpha-pentaglutamated PMX contains a glutamyl group in the D-form. In further embodiments, each glutamyl group in alpha-pentaglutamated pemetrexed other than the glutamyl group in pemetrexed is in the D-form. In further embodiments, the pentaglutamated PMX 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.

[0087] In some embodiments, the alpha polyglutamated pemetrexed is hexaglutamated (αPMX-PG5) and comprises a chain of five additional glutamyl groups attached to a glutamyl group of pemetrexed. 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 a glutamyl group of the molecule via an alpha linkage, and the remaining four, three, two, or one glutamyl groups, respectively, are attached to a glutamyl group of the molecule via a gamma linkage. In other embodiments, 1, 2, 3, or 4 of the five 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 six glutamyl groups has both alpha and gamma linkages. 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 alphahexaglutamated PMX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alphahexaglutamated pemetrexed is in the L-form. In other embodiments, the alphahexaglutamated PMX contains a glutamyl group in the D-form. In a further embodiment, each glutamyl group of alpha-hexaglutamated pemetrexed other than the glutamyl group of pemetrexed is D-type. In a further embodiment, hexaglutamated PMX 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.

[0088] In some embodiments, alpha polyglutamated pemetrexed is heptaglutamated (αPMX-PG6), thus comprising a chain of six additional glutamyl groups attached to the glutamyl groups of pemetrexed. 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 PMX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group in the alphaheptaglutamated pemetrexed is in the L-form. In other embodiments, the alphaheptaglutamated PMX contains a glutamyl group in the D-form. In further embodiments, each glutamyl group in the alphaheptaglutamated pemetrexed other than the glutamyl group in pemetrexed is in the D-form. In further embodiments, the heptaglutamated PMX 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.

[0089] In some embodiments, alpha polyglutamated pemetrexed is octaglutamated (αPMX-PG7), thus comprising a chain of seven additional glutamyl groups linked to the glutamyl groups of pemetrexed. 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 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 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 an alpha linkage. In some embodiments, the alpha-octaglutamated PMX contains two or more glutamyl groups in the L-form. In a further embodiment, each glutamyl group in the alpha-octaglutamated pemetrexed is in the L-form. In other embodiments, the alpha-octaglutamated PMX contains a glutamyl group in the D-form. In a further embodiment, each glutamyl group in the alpha-octaglutamated pemetrexed other than the glutamyl group in pemetrexed is in the D-form. In a further embodiment, the octaglutamated PMX 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.

[0090] In some embodiments, alpha polyglutamated pemetrexed is nonaglutamated (αPMX-PG8) and contains a chain of eight additional glutamyl groups linked to glutamyl groups of pemetrexed. 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, one, two, three, four, five, six, or seven of the eight additional glutamyl groups have an alpha linkage, and the remaining seven, six, five, four, three, two, or one glutamyl groups, respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, or 7 of the 8 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 9 glutamyl groups has both alpha and gamma linkages. In some embodiments, each of the 9 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha linkage. In some embodiments, 8 of the 9 glutamyl groups have alpha linkages. In some embodiments, the alpha-nonaglutamated PMX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of alpha-nonaglutamated pemetrexed is in the L-form. In other embodiments, the alpha-nonaglutamated PMX contains a glutamyl group in the D-form. In a further embodiment, each glutamyl group of alpha-nonaglutamated pemetrexed, other than the glutamyl group of pemetrexed, is D-type. In a further embodiment, nonaglutamated PMX 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.

[0091] In some embodiments, the alpha polyglutamylated pemetrexed is deca-glutamylated (αPMX-PG9) (i.e., comprises a chain of nine additional glutamyl groups attached to a glutamyl group of pemetrexed). 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 alphadecaglutamated PMX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alphadecaglutamated pemetrexed is in the L-form. In other embodiments, the alphadecaglutamated PMX contains a glutamyl group in the D-form. In a further embodiment, each glutamyl group of alphadecaglutamated pemetrexed, other than the glutamyl group of pemetrexed, is D-type. In a further embodiment, decaglutamated PMX 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.

[0092] In some embodiments, the alpha polyglutamated pemetrexed is undecaglutamated (αPMX-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, respectively, 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 PMX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group in alpha-undeca-glutamated pemetrexed is in the L-form. In other embodiments, the alpha-undeca-glutamated PMX contains a glutamyl group in the D-form. In further embodiments, each glutamyl group in alpha-undeca-glutamated pemetrexed other than the glutamyl group in pemetrexed is in the D-form. In further embodiments, the undecaglutamated PMX 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.

[0093] In some embodiments, the alpha polyglutamated pemetrexed is dodecaglutamated (αPMX-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 PMX contains two or more glutamyl groups in the L form. In further embodiments, each glutamyl group in alpha dodecaglutamated pemetrexed is in the L form. In other embodiments, the alpha dodecaglutamated PMX contains a glutamyl group in the D form. In further embodiments, each glutamyl group in alpha dodecaglutamated pemetrexed other than the glutamyl group in pemetrexed is in the D form. In further embodiments, the dodecaglutamated PMX 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.

[0094] In some embodiments, the alpha polyglutamated pemetrexed is triskydecaglutamated (αPMX-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, the alpha-Triskai deca-glutamated PMX contains two or more glutamyl groups in the L-form. In a further embodiment, each glutamyl group in the alpha-Triskai deca-glutamated pemetrexed is in the L-form. In other embodiments, the alpha-Triskai deca-glutamated PMX contains a glutamyl group in the D-form. In a further embodiment, each glutamyl group in the alpha-Triskai deca-glutamated pemetrexed other than the glutamyl group in pemetrexed is in the D-form. In further embodiments, the triskaidecaglutamated PMX 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 pemetrexed is tetradecaglutamated (αPMX-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 PMX contains two or more glutamyl groups in the L-form. In a further embodiment, each glutamyl group in the alpha-tetradecaglutamated pemetrexed is in the L-form. In other embodiments, the alpha-tetradecaglutamated PMX contains a glutamyl group in the D-form. In a further embodiment, each glutamyl group in the alpha-tetradecaglutamated pemetrexed other than the glutamyl group in pemetrexed is in the D-form. In a further embodiment, the tetradecaglutamated PMX 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.

[0096] In some embodiments, the alpha polyglutamated pemetrexed is pentadecaglutamated (αPMX-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 PMX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alpha pentadeca-glutamylated pemetrexed is in the L-form. In other embodiments, the alpha pentadeca-glutamated PMX comprises a glutamyl group in the D-form. In a further embodiment, each glutamyl group of the alpha pentadeca-glutamated pemetrexed, other than the glutamyl group of pemetrexed, is in the D-form. In a further embodiment, the pentadeca-glutamated PMX 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.

[0097] In some embodiments, the alpha polyglutamated pemetrexed is hexadecaglutamated (αPMX-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 PMX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alphahexadecaglutamated pemetrexed is in the L-form. In other embodiments, the alphahexadecaglutamated PMX comprises a glutamyl group in the D-form. In a further embodiment, each glutamyl group in the alphadecahexadecaglutamated pemetrexed other than the glutamyl group in pemetrexed is in the D-form. In a further embodiment, the hexadecaglutamated PMX 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.

[0098] In another embodiment, the alpha polyglutamated pemetrexed is heptadecaglutamated (αPMX-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 PMX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alphaheptadecaglutamated pemetrexed is in the L-form. In other embodiments, the alpha heptadeca-glutamated PMX comprises a glutamyl group in the D-form. In a further embodiment, each glutamyl group of the alpha heptadeca-glutamated pemetrexed, other than the glutamyl group of pemetrexed, is in the D-form. In a further embodiment, the heptadeca-glutamated PMX 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.

[0099] In some embodiments, the alpha polyglutamated pemetrexed is octadeca-glutamated (αPMX-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 alphaoctadecaglutamated PMX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alphaoctadecaglutamated pemetrexed is in the L-form. In other embodiments, the alpha-octadeca-glutamated PMX comprises a glutamyl group in the D-form. In a further embodiment, each glutamyl group of the alpha-octadeca-glutamated pemetrexed, other than the glutamyl group of pemetrexed, is in the D-form. In a further embodiment, the octadeca-glutamated PMX 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.

[0100] In some embodiments, the alpha polyglutamated pemetrexed is eniadecaglutamated (αPMX-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, alpha eniadecaglutamated PMX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of alpha eniadecaglutamated pemetrexed is in the L-form. In other embodiments, the alpha eniadecaglutamated PMX comprises a glutamyl group in the D-form. In a further embodiment, each glutamyl group in the alpha eniadecaglutamated pemetrexed other than the glutamyl group in pemetrexed is in the D-form. In a further embodiment, the eniadecaglutamated PMX 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.

[0101] In some embodiments, the alpha polyglutamated pemetrexed is eicosiglutamated (αPMX-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 PMX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of the alpha-icosiglutamated pemetrexed is in the L-form. In other embodiments, the alpha-icosiglutamated PMX comprises a glutamyl group in the D-form. In a further embodiment, each glutamyl group of the alpha-icosiglutamated pemetrexed, other than the glutamyl group of the pemetrexed, is in the D-form. In a further embodiment, the eicosiglutamated PMX 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.

[0102] In some embodiments, the alpha polyglutamated pemetrexed is eikoshikaihenaglutamated (αPMX-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-coccidiomycosis glutamylated PMX contains two or more L-configured glutamyl groups. In a further embodiment, each glutamyl group of alpha-cosiglutamated pemetrexed is in the L-form. In other embodiments, alpha-cosiglutamated PMX contains a glutamyl group in the D-form. In a further embodiment, each glutamyl group of alpha-cosiglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In a further embodiment, alpha-cosiglutamated PMX 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.

[0103] In some embodiments, the alpha polyglutamated pemetrexed comprises 4 to 7 glutamyl groups bound to pemetrexed (i.e., αPMX-PGn, n=4-7), and each of the 4 to 7 linked glutamyl groups has an alpha linkage. In some embodiments, the alpha polyglutamated pemetrexed comprises 4 to 7 glutamyl groups bound to pemetrexed (i.e., αPMX-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.

[0104] In one embodiment, the alpha polyglutamated pemetrexed is tetraglutamated, and each of the three glutamyl groups in the polyglutamate chain attached to pemetrexed contains an alpha linkage. In one embodiment, the alpha polyglutamated pemetrexed is tetraglutamated, and each of the three glutamyl groups in the polyglutamate chain attached to pemetrexed, 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 alpha tetraglutamated pemetrexed, other than the glutamyl group in pemetrexed, is D-type. In other embodiments, at least two of the glutamyl groups in alpha tetraglutamate pemetrexed 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.

[0105] In one embodiment, the alpha polyglutamated pemetrexed is pentaglutamated, and each of the four glutamyl groups in the polyglutamate chain attached to pemetrexed comprises an alpha linkage. In one embodiment, the alpha polyglutamated pemetrexed is pentaglutamated, and each of the four glutamyl groups in the polyglutamate chain attached to pemetrexed, other than the C-terminal glutamyl group(s), comprises an alpha linkage. In some embodiments, each of the four glutamyl groups is L-type. In some embodiments, each glutamyl group in alpha pentaglutamated pemetrexed, other than the glutamyl group in pemetrexed, is D-type. In other embodiments, at least two of the glutamyl groups in alpha pentaglutamated pemetrexed 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.

[0106] In one embodiment, the alpha polyglutamated pemetrexed is hexaglutamated, and each of the five glutamyl groups in the polyglutamate chain attached to pemetrexed comprises an alpha linkage. In one embodiment, the alpha polyglutamated pemetrexed is hexaglutamated, and each of the five glutamyl groups in the polyglutamate chain attached to pemetrexed, other than the C-terminal glutamyl group(s), comprises an alpha linkage. In some embodiments, each of the five glutamyl groups is L-type. In some embodiments, each glutamyl group in alpha hexaglutamated pemetrexed, other than the glutamyl group in pemetrexed, is D-type. In other embodiments, at least two of the glutamyl groups in alpha hexaglutamated pemetrexed 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 chains are branched.

[0107] In another embodiment, the alpha polyglutamated pemetrexed is heptaglutamated, and each of the six glutamyl groups in the polyglutamate chain attached to pemetrexed contains an alpha linkage. In another embodiment, the alpha polyglutamated pemetrexed is heptaglutamated, and each of the six glutamyl groups in the polyglutamate chain attached to pemetrexed, 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 alpha heptaglutamated pemetrexed, other than the glutamyl group in pemetrexed, is in the D-form. In other embodiments, at least two of the glutamyl groups in alpha heptaglutamated pemetrexed 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.

[0108] In some embodiments, alpha polyglutamylated pemetrexed (αPPMX) contains a total of 1 to 15, 1 to 10, 2 to 15, 2 to 10, 3 to 15, 3 to 10, 3 to 6, 3 to 5, 4 to 10, 4 to 7, or 4 to 6 glutamyl groups, including the glutamyl group of pemetrexed, or any range therebetween. In some embodiments, each glutamyl group in αPPMX other than the glutamyl group of pemetrexed has an alpha linkage. In some embodiments, each glutamyl group in αPPMX other than the C-terminal glutamyl group(s) and the glutamyl group of pemetrexed has an alpha linkage. In some embodiments, each glutamyl group in αPPMX 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 αPPMX have alpha linkages. In some embodiments, αPPMX contains glutamyl groups in L- and D-forms. In further embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPPMX 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 pemetrexed is in the L-form. In some embodiments, each glutamyl group in αPPMX other than the glutamyl groups of pemetrexed is in the D-form. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the glutamyl groups in αPPMX are in the L-form. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPPMX are in the D-form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0109] In some embodiments, alpha polyglutamylated pemetrexed (αPPMX) contains a total of 2 to 20, 2 to 15, 2 to 10, 2 to 5, or any range therebetween, including the glutamyl group of pemetrexed. In some embodiments, each glutamyl group in αPPMX other than the glutamyl group of pemetrexed has an alpha linkage. In some embodiments, each glutamyl group in αPPMX other than the C-terminal glutamyl group(s) and the glutamyl group of pemetrexed has an alpha linkage. In some embodiments, each glutamyl group in αPPMX 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, αPPMX comprises 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 αPPMX other than the glutamyl groups in pemetrexed 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 αPPMX is in the L-form. In some embodiments, each glutamyl group in αPPMX other than the glutamyl groups in pemetrexed 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 αPPMX 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 αPPMX are in the D-form.

[0110] In some embodiments, the alpha polyglutamated pemetrexed 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 pemetrexed. 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 pemetrexed has gamma linkages. In some embodiments, at least one glutamyl group has both alpha and gamma linkages. In some embodiments, the glutamyl groups in pemetrexed have alpha linkages. In some embodiments, the glutamyl group in pemetrexed has both alpha and gamma linkages.

[0111] 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 pemetrexed are in the L-form, the D-form, or the L- and D-form. In some embodiments, each glutamyl group in the alpha polyglutamated pemetrexed is in the L-form. In other embodiments, each glutamyl group in the alpha polyglutamated pemetrexed other than the glutamyl group in pemetrexed is in the D-form. In alternative embodiments, at least two of the glutamyl groups in the alpha polyglutamated pemetrexed are in the L-form and at least one of the glutamyl groups in the alpha polyglutamated pemetrexed 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 pemetrexed 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 pemetrexed are in the D-form.

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

[0113] In further embodiments, provided compositions comprise alpha polyglutamated pemetrexed comprising glutamyl groups with 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 alpha linkages. In some embodiments, the alpha polyglutamated pemetrexed comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 L-glutamyl groups. In some embodiments, the alpha polyglutamated pemetrexed comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 D-glutamyl groups. In some embodiments, the alpha polyglutamated pemetrexed 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 pemetrexed contains at least one glutamyl group with both alpha and gamma linkages. In some embodiments, the alpha polyglutamated pemetrexed contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or more than 10 glutamyl groups with both alpha and gamma linkages.

[0114] In some embodiments, the alpha polyglutamated pemetrexed 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 pemetrexed 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 pemetrexed 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 pemetrexed 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 pemetrexed contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma glutamyl group linkages and further contains 1, 2, 3, 4, 5, 6, or 1-10 L-gamma glutamyl group linkages. In other embodiments, the alpha polyglutamated pemetrexed contains at least one glutamyl group with both alpha and gamma linkages. In some embodiments, the alpha polyglutamated pemetrexed contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or more than 10 glutamyl groups with both alpha and gamma linkages.

[0115] In some embodiments, the alpha polyglutamated pemetrexed 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 pemetrexed compositions to act as substrates for FPGS (e.g., human FPGS, or rat liver FPGS) are readily available and can be performed routinely.

[0116] In some embodiments, the rate of hepatocyte uptake of a naked alphaPPMX composition disclosed herein (e.g., alphaPPMX not conjugated to a delivery vehicle) is significantly reduced compared to the rate of uptake of pemetrexed under physiological conditions. In some embodiments, the rate of hepatocyte uptake of a naked alphaPPMX composition is less than 30%, 20%, 15%, or 10% of the rate of pemetrexed. In further embodiments, the rate of efflux (transport) of an alphaPPMX composition disclosed herein from hepatocytes occurs at a significantly lower rate (less than 30%, 20%, 15%, or 10%) compared to pemetrexed.

[0117] In some embodiments, the alpha polyglutamated pemetrexed compositions provided herein have greater cytotoxicity against hyperproliferative cells than pemetrexed. 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 pemetrexed is hexaglutamated pemetrexed.

[0118] In some embodiments, the alpha polyglutamated pemetrexed compositions provided herein have fewer toxic side effects than pemetrexed. In some embodiments, the alpha polyglutamated pemetrexed compositions provided herein are less toxic than pemetrexed to non-hyperproliferative cells. In some embodiments, the alpha polyglutamated pemetrexed compositions provided herein are less toxic than pemetrexed to neutrophils, liver cells, or colon epithelial cells. 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 pemetrexed is hexaglutamated pemetrexed.

[0119] In some embodiments, the alpha polyglutamated pemetrexed compositions provided herein have fewer toxic side effects than pemetrexed. In some embodiments, the alpha polyglutamated pemetrexed compositions provided herein result in less frequent or less severe toxic side effects than pemetrexed 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 pemetrexed compositions provided herein result in less frequent or less severe hematologic or liver toxic side effects than pemetrexed. 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 comprises administering 40 mg / kg or 80 mg / kg of the alpha polyglutamated pemetrexed composition once a week for four weeks. In some embodiments, the alpha polyglutamated pemetrexed is hexaglutamated pemetrexed.

[0120] In some embodiments, treatment with the alpha polyglutamated pemetrexed 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 pemetrexed compositions provided herein do not significantly reduce mean neutrophil, mean white blood cell, or mean platelet counts. In some embodiments, the alpha polyglutamated pemetrexed compositions provided herein do not significantly increase serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. In some embodiments, the alpha polyglutamated pemetrexed compositions provided herein do not significantly reduce serum albumin levels. In some embodiments, the in vivo assay comprises administering 40 mg / kg or 80 mg / kg of the alpha polyglutamated pemetrexed composition once a week for four weeks. In some embodiments, the alpha polyglutamated pemetrexed is hexaglutamated pemetrexed.

[0121] In some embodiments, the alpha polyglutamated pemetrexed composition does not contain fluorine atoms. In some embodiments, the alpha polyglutamated pemetrexed composition does not contain 4-fluoroglutamyl groups.

[0122] Alpha polyglutamated pemetrexed (αPPMX) compositions and their uses are further described in U.S. Patent Application Nos. 62 / 374,458, 15 / 675,695, 15 / 675,701, and 62 / 583,432, and International Application Nos. PCT / US2017 / 046666 and PCT / US2017 / 046667, the contents of each of which are incorporated herein by reference in their entirety.

[0123] A. Polyglutamated Pemetrexed Analogs and Derivatives The present disclosure also encompasses alpha polyglutamated pemetrexed derivatives and analogs. The compositions and methods disclosed herein are contemplated for application to any and all known polyglutamated pemetrexed derivatives or analogs. In some embodiments, polyglutamated pemetrexed 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 pemetrexed, in which the glutamyl group of pemetrexed is not linked to the remainder of the pemetrexed molecule via a gamma peptide bond. In some embodiments, the analog is a variant of pemetrexed, in which the glutamyl group in pemetrexed is in the D-form. In some embodiments, the polyglutamated form of pemetrexed or the polyglutamated pemetrexed analog or derivative is non-fluorinated.

[0124] In further embodiments, the alpha polyglutamated pemetrexed 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.

[0125] B.PMX-PG synthesis The pemetrexed polyglutamate compositions provided herein can be obtained by the following synthetic methods known in the art: Procedures for synthesizing pemetrexed (including different pharmaceutically acceptable salts or acids (e.g., pemetrexed disodium) and crystalline and amorphous forms) and intermediates for synthesizing pemetrexed include, but are not limited to, those disclosed in U.S. Patent Nos. 8,507,508, 8,362,245, 7,138,521, 6,262,262, 6,066,732, 5,416 ... 11, 5,344,932; U.S. Patent Application Publication No. 2013 / 0165654A1; European Patent Nos. 0905128, 2882753B1, 0905128, and 2409978B1; International Publication Nos. 2014 / 024164A1, 2012 / 056285A1, 2008 / 021410A1, and 2001 / 14379A2, and Barnett et al., Org. Proc. Res & Develop. 3:184-188 (1999); Taylor et al., J. Org. Chem. 68:9938-9947 (2003); Taylor et al., Tetrahedron Lett. 40:4023 (1999); Barnett et al., Org. Proc. Res. & Develop. 3:184-188 (1999) and Kjell et al., Org. Proc. Res. Dev. 9:738 (2005).

[0126] The addition of glutamyl residues to glutamyl residues of pemetrexed can be carried out using synthetic methods known in the art. In some embodiments, glutamyl residues are added sequentially to glutamyl residues of pemetrexed. In further embodiments, polyglutamates are added to glutamyl residues of pemetrexed using "click chemistry" or other bioconjugate chemistries known to those skilled in the art. Alternatively, a peptide of the desired length of glutamyl residues can be generated and added to a precursor of pemetrexed that does not contain glutamyl residues. 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 pemetrexed precursor is coupled to the peptide, and the molecule is cleaved from the resin.

[0127] C. Pemetrexed-PG complex The inventors have surprisingly found that polyglutamylated pemetrexed (αPPMX) can be complexed with other compositions, including therapeutic agents, such as cytotoxic compounds, such as platinum-based compounds. Thus, in some embodiments, the present disclosure provides complexes of αPPMX (e.g., the αPPMX disclosed herein) with a therapeutic agent, or a salt or acid thereof.

[0128] In some embodiments, the αPPMX / complex comprises αPPMX and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound such as a chemotherapeutic agent. In further embodiments, the αPPMX / complex comprises a platinum-based drug such as a platinum-based chemotherapeutic agent (e.g., cisplatin, carboplatin, and oxaliplatin). In other embodiments, the αPPMX / complex comprises a taxane-based chemotherapeutic agent (e.g., paclitaxel and docetaxel). In other embodiments, the αPPMX / complex comprises a cyclodextrin. In further embodiments, the αPPMX / complex is encapsulated in a liposome.

[0129] In some embodiments, the present disclosure provides compositions comprising a complex of αPPMX and a therapeutic agent, or a salt or acid thereof. In further embodiments, the αPPMX / therapeutic agent complex comprises one or more αPPMXs containing 2-150, 2-100, 2-75, 2-50, 2-24, 2-30, 2-20, 2-19, 2-15, 2-10, or 2-5 glutamyl groups. In some embodiments, the αPPMX / therapeutic agent complex comprises one or more αPPMXs containing 3-10, 3-9, 3-8, or 3-7 glutamyl groups, or any range therebetween. In other embodiments, the αPPMX / therapeutic agent complex comprises one or more αPPMXs containing 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5 glutamyl groups, or any range therebetween. In one particular embodiment, the complex comprises one or more αPPMXs containing 3-10 glutamyl groups. In further embodiments, the αPPMX / therapeutic agent conjugate comprises one or more αPPMX containing 3-7 glutamyl groups. In another embodiment, the αPPMX / therapeutic agent conjugate comprises one or more αPPMX containing 5 glutamyl groups. In another embodiment, the αPPMX / therapeutic agent conjugate comprises one or more αPPMX containing 6 glutamyl groups. In some embodiments, the therapeutic agent is a cytotoxic compound, or a salt or acid thereof. In further embodiments, the therapeutic agent is a chemotherapeutic agent, or a salt or acid thereof. In another embodiment, the chemotherapeutic agent is a platinum-based drug. In another embodiment, the chemotherapeutic agent is a taxane-based drug. In further embodiments, the molar ratio of αPPMX / therapeutic agent in the conjugate ranges from 1-10:1. In some embodiments, the molar ratio of αPPMX / 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 αPPMX / 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 αPPMX / therapeutic agent complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0130] In alternative embodiments, the αPPMX complex comprises αPPMX and cyclodextrin. In some embodiments, the molar ratio of αPPMX (e.g., αPPMX 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 αPPMX 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 αPPMX 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 αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / cyclodextrin complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0131] In some embodiments, the present disclosure provides compositions comprising an αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / platinum-based agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of αPPMX / 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 αPPMX / 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 αPPMX / platinum-based drug complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0132] In further embodiments, the αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / platinum-based analog complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0133] In further embodiments, the present disclosure provides a complex comprising αPPMX and cisplatin, or a salt or acid thereof. In some embodiments, the molar ratio of αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / cisplatin (or a salt or acid of cisplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In some embodiments, the molar ratio of αPPMX / cisplatin (or a salt or acid of cisplatin) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPPMX / cisplatin (or a salt or acid of cisplatin) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0134] In another embodiment, the present disclosure provides a conjugate comprising αPPMX and carboplatin, or a salt or acid thereof. In some embodiments, the molar ratio of αPPMX / carboplatin (or a carboplatin 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 αPPMX / carboplatin (or a carboplatin 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 αPPMX / carboplatin (or a carboplatin 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 αPPMX / 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 αPPMX / 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 αPPMX / carboplatin (or carboplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0135] In another embodiment, the present disclosure provides a complex comprising αPPMX and oxaliplatin, or a salt or acid thereof. In some embodiments, the molar ratio of αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / oxaliplatin (or oxaliplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0136] In a further embodiment, the present disclosure provides a conjugate comprising αPPMX and a platinum-based chemotherapeutic agent (platinum) selected from the group consisting of nedaplatin, heptaplatin, lobaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexorumaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamines, satraplatin, enloplatin, JM216, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or a salt or acid thereof. In other embodiments, the αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / platinum (or platinum salt or acid) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPPMX / 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 αPPMX / 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 αPPMX / 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).

[0137] In some embodiments, the present disclosure provides compositions comprising an αPPMX / 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 αPPMX / taxane in the conjugate is in the range of 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX 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 αPPMX / taxane complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0138] In further embodiments, the present disclosure provides a complex comprising αPPMX and paclitaxel (PTX) or a salt or acid thereof. In other embodiments, the αPPMX / taxane chemotherapeutic agent complex comprises a paclitaxel (PTX) analog, or a salt or acid thereof. In some embodiments, the molar ratio of αPPMX / paclitaxel (or 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 αPPMX / paclitaxel (or 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 αPPMX / paclitaxel (or 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 αPPMX / 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 αPPMX / 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 αPPMX / paclitaxel (or paclitaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0139] In further embodiments, the present disclosure provides a complex comprising αPPMX and docetaxel (DTX) or a salt or acid thereof. In other embodiments, the αPPMX / taxane chemotherapeutic agent complex comprises a docetaxel (DTX) analog, or a salt or acid thereof. In some embodiments, the molar ratio of αPPMX / docetaxel (or 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 αPPMX / docetaxel (or 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 αPPMX / docetaxel (or 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 αPPMX / 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 αPPMX / docetaxel (or 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 αPPMX / docetaxel (or docetaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0140] In further embodiments, the present disclosure provides a complex comprising αPPMX and larotaxel (LTX) or a salt or acid thereof. In other embodiments, the αPPMX / taxane chemotherapeutic agent complex comprises an analog of larotaxel (LTX), or a salt or acid thereof. In some embodiments, the molar ratio of αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / 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 αPPMX / larotaxel (or larotaxel salt or acid) 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 conjugate comprising αPPMX and cabazitaxel (CTX) or a salt or acid thereof. In other embodiments, the αPPMX / taxane chemotherapeutic agent conjugate comprises an analog of cabazitaxel (CTX), or a salt or acid thereof. In some embodiments, the molar ratio of αPPMX / cabazitaxel (or a 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 αPPMX / cabazitaxel (or a 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 αPPMX / cabazitaxel (or a 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 αPPMX / 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 αPPMX / 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 αPPMX / cabazitaxel (or cabazitaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0142] In further embodiments, the present disclosure provides a complex comprising αPPMX 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 αPPMX and pemetrexed (PMX), or a salt or acid thereof. In some embodiments, the present disclosure provides a complex comprising αPPMX and an antimetabolite selected from the group consisting of gemcitabine, fluorouracil, capecitabine, antifolate (e.g., methotrexate, raltitrexed), 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 αPPMX to 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 αPPMX to 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 αPPMX to 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 αPPMX / 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 αPPMX / 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 αPPMX / anti-metabolite (or salt or acid of the anti-metabolite) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0143] In a further embodiment, the present disclosure provides a complex of αPPMX (e.g., the αPPMX disclosed herein) and cyclodextrin. Cyclodextrins (CDs) are a group of cyclic oligosaccharides that have been shown to improve the physicochemical properties of many drugs through complexation. CDs are cyclic oligosaccharides composed of several D-glucose units linked by α-(1,4) bonds. This cyclic structure provides a hydrophobic interior cavity, giving CDs a shortened, pyramidal shape. Many hydroxyl groups are located on the ends of the ring, making CDs both lipophilic and water-soluble. As a result, CDs can form complexes with a wide variety of hydrophobic drugs, thereby altering the physicochemical properties of these complexed drugs.

[0144] The terms "cyclodextrin" or "CD," unless otherwise specified, generally refer to parent or derivatized cyclic oligosaccharides capable of complexing with pemetrexed-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.

[0145] As used herein, there are no particular limitations on the cyclodextrin component of the αPPMX / cyclodextrin complex, so long as the cyclodextrin is capable of forming a complex with αPPMX. 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 αPPMX and / or liposome encapsulation.

[0146] 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 of the αPPMX / 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., C-C alkyl or C-C alkyl ether moieties) substituted with one or more charged moieties.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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).

[0151] 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.

[0152] 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 average 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.

[0153] 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, and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and up to all α-D-glucopyranoside units in the modified cyclodextrin can be similarly combined. 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.

[0154] Additional cyclodextrin derivatives that can be complexed with therapeutic agents in the disclosed liposomal compositions include sugammadex or Org-25969, in which the 6-hydroxy group on γ-CD has been replaced with a carboxythioacetate ether linkage, and hydroxybutenyl-β-CD. Alternative forms of cyclodextrin include 2,6-di-O-methyl-β-CD (DIMEB), 2-hydroxypropyl-3-cyclodextrin (HP-β-CD), randommethylated-β-cyclodextrin (RAMEB), sulfobutyl ether Examples of suitable cyclodextrins include beta-cyclodextrin (SBE-β-CD), sulfobutylether-γ-cyclodextrin (SBEγCD), sulfobutylated-beta-cyclodextrin sodium salt, (2-hydroxypropyl)-alpha-cyclodextrin, (2-hydroxypropyl)-beta-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, 2,6-di-O-methyl)-beta-cyclodextrin (DIMEB-50 heptakis), 2,3,6-tri-O-methyl)-beta-cyclodextrin (TRIMEB heptakis), methyl-beta-cyclodextrin, octakis(6-deoxy-6-iodo)-γ-cyclodextrin, and octakis(6-deoxy-6-bromo)-gamma-cyclodextrin.

[0155] In some embodiments, the cyclodextrin has high solubility in water to facilitate encapsulation 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).

[0156] In some embodiments, a large binding constant between the cyclodextrin and αPPMX 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 (see, e.g., Albers et al., Crit. Rev. Therap. Drug Carrier Syst. 12:311-337 (1995); Stella et al., Toxicol. Pathol. 36:30-42 (2008)). If the binding constant is pH dependent, the cyclodextrin can be selected to have a large 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 higher. In some embodiments, the binding constant between the cyclodextrin and the therapeutic agent is between 100 and 1,200, 200 and 1,000, 300 and 750, and any range therebetween.

[0157] In some embodiments, the cyclodextrin of the αPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is underivatized.

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

[0159] In some embodiments, the cyclodextrin derivative of the αPPMX / cyclodextrin complex and / or cyclodextrin / therapeutic agent complex has Formula II: [ka] wherein n is 4, 5, or 6; R1, R2, R3, R4, R5, R6, R7, R8, and R9 each independently represent -O- or -O-(C2-C6 alkylene)-SO3 - group; and at least one of R1 and R2 is independently -O-(C2-C6 alkylene)-SO3 - groups; and S1, S2, S3, S4, S5, S6, S7, S8, and S9 are each independently a pharmaceutically acceptable cation. In a further embodiment, the pharmaceutically acceptable cation is Li + , Na + , or K + Alkali metals such as Ca 2+ , or Mg 2+ and ammonium ions and amine cations such as cations of (C-C)-alkylamines, piperidines, pyrazines, (C-C)-alkanolamines, and (C-C)-cycloalkanolamines. In some embodiments, at least one of R and R is independently selected from -O-(CH). man SO3- group, -O-(C2-C6 alkylene)-SO3- group, where m is 2 to 6, preferably 2 to 4 (e.g., -O-CH2CH2CH2SO3- or -O-CH2CH2CH2CH2SO3-); and S1, S2, S3, S4, S5, S6, S7, S8, and S9 are each independently H or a pharmaceutically acceptable cation, including, for example, an alkali metal (e.g., Li + , Na + , K. + ), alkaline earth metals (e.g., Ca 2+ , Mg 2+ ), ammonium ions and amine cations such as the cations of (C1-C6)-alkylamines, piperidines, pyrazines, (C1-C6)-alkanolamines and (C4-C8)-cycloalkanolamines.

[0160] In some embodiments, the cyclodextrin derivative of the αPPMX / cyclodextrin complex and / or cyclodextrin / therapeutic agent complex is a cyclodextrin disclosed in U.S. Patent Nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, 6,869,939; and WO 02005 / 117911, the contents of each of which are hereby expressly incorporated by reference.

[0161] In some embodiments, the cyclodextrin derivative of the αPPMX / 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.

[0162] In some embodiments, the cyclodextrin derivative of the αPPMX / 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.

[0163] In a further embodiment, the αPPMX / cyclodextrin complex and / or cyclodextrin / therapeutic agent complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0164] III. αPPMX Delivery Carrier In alternative embodiments, the present disclosure provides αPPMX delivery systems and their use for delivering a payload of αPPMX to a cell(s) in vitro or in vivo. In some embodiments, αPPMX 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-αPPMX 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.

[0165] A. Liposomes In some embodiments, the present disclosure provides liposome compositions comprising liposomes encapsulating (loaded with) alpha polyglutamated pemetrexed (e.g., αPPMX as disclosed herein). In some embodiments, the liposomes in the liposome composition comprise αPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups (including the glutamyl group of pemetrexed). In some embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains two or more L-glutamyl groups. In other embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains D-glutamyl groups. In further embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains D-glutamyl groups and two or more L-glutamyl groups. In further embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains two or more glutamyl groups with gamma carboxyl linkages. In some embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX 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 α-pentaglutamated PMX. In further embodiments, the liposomes comprise L-α pentaglutamated PMX, D-α pentaglutamated PMX, or L- and D-α pentaglutamated PMX. In some embodiments, the liposome composition comprises liposomes containing hexaglutamated PMX (Lp-αPPMX). In further embodiments, the liposomes comprise L-α hexaglutamated PMX, D-α hexaglutamated PMX, or L- and D-α hexaglutamated PMX. 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-αPPMX composition is not PEGylated. In some embodiments, the Lp-αPPMX composition is non-targeted (NTLp-αPPMX).In other embodiments, the Lp-αPPMX composition is targeted (TLp-αPPMX). 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 pemetrexed, or any range therebetween, is encapsulated (encapsulated) in Lp-αPPMX. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or greater than 75% of alpha polyglutamated pemetrexed is encapsulated in Lp-αPPMX during the liposome preparation process.

[0166] In some embodiments, provided liposomes further comprise an immunostimulatory agent, a detectable marker, or both, disposed on the exterior surface of the liposome. The immunostimulatory agent or detectable marker can be ionically or covalently bound to the exterior surface of the liposome, optionally including, for example, binding to a steric stabilizing component of the liposome.

[0167] 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. Specific adjuvants include, 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 quinoline. A; IFN gamma, IFN alpha, FLT3 ligand; and immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3) are commercially available. 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 immunostimulatory agent can 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 immunostimulatory agent 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.

[0168] 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.

[0169] 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.

[0170] In some embodiments, the liposome further comprises an agent that increases uptake of the liposome into a desired intracellular compartment, including the cytosol.

[0171] 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.

[0172] 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).

[0173] 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.

[0174] 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.

[0175] 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 steric 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.

[0176] In some embodiments, the liposome composition comprises PEGylated liposomes (PLp-αPPMX). In some embodiments, the PEGylated liposomes in the liposome composition comprise αPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains two or more L-glutamyl groups. In other embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains D-glutamyl groups. In further embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains D-glutamyl groups and two or more L-glutamyl groups. In further embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX 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 PMX. In further embodiments, the liposomes comprise L-α-pentaglutamated PMX, D-α-pentaglutamated PMX, or L- and D-α-pentaglutamated PMX. In some embodiments, the liposome composition comprises PEGylated liposomes comprising α-hexaglutamated PMX. In further embodiments, the liposomes comprise L-α-hexaglutamated PMX, D-α-hexaglutamated PMX, or L- and D-α-hexaglutamated PMX. 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-αPPMX composition is non-targeted (NTPLp-αPPMX). In other embodiments, the PLp-αPPMX composition is targeted (TPLp-αPPMX). In further embodiments, the liposome composition comprises PEGylated liposomes containing 30-70%, 30-60%, or 30-50% liposome-encapsulated alpha polyglutamated pemetrexed, 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 alpha polyglutamated pemetrexed is encapsulated in PLp-αPPMX. 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.

[0177] In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamated pemetrexed in the composition has 4 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamated pemetrexed in the provided liposome compositions is tetraglutamated. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamated pemetrexed in the provided liposome compositions is pentaglutamated. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamated pemetrexed in the provided liposome compositions is hexaglutamated.

[0178] In some embodiments, the alpha polyglutamated pemetrexed composition (e.g., delivery vehicles such as polyglutamates and liposomes containing polyglutamates) is in an aqueous solution. In some embodiments, the αPPMX composition is in a volume of 1 square meter (m 2 ) as a liposome composition. 2In a further embodiment, the αPPMX composition is administered as a liposome composition at a dose of about 0.1 to about 1000 mg of αPPMX per square meter of body surface area, or any range therebetween.

[0179] (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, the alpha-polyglutamated PMX 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 interior space separated from the external medium by one or more lipid bilayers, or any microcapsule structure with a semipermeable membrane with a lipophilic core separating the interior. The lipid bilayer can be any structure of amphiphilic molecules characterized by hydrophilic and hydrophobic portions. Typically, the amphiphilic molecules in the bilayer are arranged in a two-dimensional sheet, with the hydrophobic portions facing the interior of the sheet, while the hydrophilic portions facing the exterior. 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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 pemetrexed 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] In this specification, anionic and neutral lipids are collectively referred to as non-cationic lipids. Such lipids may contain phosphorus, but they are not so limited. Examples of non-cationic lipids include lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), lecithin, ... 1-Ethanolamine (DSPE), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans These include PE, palmitoyloleoylphosphatidylethanolamine (POPE), 1-stearoyl-2-oleoylphosphatidiethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, and cholesterol.

[0190] 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.

[0191] In some embodiments, the αPPMX 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 2011 / 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-dimemylhexacosa-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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] In some embodiments, the αPPMX 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, but not limited to, polylysine, polyornithine, and / or polyarginine, and cationic peptides described in WO 2012 / 013326, which is incorporated herein by reference in its entirety. In another embodiment, the αPPMX is formulated in a lipid-polycation complex, which further includes a neutral lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0196] 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.

[0197] 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.

[0198] 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.

[0199] In some embodiments, the liposomal alpha polyglutamated pemetrexed composition is pegylated (i.e., a pegylated liposomal alpha polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate (PLp-αPPMX or PLp-αPPMX)). In some embodiments, the PLp-αPPMX or PLp-αPPMX is water-soluble. That is, the PLp-αPPMX or PLp-αPPMX is in the form of an aqueous solution.

[0200] 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.

[0201] 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.

[0202] 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).

[0203] 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.

[0204] 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).

[0205] (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 pemetrexed provided herein. In further embodiments, the interior space of the liposome comprises a tonicity agent. 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.

[0206] 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% (wt%) trehalose, or any combination of one or more lyoprotectants or cryoprotectants at a total concentration of 5%-20%. In some embodiments, the pH of the solution containing the liposome composition is 6-7.5, 6.5-7.5, 6.7-7.5, or 6.3-7.0, or any range therebetween. In some embodiments, the interior space contains a buffer solution. In some embodiments, the buffer is selected from HEPES, citrate, or sodium phosphate (e.g., monobasic sodium phosphate and / or dibasic sodium phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., monobasic sodium phosphate and / or dibasic sodium phosphate). In some embodiments, the buffer is 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.

[0207] 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 some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., monobasic sodium phosphate and / or dibasic sodium phosphate). In some embodiments, the buffer is at a concentration of 15-200 mM, or any range therebetween. In still further embodiments, the buffer is at a concentration of 5-200 mM, 15-200 mM, 5-100 mM, 15-100 mM, 5-50 mM, 15-50 mM, 5-25 mM, 5-20 mM, 5-15 mM, or any range therebetween. In some embodiments, the buffer is HEPES at a concentration of 15-200 mM, or any range therebetween. In some embodiments, the buffer is citrate at a concentration of 15-200 mM, or any range therebetween. In some embodiments, the buffer is sodium phosphate at a concentration of 15-200 mM, or any range therebetween. In further embodiments, the interior space of the liposome contains sodium acetate and / or calcium acetate.In some embodiments, the interior space of the liposome comprises a total concentration of sodium acetate and calcium acetate of 5 mM to 500 mM, or 50 mM to 500 mM, or any range therebetween.

[0208] In further embodiments, the present disclosure provides liposome compositions comprising liposomes encapsulating (i.e., loaded with) alpha polyglutamated pemetrexed (e.g., αPPMX as disclosed herein). In some embodiments, the liposomes in the liposome composition comprise αPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups (including the glutamyl group of pemetrexed). In some embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains two or more L-glutamyl groups. In other embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains D-glutamyl groups. In further embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains D-glutamyl groups and two or more L-glutamyl groups. In further embodiments, the alpha polyglutamated pemetrexed in Lp-αPPMX contains two or more glutamyl groups with gamma carboxyl bonds. In some embodiments, the liposome composition comprises liposomes containing α pentaglutamated PMX. In further embodiments, the liposomes contain L-α pentaglutamated PMX, D-α pentaglutamated PMX, or L- and D-α pentaglutamated PMX. In some embodiments, the liposome composition comprises liposomes containing α hexaglutamated PMX (Lp-αPPMX). In further embodiments, the liposomes contain L-α hexaglutamated PMX, D-α hexaglutamated PMX, or L- and D-α hexaglutamated PMX.

[0209] In some embodiments, the targeted PEGylated liposomal alpha-polyglutamated (e.g., pentaglutamated or hexaglutamated) pemetrexed comprises a vehicle comprising a liposome comprising an interior space; aqueous alpha-polyglutamated pemetrexed disposed within the interior space; and a targeting moiety comprising a protein having specific affinity for at least one folate receptor, the targeting moiety being disposed on the exterior surface of the liposome. In some embodiments, the vehicle is an aqueous solution. In some embodiments, the interior space, the exterior space (e.g., the vehicle), or both the interior space and the vehicle comprise one or more of the above-listed lyoprotectants or cryoprotectants. In some embodiments, the cryoprotectant is mannitol, trehalose, sorbitol, or sucrose.

[0210] In some embodiments, liposomes encapsulating alpha polyglutamated pemetrexed (i.e., Lp-αPPMX, including PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) have an interior space containing fewer than 500,000 or fewer than 200,000 alpha polyglutamated pemetrexed molecules. In some embodiments, the liposome interior space contains 10 to 100,000 alpha polyglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposome interior space contains 10,000 to 100,000 alpha polyglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposomes are not PEGylated and have an interior space containing fewer than 500,000 or fewer than 200,000 alpha polyglutamated pemetrexed molecules. In some embodiments, the liposomes are non-PEGylated and the interior space of the liposomes contains 10 to 100,000 alpha polyglutamated pemetrexed molecules, or any range therebetween. In further embodiments, the liposomes are non-PEGylated and the interior space of the liposomes contains 10,000 to 100,000 alpha polyglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposomes are targeted and non-PEGylated (TLp-αPPMX) and have an interior space containing less than 500,000 or less than 200,000 alpha polyglutamated pemetrexed molecules. In some embodiments, the liposomes are targeted and non-PEGylated and the interior space of the liposomes contains 10 to 100,000 alpha polyglutamated pemetrexed molecules, or any range therebetween. In a further embodiment, the liposome is targeted and non-PEGylated, and the interior space of the liposome contains 10,000 to 100,000 alpha polyglutamated pemetrexed molecules, or any range therebetween.In some embodiments, the liposomes are non-targeted and non-PEGylated (NTLp-αPPMX) and have an interior space containing fewer than 500,000 or fewer than 200,000 alpha polyglutamated pemetrexed molecules. In some embodiments, the liposomes are non-targeted and non-PEGylated and the interior space of the liposomes contains 10 to 100,000 alpha polyglutamated pemetrexed molecules, or any range therebetween. In further embodiments, the liposomes are non-targeted and non-PEGylated and the interior space of the liposomes contains 10,000 to 100,000 alpha polyglutamated pemetrexed molecules, or any range therebetween.

[0211] In some embodiments, the liposomes encapsulate alpha polyglutamated pemetrexed containing 2 to 10 glutamyl groups (i.e., Lp-αPPMX, including PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) and have an interior space containing fewer than 500,000 or fewer than 200,000 alpha polyglutamated pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome interior space contains 10 to 100,000 alpha polyglutamated pemetrexed molecules containing 2 to 10 glutamyl groups, or any range therebetween. In further embodiments, the liposome interior space contains 10,000 to 100,000 alpha polyglutamated pemetrexed molecules containing 2 to 10 glutamyl groups, or any range therebetween. In some embodiments, the liposomes are not PEGylated and have an interior space containing fewer than 500,000 or fewer than 200,000 alpha polyglutamated pemetrexed molecules containing 2-10 glutamyl groups. In some embodiments, the liposomes are not PEGylated and the interior space contains 10-100,000 alpha polyglutamated pemetrexed molecules containing 2-10 glutamyl groups, or any range therebetween. In further embodiments, the liposomes are not PEGylated and the interior space contains 10,000-100,000 alpha polyglutamated pemetrexed molecules containing 2-10 glutamyl groups, or any range therebetween. In some embodiments, the liposomes are targeted and non-PEGylated (TLp-αPPMX) and have an interior space containing fewer than 500,000 or fewer than 200,000 alpha polyglutamated pemetrexed molecules containing 2-10 glutamyl groups. In some embodiments, the liposomes are targeted and non-PEGylated and the interior space of the liposomes contains 10-100,000 or any range therebetween alpha polyglutamated pemetrexed molecules containing 2-10 glutamyl groups.In further embodiments, the liposomes are targeted and non-PEGylated, and the interior space of the liposomes contains 10,000 to 100,000 alpha polyglutamated pemetrexed molecules containing 2 to 10 glutamyl groups, or any range therebetween. In some embodiments, the liposomes are non-targeted and non-PEGylated (NTLp-αPPMX), and have an interior space containing fewer than 500,000 or fewer than 200,000 alpha polyglutamated pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposomes are non-targeted and non-PEGylated, and the interior space of the liposomes contains 10 to 100,000 alpha polyglutamated pemetrexed molecules containing 2 to 10 glutamyl groups, or any range therebetween. In a further embodiment, the liposome is non-targeted and non-PEGylated, and the interior space of the liposome contains 10,000 to 100,000 alpha polyglutamated pemetrexed molecules containing 2 to 10 glutamyl groups, or any range therebetween.

[0212] In some embodiments, the liposomes encapsulate alpha-polyglutamated pemetrexed (i.e., Lp-αPPMX, including PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) and have an interior space containing fewer than 500,000 or fewer than 200,000 alpha-tetraglutamated pemetrexed molecules. In some embodiments, the liposome interior space contains 10 to 100,000 alpha-tetraglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposome interior space contains 10,000 to 100,000 alpha-tetraglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposomes are not PEGylated and have an interior space containing fewer than 500,000 or fewer than 200,000 alpha-tetraglutamated pemetrexed molecules. In some embodiments, the liposomes are non-PEGylated and the interior space of the liposomes contains 10 to 100,000 alpha-tetraglutamated pemetrexed molecules, or any range therebetween. In further embodiments, the liposomes are non-PEGylated and the interior space of the liposomes contains 10,000 to 100,000 alpha-tetraglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposomes are targeted and non-PEGylated (TLp-αPPMX) and have an interior space containing less than 500,000 or less than 200,000 alpha-tetraglutamated pemetrexed molecules. In some embodiments, the liposomes are targeted and non-PEGylated and the interior space of the liposomes contains 10 to 100,000 alpha-tetraglutamated pemetrexed molecules, or any range therebetween. In a further embodiment, the liposome is targeted and non-pegylated, and the interior space of the liposome contains 10,000 to 100,000 alpha tetraglutamated pemetrexed molecules, or any range therebetween.In some embodiments, the liposomes are non-targeted and non-PEGylated (NTLp-αPPMX) and have an interior space containing fewer than 500,000 or fewer than 200,000 alpha-tetraglutamated pemetrexed molecules. In some embodiments, the liposomes are non-targeted and non-PEGylated and the interior space of the liposomes contains 10 to 100,000 alpha-tetraglutamated pemetrexed molecules, or any range therebetween. In further embodiments, the liposomes are non-targeted and non-PEGylated and the interior space of the liposomes contains 10,000 to 100,000 alpha-tetraglutamated pemetrexed molecules, or any range therebetween.

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

[0214] In some embodiments, the liposomes encapsulate alphahexaglutamated pemetrexed (i.e., Lp-αPPMX, including PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) and have an interior space containing fewer than 500,000 or fewer than 200,000 alphahexaglutamated pemetrexed molecules. In some embodiments, the liposome interior space contains 10 to 100,000 alphahexaglutamated pemetrexed molecules, or any range therebetween. In further embodiments, the liposome interior space contains 10,000 to 100,000 alphahexaglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposomes are non-PEGylated and have an interior space containing fewer than 500,000 or fewer than 200,000 alphahexaglutamated pemetrexed molecules. In some embodiments, the liposomes are non-PEGylated and have an interior space containing 10 to 100,000 alphahexaglutamated pemetrexed molecules, or any range therebetween. In further embodiments, the liposomes are non-PEGylated and have an interior space containing 10,000 to 100,000 alphahexaglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposomes are targeted, non-PEGylated (TLp-αPPMX), and have an interior space containing fewer than 500,000 or fewer than 200,000 alphahexaglutamated pemetrexed molecules. In some embodiments, the liposomes are targeted and non-PEGylated, and the interior space of the liposomes contains 10 to 100,000 alphahexaglutamated pemetrexed molecules, or any range therebetween. In further embodiments, the liposomes are targeted and non-PEGylated, and the interior space of the liposomes contains 10,000 to 100,000 alphahexaglutamated pemetrexed molecules, or any range therebetween.In some embodiments, the liposomes are non-targeted and non-PEGylated (NTLp-αPPMX) and have an interior space containing fewer than 500,000 or fewer than 200,000 alphahexaglutamated pemetrexed molecules. In some embodiments, the liposomes are non-targeted and non-PEGylated and the interior space of the liposomes contains 10 to 100,000 alphahexaglutamated pemetrexed molecules, or any range therebetween. In further embodiments, the liposomes are non-targeted and non-PEGylated and the interior space of the liposomes contains 10,000 to 100,000 alphahexaglutamated pemetrexed molecules, or any range therebetween.

[0215] In some embodiments, the present disclosure provides liposomal alpha polyglutamated pemetrexed compositions, wherein liposomes encapsulate alpha polyglutamated pemetrexed, or a salt or acid thereof, and one or more aqueous pharmaceutically acceptable carriers. In some embodiments, the interior space of the liposomes contains trehalose. In some embodiments, the interior space of the liposomes contains 1% to 50% (wt%) trehalose. In some embodiments, the interior space of the liposomes contains HBS at a concentration of 1 to 200 mM and a pH of 2 to 8. In some embodiments, the interior space of the liposomes has a pH of 5 to 8, or any range therebetween. In some embodiments, the interior space of the liposomes has a pH of 6 to 7, or any range therebetween. In some embodiments, the interior space of the liposomes contains a combined concentration of sodium acetate and calcium acetate of 50 mM to 500 mM, or any range therebetween.

[0216] A. Non-polyglutamylated polyglutamylated antifolates In some embodiments, liposomal alpha polyglutamated pemetrexed (i.e., Lp-αPPMX, including PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) compositions comprise alpha polyglutamated pemetrexed (e.g., αPPMX disclosed herein) and one or more non-polyglutamylatable polyglutamate antifolate compositions.

[0217] In some embodiments, Lp-αPPMX (e.g., PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) compositions comprise alpha polyglutamated pemetrexed (e.g., the αPPMX disclosed herein) and pemetrexed (PMX). In some embodiments, Lp-αPPMX (i.e., liposomal alpha polyglutamated pemetrexed) comprises alpha polyglutamated pemetrexed and a polyglutamylatable antifolate selected from the group consisting of pemetrexed (PMX), methotrexate (MTX), lometrexol (LMX), raltitrexed (RTX), pralatrexate, AG2034, GW1843, aminopterin, and LY309887. In some embodiments, Lp-αPPMX comprises alpha polyglutamated pemetrexed and lometrexol. In some embodiments, Lp-αPPMX comprises alpha polyglutamated pemetrexed and pemetrexed. In some embodiments, Lp-αPPMX comprises alpha polyglutamated pemetrexed and leucovorin. In some embodiments, Lp-αPPMX comprises alpha polyglutamated pemetrexed and a triazine antifolate derivative (e.g., a sulfonyluride triazine such as NSC127755). In some embodiments, Lp-αPPMX comprises alpha polyglutamated pemetrexed and a serine hydroxymethyltransferase (SHMT2) inhibitor. In some embodiments, the SHMT2 inhibitor is an antifolate (e.g., a polyglutamylatable or non-polyglutamylatable antifolate). In some embodiments, the SHMT2 inhibitor is an antifolate.

[0218] B. Non-polyglutamylatable antifolates In some embodiments, Lp-αPPMX (e.g., PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) comprises alpha polyglutamated pemetrexed (e.g., αPPMX disclosed herein) and a so-called "non-polyglutamylatable" antifolate. In some embodiments, the liposome comprises alpha polyglutamated pemetrexed and a non-polyglutamylatable antifolate that inhibits one or more enzymes in the folate cycle metabolic pathway. In further embodiments, the non-polyglutamylatable antifolate inhibits one or more enzymes selected from thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide (GAR) transformylase, and aminoimidazolecarboxamide ribonucleotide (AICAR) transformylase. In some embodiments, the liposomes comprise alpha polyglutamated pemetrexed and a non-polyglutamate antifolate that inhibits DHFR. In some embodiments, the liposomes comprise alpha polyglutamated pemetrexed and a non-polyglutamate antifolate that inhibits TS. In some embodiments, the liposomes comprise alpha polyglutamated pemetrexed and a non-polyglutamate antifolate that inhibits GAR or AICAR transformylase. In further embodiments, the non-polyglutamate antifolate is selected from the group consisting of trimetrexate (TMQ), piritrexim (BW301U), and talotrexin (PT523). In further embodiments, the non-polyglutamylatable antifolate is selected from the group consisting of nolatrexed (AG337), previtrexed (ZD9331, BGC9331), BGC945 (ONX0801).

[0219] C platinum In some embodiments, liposomes comprising alpha polyglutamated pemetrexed (e.g., Lp-αPPMX, such as PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTL...

Claims

1. A liposome composition comprising liposomes encapsulating alpha polyglutamated pemetrexed, the alpha polyglutamylated pemetrexed comprises 2-10 glutamyl groups with alpha carboxyl linkages; the liposome is pegylated and comprises a targeting moiety having specific affinity for a surface antigen on a target cell; the liposomes further encapsulate one or more non-polyglutamylatable or non-polyglutamylatable antifolates; Liposomal compositions.

2. The liposome composition of claim 1, wherein the alpha polyglutamylated pemetrexed contains 4-6 glutamyl groups with alpha carboxyl group linkages.

3. the non-polyglutamylated polyglutamylatable antifolate is selected from the group consisting of methotrexate (MTX), pemetrexed (PMX), lometrexol (LMX), raltitrexed (RTX), pralatrexate, AG2034, GW1843, aminopterin, and LY309887; or the non-polyglutamylatable antifolate is selected from the group consisting of trimetrexate (TMQ), piritrexim (BW301U), tarotrexin (PT523), nolatrexide (AG337), previtrexed (ZD9331, BGC9331), and BGC945 (ONX0801); The liposome composition of claim 1.

4. (a) two or more glutamyl groups of the alpha polyglutamylated pemetrexed have an alpha carboxyl group bond; (b) each glutamyl group of the alpha polyglutamylated pemetrexed has an alpha carboxyl group bond; (c) at least one glutamyl group has both an alpha carboxyl group linkage and a gamma carboxyl group linkage; (d) at least two glutamyl groups of the alpha polyglutamylated pemetrexed are in the L-configuration; (e) each glutamyl group of the alpha polyglutamylated pemetrexed is in the L-configuration; (f) at least one glutamyl group of the alpha polyglutamylated pemetrexed is in the D form; (g) each glutamyl group of said alpha polyglutamylated pemetrexed other than the glutamyl group of pemetrexed is in the D form; or (h) at least two of the glutamyl groups of the alpha polyglutamylated pemetrexed are in the L-form and at least one of the glutamyl groups is in the D-form; The liposome composition of claim 1.

5. 2. The liposome composition of claim 1, wherein the liposome comprises an alpha polyglutamated pemetrexed, including alpha tetraglutamated pemetrexed, alpha pentaglutamated pemetrexed, or alpha hexaglutamated pemetrexed.

6. The liposome composition of claim 1 , wherein the polyglutamate is linear or branched.

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

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

9. 2. The liposome composition of claim 1, wherein the targeting moiety is a polypeptide or 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.

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

11. The liposomes are formed from liposome components including at least one of anionic lipids and neutral lipids, and at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide, and optionally the liposome components include at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC, and optionally one or more liposome components include at least one selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM 1); 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, optionally wherein the steric stabilizer is PEG, the PEG having a number average molecular weight (Mn) of 200 to 5000 Daltons.

12. The liposome is anionic. the liposome is cationic; The liposome is neutral. the liposome has a zeta potential of zero or less; the liposome has a zeta potential of 0 to -150 mV; or The liposome has a zeta potential of -30 to -50 mV. The liposome composition of claim 1.

13. 2. The liposome composition of claim 1, wherein the liposome has an interior space containing the alpha polyglutamated pemetrexed and an aqueous pharma- ceutically acceptable carrier, the aqueous pharma-ceutically acceptable carrier comprising an isotonicity agent such as dextrose, mannitol, glycerol, potassium chloride, sodium chloride at a concentration greater than 1%, 1%-50% trehalose, 5% dextrose suspended in HEPES buffer, or sodium acetate and calcium acetate at a total concentration of 50 mM-500 mM, optionally the liposome interior space has a pH of 5-8 or a pH of 6-7, or any range therebetween, and optionally the liposome comprises less than 500,000 or less than 200,000 of the alpha polyglutamated pemetrexed molecules, or 10-100,000 or any range therebetween.

14. The liposome further comprises one or more of an immunostimulant, a detectable marker, and a maleimide, wherein the immunostimulant, the detectable marker, or the maleimide is attached to the PEG or the outer surface of the liposome, and the immunostimulant is selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan, resolvin (e.g., resolvin D, e.g., D ｎ－６ＤＰＡ Or D ｎ－３ＤＰＡ , resolvin E, or T-series resolvins), and oxidized low density lipoproteins (e.g., OXPAC, PGPC), and Toll-like receptor (TLR) modulators such as erythropoietin lipids (e.g., E5564), optionally further comprising at least one cryoprotectant selected from the group consisting of mannitol, trehalose, sorbitol, and sucrose, or further comprising carboplatin and / or pembrolizumab.

15. A pharmaceutical composition comprising the liposomal alpha polyglutamated pemetrexed composition of claim 1.

16. A composition comprising the liposome composition according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 15 for the treatment of cancer, immune system disorders, or infectious diseases.

17. 17. The composition of claim 16 for treating a cancer selected from the group consisting of breast cancer, head and neck cancer, lung cancer, gastric cancer and bladder cancer.

18. 15. A method of making an alpha polyglutamated pemetrexed composition comprising the liposomal alpha polyglutamated pemetrexed composition of any one of claims 1 to 14, comprising forming a mixture comprising liposome components and alpha polyglutamated pemetrexed in a solution; homogenizing the mixture in the solution to form liposomes; and processing the mixture to form liposomes comprising alpha polyglutamated pemetrexed, optionally wherein the processing comprises one or more of the following steps: thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw method, reverse phase evaporation, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, lyophilized double emulsion, 3D printing, membrane contactor method, and stirring.

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