Gamma polyglutamylated pemetrexed and uses thereof

Abstract

To provide compositions to treat diseases including hyperproliferative diseases such as cancer, disorders of the immune system such as inflammation and rheumatoid arthritis, and infectious disease such as HIV and malaria.SOLUTION: Provided are gamma polyglutamated pemetrexed compositions including delivery vehicles such as liposomes containing the gamma polyglutamated pemetrexed, and gamma polyglutamated pemetrexed compositions to treat hyperproliferative disorders (e.g., cancer) and disorders of the immune system (e.g., inflammation and autoimmune diseases such as rheumatoid arthritis). Also provided are methods of making the compositions.SELECTED DRAWING: None

Description

[Background technology]

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

[0002] Pemetrexed disodium is the active ingredient in the antineoplastic product 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 Pemetrexed is also known as N5Na2O6·7H2O. It is 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 also demonstrated activity in clinical trials in a variety of tumor types, including lung cancer, breast cancer, colorectal 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 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. Similarly, monoglutamate-type polyglutamic antifolates, such as pemetrexed, do not transport across cell membranes. Once inside cells, intracellular folate is converted to polyglutamate by the enzyme folylpolygammaglutamate synthase (FPGS).

[0004] Pemetrexed is a multitargeted antifolate drug that acts by disrupting folate-dependent metabolic processes essential for cellular homeostasis and replication. Pemetrexed inhibits at least 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, leading to disruption of cellular homeostasis and an imbalance of purine and pyrimidine precursors, which prevents cells from undergoing 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) gamma and beta, as well as by the proton-coupled folate transporter (PCFT), which is most active in subnormal pH environments. RFC is the primary pemetrexed transporter at physiological pH and is widely expressed in both normal and diseased cells. As a result, pemetrexed treatment is often affected by dose-limiting toxicity, a major obstacle in cancer chemotherapy. Once inside the cell, pemetrexed is polyglutamylated by FPGS, which can add up to six L-glutamyl groups to pemetrexed in the L-gamma carboxyl linkage. L-gamma polyglutamation 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 promotes the accumulation of polyglutamylated pemetrexed, which, unlike pemetrexed (monoglutamate), is not freely transported out of cells by cellular efflux pumps.

[0006] Pemetrexed specifically acts on DNA and RNA synthesis, resulting in significant toxic effects on rapidly dividing cells, such as malignant and myeloid cells. Myelosuppression is typically the dose-limiting toxicity of pemetrexed therapy, limiting its clinical application. Pretreatment with folic acid and vitamin B6 is currently used to ameliorate the most frequent side effects associated with pemetrexed treatment, including myelosuppression, fatigue, and skin 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 synthase activity, (c) decreased folylpoly-gamma-glutamate synthase (FPGS) activity, and (d) increased gamma-glutamyl hydrolase (GGH) activity, which cleaves gamma-polyglutamate chains attached to folate and antifolates, making them more susceptible to efflux transport out of the cell.

[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 use of gamma polyglutamated pemetrexed compositions provides a strategy to overcome the pharmacological challenges associated with dose-limiting toxicity and therapeutic resistance associated with pemetrexed therapy. The provided methods deliver potently cytotoxic gamma polyglutamated pemetrexed to target cells while (a) minimizing / reducing exposure to normal tissue cells, (b) optimizing / improving the cytotoxic effect of pemetrexed-based drugs on target cells, such as cancer cells, and (c) minimizing / reducing the effects of efflux pumps, altered activity of enzymes in the folate metabolic pathway, and other resistance mechanisms that limit the therapeutic efficacy of pemetrexed. Summary of the Invention

[0010] The present disclosure relates generally to gamma polyglutamated pemetrexed (PMX) compositions and methods of making and using the compositions to treat diseases, including hyperproliferative diseases such as cancer, inflammation and immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0011] In some embodiments, the present disclosure provides: [1] A composition comprising gamma polyglutamated pemetrexed; [2] The composition according to item [1], wherein the gamma polyglutamylated pemetrexed contains 1 to 10 glutamyl groups having gamma carboxyl group bonds; [3] The composition according to item [1] or [2], wherein the gamma polyglutamated pemetrexed contains 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups with gamma carboxyl group bonds; [4] The composition according to any one of items [1] to [3], wherein the gamma polyglutamated pemetrexed is gamma tetraglutamated pemetrexed; [5] The composition according to any one of items [1] to [3], wherein the gamma polyglutamated pemetrexed is gamma pentaglutamated pemetrexed; [6] The composition according to any one of items [1] to [3], wherein the gamma polyglutamated pemetrexed is gamma hexaglutamated pemetrexed; [7] The composition according to any one of items [1] to [6], which is the following composition: (a) gamma polyglutamated pemetrexed contains two or more L-glutamyl groups with gamma carboxyl linkages; (b) each of the glutamyl groups of the gamma polyglutamated pemetrexed is in the L-configuration and has a gamma carboxyl group bond; (c) at least one glutamyl group of the gamma polyglutamated pemetrexed is in the D-form and has a gamma carboxyl group bond; (d) each glutamyl group of gamma-polyglutamated pemetrexed other than the glutamyl group of pemetrexed is D-type and has a gamma carboxyl group bond; or (e) Gamma polyglutamated pemetrexed contains two or more L-glutamyl groups and at least one D-glutamyl group with a gamma carboxyl group bond. [8] The composition according to item [4], (a) each glutamyl group is in the L-configuration and has a gamma carboxyl linkage, or (b) each glutamyl group other than the glutamyl group of pemetrexed is in the D-configuration and each glutamyl group has a gamma carboxyl linkage. [9] The composition according to item [5], wherein (a) each glutamyl group is in the L-configuration and has a gamma carboxyl group bond, or (b) each glutamyl group other than the glutamyl group of pemetrexed is in the D-configuration and each glutamyl group has a gamma carboxyl group bond;

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

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

[10] , wherein the gamma-polyglutamated aminopterin can be polyglutamated by FGPS under physiological conditions, and / or the polyglutamated PMX has a lower uptake rate (less than 30%) by hepatocytes than PMX;

[12] A liposomal composition containing gamma polyglutamylated pemetrexed (Lp-γPPMX) according to any one of items [1] to

[11] ;

[13] The Lp-γPPMX composition according to item

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

[14] The Lp-γPPMX composition according to item

[12] or

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

[15] The Lp-γPPMX composition according to item

[12] or

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

[28] The Lp-γPPMX composition according to item

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

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

[27] to

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

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

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

[33] , wherein the liposome is cationic;

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

[12] to

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

[40] The Lp-γPPMX 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-γPPMX composition according to item

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

[42] The Lp-γPPMX composition according to item

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

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

[39] to

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

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

[39] to

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

[45] The Lp-γPPMX 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 a similar substance at a concentration of 1 to 200 mM and a pH of 2 to 8;

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[50] or

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

[53] The Lp-γPPMX 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-γPPMX 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 an Lp-γPPMX composition that binds to surface antigens with an equilibrium dissociation constant (Kd) in the range of

[55] The Lp-γPPMX composition according to any one of Items

[50] to

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

[56] The Lp-γPPMX composition according to any one of Items

[50] to

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

[57] The Lp-γPPMX 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-γPPMX composition according to any one of Items

[39] to

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

[59] The Lp-γPPMX 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-γPPMX composition according to item

[58] or

[59] , wherein the immunostimulant is fluorescein, fluorescein isothiocyanate (FITC), DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan, resolvin (e.g., D n-6DPA Or D n-3DPA an Lp-γPPMX composition, wherein the at least one selected from the group consisting of a toll-like receptor (TLR) modulator, such as resolvin D, resolvin E, or T-series resolvin (e.g., OXPAC, PGPC), and an eritoran lipid (e.g., E5564);

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

[58] to

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

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

[58] to

[61] , further comprising a hapten;

[63] The Lp-γPPMX composition according to item

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

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

[12] to

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

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

[64] ;

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

[49] ;

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

[12] to

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

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

[12] to

[67] .

[69] A pharmaceutical composition comprising the gamma 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 of such treatment or prevention, 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 of such treatment or prevention, the method comprising administering to the subject the liposomal gamma polyglutamated pemetrexed composition according to any one of

[12] to

[69] ;

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

[69] ;

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

[12] to

[69] ;

[76] The method according to item

[74] or

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

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

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

[78] A method for treating cancer, comprising administering an effective amount of the liposomal gamma polyglutamated pemetrexed composition according to any one of items

[12] to

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

[79] The method according to item

[77] or

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

[80] The method according to item

[77] or

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

[81] The method according to item

[77] or

[78] , wherein the cancer is 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-γPPMX 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 gamma polyglutamated pemetrexed composition according to any one of items

[12] 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 gamma polyglutamated pemetrexed composition according to any one of items [8] 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 gamma polyglutamated pemetrexed composition according to any one of items

[12] to

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

[90] A method for delivering gamma polyglutamated pemetrexed to a tumor expressing a folate receptor on its surface, comprising administering the Lp-γPPMX composition according to any one of Items [1] to

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

[91] A method for preparing a gamma polyglutamated pemetrexed composition, including the liposomal gamma polyglutamated pemetrexed composition according to any one of Items

[12] to

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

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

[12] to

[69] , comprising the steps of: forming a mixture in a solution containing liposome components and gamma polyglutamated pemetrexed; homogenizing the mixture in the solution to form liposomes; and treating the mixture to form liposomes that entrap and / or encapsulate gamma 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);

[93] The method according to item

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

[94] The method according to item

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

[0012] In some embodiments, the present disclosure provides gamma polyglutamylated pemetrexed (γPPMX) compositions, wherein at least two glutamyl residues of the gamma polyglutamylated pemetrexed have gamma carboxyl linkages. In some embodiments, the γPPMX contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl groups in pemetrexed). In some embodiments, the γPPMX contains two or more L-glutamyl groups. In other embodiments, the γPPMX contains a D-glutamyl group. In further embodiments, the γPPMX contains a D-glutamyl group and two or more L-glutamyl groups.

[0013] In one embodiment, the γPPMX composition comprises a chain of three glutamyl groups bound to the glutamyl group of pemetrexed (i.e., tetraglutamylated pemetrexed). In some embodiments, the tetraglutamylated PMX comprises two or more L-glutamyl groups. In other embodiments, the tetraglutamylated PMX comprises a D-glutamyl group. In further embodiments, the tetraglutamylated PMX comprises a D-glutamyl group and two or more L-glutamyl groups.

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

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

[0016] In further embodiments, the present disclosure provides compositions comprising delivery vehicles, such as liposomes, loaded (i.e., encapsulated) and / or otherwise bound to gamma polyglutamated pemetrexed, as well as methods for making and using γPPMX-loaded / bound delivery vehicle compositions (DV-γPPMX) to deliver gamma 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 hyperproliferative diseases such as cancer, immune system disorders such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria. In some embodiments, the gamma polyglutamated pemetrexed in DV-γPPMX contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, more than 5, or more than 20 glutamyl groups (including the glutamyl groups in pemetrexed). The DV-γPPMX-loaded / bound delivery vehicle composition provides selective delivery of a more cytotoxic payload (e.g., polyglutamylated pemetrexed) compared to the cytotoxicity of pemetrexed (PMX) administered in its monoglutamate form, thereby improving the efficacy and safety of pemetrexed delivery to cancer cells.

[0017] In further embodiments, the present disclosure provides a composition (Lp-γPPMX) comprising liposomes encapsulating (loaded with) gamma polyglutamated pemetrexed. In some embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 20 glutamyl groups (including the glutamyl groups in pemetrexed). In some embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains two or more L-glutamyl groups. In other embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains D-glutamyl groups. In further embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains D-glutamyl groups and two or more L-glutamyl groups.

[0018] In one embodiment, the Lp-γPPMX composition comprises gamma-polyglutamylated PMX containing a chain of three glutamyl groups linked to the glutamyl group of 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 D-glutamyl groups. In further embodiments, the tetraglutamylated PMX contains D-glutamyl groups and two or more L-glutamyl groups.

[0019] In one embodiment, the Lp-γPPMX composition comprises gamma polyglutamylated PMX comprising a chain of four gamma glutamyl groups linked to the glutamyl group of pemetrexed (e.g., gamma pentaglutamylated pemetrexed). In some embodiments, the gamma pentaglutamylated PMX comprises two or more L-glutamyl groups. In other embodiments, the gamma pentaglutamylated PMX comprises a D-glutamyl group. In further embodiments, the gamma pentaglutamylated PMX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0020] In one embodiment, the Lp-γPPMX composition comprises gamma polyglutamylated PMX, which comprises a chain of five gamma glutamyl groups linked to the glutamyl group of pemetrexed (e.g., gamma hexaglutamylated pemetrexed). In some embodiments, the gamma hexaglutamylated PMX comprises two or more L-glutamyl groups. In other embodiments, the gamma hexaglutamylated PMX comprises a D-glutamyl group. In further embodiments, the gamma hexaglutamylated PMX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0021] In some embodiments, the Lp-γPPMX composition is cationic. In some embodiments, the Lp-γPPMX 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-γPPMX 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-γPPMX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of gamma polyglutamylated PMX. In some embodiments, during the process of making Lp-γPPMX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the gamma polyglutamylated PMX starting material is encapsulated in cationic Lp-γPPMX. In further embodiments, the gamma polyglutamylated pemetrexed encapsulated by the liposomes is present in a HEPES buffer within the liposomes.

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

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

[0024] In some embodiments, the liposomal gamma polyglutamated pemetrexed composition is non-targeted (NTLp-γPPMX). That is, the NTLp-γPPMX composition does not have specific affinity for an epitope expressed on the surface of a target cell of interest (e.g., an epitope on a surface antigen). In further embodiments, the non-targeted liposomal gamma polyglutamated pemetrexed composition is pegylated (NTPLp-γPPMX).

[0025] In other embodiments, the liposomal gamma polyglutamated pemetrexed compositions are targeted (TLp-γPPMX). That is, the TLp-γPPMX compositions include a targeting moiety that has specific affinity for an epitope (surface antigen) on a target cell of interest. In some embodiments, the targeting moiety of TLp-γPPMX or TPLp-γPPMX is not covalently attached to the liposome. In other embodiments, the targeting moiety of TLp-γPPMX or TPLp-γPPMX is attached to one or both of the PEG and the exterior surface of the liposome. The targeted liposomal gamma polyglutamated pemetrexed compositions (TLp-γPPMX and TPLp-γPPMX) provide further improvements over the efficacy and safety profile of pemetrexed by specifically delivering gamma polyglutamated (e.g., gamma pentaglutamated and / or gamma hexaglutamated) pemetrexed to target cells, such as cancer cells. In some embodiments, the targeted liposomal gamma polyglutamated pemetrexed composition is PEGylated (TPLp-γPPMX). In some embodiments, the targeting moiety of TLp-γPPMX or TPLp-γPPMX is bound to one or both of the PEG and the outer surface of the liposome. In some embodiments, the targeting moiety of TLp-γPPMX or TPLp-γPPMX is bound to the liposome via a covalent bond. The function of the targeting moiety of TLp-γPPMX and / or TPLp-γPPMX composition includes, but is not limited to, targeting the liposome to a target cell of interest in vivo or in vitro; interacting with a surface antigen to which the targeting moiety has specific affinity; and delivering the liposomal payload (γPPMX) to the cell. Suitable targeting moieties are known in the art and include, but are not limited to, antibodies, antigen-binding antibody fragments, scaffold proteins, polypeptides, and peptides. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is a polypeptide comprising at least 3, 5, 10, 15, 20, 30, 40, 50, or 100 amino acid residues.

[0026] In some embodiments, the targeting moiety of TLp-γPPMX or TPLp-γPPMX is an antibody or antigen-binding antibody fragment. In further embodiments, the targeting moiety comprises one or more of an antibody, a humanized antibody, an antigen-binding fragment of an antibody, a single-chain antibody, a single-domain antibody, a bispecific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody. In some embodiments, the targeting moiety of TLp-γPPMX or TPLp-γPPMX has specific affinity for an epitope that is selectively expressed on target cells, such as tumor cells, compared to normal or non-tumor cells. In some embodiments, the targeting moiety has specific affinity for an epitope on a tumor cell surface antigen that is present on tumor cells but absent or inaccessible on non-tumor cells. In some embodiments, the targeting moiety has a specific affinity of 0.5×10 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 TLp-γPPMX or TPLp-γPPMX 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 gamma (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 Lp-γPPMX composition comprises one or more of an immunostimulant, a detectable marker, and a maleimide disposed on at least one of the PEG or outer surface of the liposome. In some embodiments, the liposomal γPPMX composition (e.g., Lp-γPPMX, PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX, or TPLp-γPPMX) is cationic. In other embodiments, the liposomal γPPMX composition (e.g., Lp-γPPMX, PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX, or TPLp-γPPMX) is anionic or neutral. In further embodiments, the liposomes of the liposomal γPPMX composition (e.g., Lp-γPPMX, PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX, or TPLp-γPPMX) have diameters ranging from 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, 50 nm to 150 nm, or any range therebetween. In some embodiments, the liposomes of the liposomal γPPMX composition have diameters ranging from 30 nm to 175 nm or 50 nm to 150 nm, or any range therebetween. In further embodiments, the liposomes of the liposomal γPPMX composition have diameters ranging from 80 nm to 120 nm, or any range therebetween. In some embodiments, the liposomal γPPMX composition is PEGylated (e.g., PLp-γPPMX, NTPLp-γPPMX, or TPLp-γPPMX). In some embodiments, the liposomal γPPMX composition comprises a targeting moiety (e.g., TLp-γPPMX or TPLp-γPPMX). In further embodiments, the liposomal γPPMX composition is pegylated and targeted (e.g., TPLp-γPPMX). In some embodiments, the liposomal γPPMX composition comprises gamma-polyglutamated pemetrexed containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomal γPPMX composition comprises gamma-tetraglutamated pemetrexed.In some embodiments, the liposomal γPPMX composition comprises gamma-pentaglutamated pemetrexed. In other embodiments, the liposomal γPPMX composition comprises gamma-hexaglutamated pemetrexed.

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

[0030] In some embodiments, the liposome composition is comprised of gamma-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 gamma-tetraglutamated PMX. In some embodiments, the Lp-γPPMX composition comprises gamma-tetraglutamated pemetrexed and 1% to 98.5% w / w of gamma-tetraglutamated PMX. In some embodiments, the liposomes comprise gamma-tetraglutamated pemetrexed, and during the process of making Lp-γPPMX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of the gamma-tetraglutamated pemetrexed starting material is encapsulated (encapsulated) in Lp-γPPMX.

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

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

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

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

[0035] In some embodiments, the pharmaceutical composition comprises liposomal γPPMX (e.g., Lp-γPPMX, PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX, and TPLp-γPPMX). In some embodiments, the liposomal γPPMX composition is pegylated (e.g., NTPLp-γPPMX and TPLp-γPPMX). In some embodiments, the liposomal γPPMX 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-γPPMX or TPLp-γPPMX). In further embodiments, the pharmaceutical composition comprises pegylated liposomal γPPMX 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-γPPMX). In some embodiments, the pharmaceutical composition comprises cationic liposomal γPPMX. In other embodiments, the pharmaceutical composition comprises anionic or neutral liposomal γPPMX. In a further embodiment, the pharmaceutical composition comprises liposomal γPPMX having a diameter in the range of 20 nm to 500 nm, or in the range of 20 nm to 500 nm, or any range therebetween. In a further embodiment, the liposomal γPPMX composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

[0036] In further embodiments, the disclosure provides methods of killing cells, the methods comprising contacting the cells with a composition comprising a gamma polyglutamylated pemetrexed (γPPMX) composition (e.g., γPPMX disclosed herein). In some embodiments, the contacted cells are mammalian cells. In further embodiments, the contacted cells are human cells. In some embodiments, the contacted cells are hyperproliferative cells. In further embodiments, the hyperproliferative cells are cancer cells. In further embodiments, the cancer cells contacted 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 cancer cells are primary cells or cells from a cell line obtained / derived from lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from breast cancer (e.g., HER2+ or triple-negative breast cancer). In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from colorectal cancer. In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from ovarian cancer. In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from endometrial cancer. In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from pancreatic cancer. In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from liver cancer. In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from head and neck cancer. In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from osteosarcoma. In some embodiments, the method is performed in vivo.In other embodiments, the method is performed in vitro. In some embodiments, γPPMX comprises 4, 5, 6, 2-10, 4-6, or more than 5 gamma-glutamyl groups. In some embodiments, γPPMX comprises a gamma-glutamyl group in the D-form. In some embodiments, γPPMX comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma-glutamyl groups in the D-form. In some embodiments, γPPMX comprises a gamma-glutamyl group in the L-form. In some embodiments, γPPMX comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma-glutamyl groups in the L-form. In some embodiments, γPPMX comprises gamma-glutamyl groups in both the L- and D-forms. In some embodiments, γPPMX comprises 2, 3, 4, 5, or more than 5 gamma-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 gamma-glutamyl groups in the D-form. In some embodiments, the γPPMX composition comprises gamma-tetraglutamated pemetrexed. In some embodiments, the γPPMX composition comprises gamma-pentaglutamated pemetrexed. In other embodiments, the γPPMX composition comprises gamma-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 gamma polyglutamylated pemetrexed (e.g., Lp-γPPMX, such as PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX, or TPLp-γPPMX). In some embodiments, the contacted cell is a mammalian cell. In still further embodiments, the contacted cell is a human cell. In some embodiments, the contacted cell is a hyperproliferative cell. In further embodiments, the contacted hyperproliferative cell is a cancer cell. In further embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from a cancer selected from the group consisting of 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 cells are primary cells or cells from a cell line obtained / derived from lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from breast cancer (e.g., HER2+ or triple-negative breast cancer). In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from colorectal cancer. In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from ovarian cancer. In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from endometrial cancer. In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from pancreatic cancer. In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from liver cancer. In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from head and neck cancer. In some embodiments, the cancer cells are primary cells or cells from a cell line obtained / derived from osteosarcoma.In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the liposome comprises γPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome comprises gamma tetraglutamated pemetrexed. In some embodiments, the liposome comprises gamma pentaglutamated pemetrexed. In other embodiments, the liposome comprises gamma hexaglutamated pemetrexed.

[0038] In some embodiments, the liposomes comprise γPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the liposomes comprise γPPMX containing γ-glutamyl groups in the D-form. In some embodiments, the liposomes comprise γPPMX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form. In some embodiments, the liposomes comprise γPPMX containing γ-glutamyl groups in the L-form. In some embodiments, the liposomes comprise γPPMX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposomes comprise γPPMX containing γ-glutamyl groups in both the L- and D-forms. In some embodiments, the liposomes comprise γPPMX containing 2, 3, 4, 5, or more than 5 gamma glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 gamma glutamyl groups in the D-form. In some embodiments, the liposomes comprise gamma pentaglutamated pemetrexed. In other embodiments, the liposomes comprise gamma hexaglutamated pemetrexed.

[0039] In further embodiments, the present disclosure provides methods of treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a delivery vehicle (e.g., an immunoconjugate or liposome) comprising gamma polyglutamated pemetrexed. 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-γPPMX, such as PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX, or TPLp-γPPMX). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is not pegylated. In further embodiments, the administered delivery vehicle comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a cancer cell.In further embodiments, the delivery vehicle comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, 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 (GC C), 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, EGFRv III, 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, C D40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, Crypto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrins (e.g., integrin α). v β3, α v β5 or α vβ6), C242 antigen, 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(s) determined to be derived from or expressed on a particular target cancer (tumor), such as a neoantigen. In some embodiments, the targeting moiety has specific affinity for a cell surface antigen(s) determined to be derived from or expressed on a particular target tumor, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the administered delivery vehicle comprises γPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the administered delivery vehicle comprises γPPMX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form. In some embodiments, the administered delivery vehicle comprises γPPMX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the administered delivery vehicle comprises γPPMX containing 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D-form. In some embodiments, the administered delivery vehicle comprises gamma-tetraglutamated pemetrexed. In some embodiments, the administered delivery vehicle comprises gamma-pentaglutamated pemetrexed. In other embodiments, the administered delivery vehicle comprises gamma hexaglutamated pemetrexed. In some embodiments, the administered delivery vehicle comprises L-gamma polyglutamated pemetrexed. In some embodiments, the administered delivery vehicle comprises D-gamma polyglutamated pemetrexed. In further embodiments, the administered delivery vehicle comprises L- and D-gamma polyglutamated pemetrexed.In some embodiments, the cancer is selected from the group consisting of non-hematologic tumors, including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma, brain cancer, central nervous system cancer, and melanoma; and hematologic tumors, such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. In some embodiments, the cancer is lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer is breast cancer (e.g., HER2++ or triple-negative breast cancer). In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is osteosarcoma.

[0040] In further embodiments, the present disclosure provides methods of treating cancer, the methods comprising administering an effective amount of liposomes comprising gamma polyglutamylated pemetrexed (e.g., Lp-γPPMX, such as PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX, or TPLp-γPPMX) 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 having specific affinity for an epitope of a cell surface antigen selected from the group consisting of GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α, folate receptor-β, or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (G CC), 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, EGFR vIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD 2, 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, CD105, CD133, CD138, Crypto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrins (e.g., integrin α). v β3, α v β5 or α vβ6), C242 antigen, 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 liposome contains a targeting moiety with specific affinity for an epitope of a cell surface antigen(s) determined to be derived from or expressed on a particular tumor of interest, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or antigen-binding antibody fragment. In some embodiments, the liposomes comprise γPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise γPPMX containing γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPPMX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPPMX containing γ-glutamyl groups in the L-form and D-form. In some embodiments, the liposomes of the administered liposome composition comprise γPPMX containing 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D-form. In some embodiments, the administered liposome composition comprises tetraglutamylated γPPMX. In some embodiments, the liposome composition administered comprises pentaglutamylated γPPMX. In some embodiments, the liposome composition administered comprises hexaglutamylated γPPMX.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.

[0041] In a further embodiment, the present disclosure provides a method of treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a liposome composition comprising gamma polyglutamylated pemetrexed and a liposome comprising a targeting moiety having specific affinity for an epitope of an antigen on the surface of the cancer.In some embodiments, the liposome comprises a targeting moiety that has specific affinity for an epitope of a cell surface antigen selected from the group consisting of GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α, folate receptor-β, or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T 4 (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, SM O, 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, CD105, 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, integrins (e.g., integrin αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the liposomes contain a targeting moiety that has specific affinity for an epitope of a cell surface antigen(s) determined to be derived from or expressed on a particular target cancer (tumor), such as a neoantigen. In some embodiments, the targeting moiety is an antibody or antigen-binding antibody fragment. In some embodiments, the liposomes contain γPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the liposomes contain γPPMX containing γ-glutamyl groups in the L-form. In some embodiments, the liposomes contain γPPMX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposomes contain γPPMX containing γ-glutamyl groups in the D-form. In some embodiments, the liposomes contain γPPMX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form. In some embodiments, the liposomes comprise gamma-tetraglutamated pemetrexed. In some embodiments, the liposomes comprise gamma-pentaglutamated pemetrexed. In other embodiments, the liposomes comprise gamma-hexaglutamated pemetrexed.

[0042] In some embodiments, the administered liposome composition comprises PEGylated liposomes (e.g., TPLp-γPPMX). In some embodiments, the administered liposome composition comprises non-PEGylated liposomes. In some embodiments, the administered liposomes of the liposome composition comprise γPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 gamma-glutamyl groups. In some embodiments, the administered liposomes of the liposome composition comprise γPPMX containing γ-glutamyl groups in the D-form. In some embodiments, the administered liposomes of the liposome composition comprise γPPMX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma-glutamyl groups in the D-form. In some embodiments, the administered liposomes of the liposome composition comprise γPPMX containing γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPPMX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPPMX containing γ-glutamyl groups in the L-form and the D-form. In some embodiments, the liposomes of the administered liposome composition comprise γPPMX containing 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D-form. In some embodiments, the liposomes of the administered liposome composition comprise gamma-tetraglutamated pemetrexed. In some embodiments, the administered liposome composition comprises gamma-pentaglutamated pemetrexed. In some embodiments, the administered liposomes of the administered liposome composition comprise gamma-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, leukemia, and lymphoma.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.

[0043] In further embodiments, the present disclosure provides methods for treating cancer, the method comprising administering an effective amount of a liposome composition to a subject having or at risk of having a cancer that expresses a folate receptor on its cell surface, the liposome composition comprising (a) gamma polyglutamated pemetrexed (γPPMX) 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), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α) and folate receptor beta (FR-β). In some embodiments, the administered liposome composition comprises PEGylated liposomes (e.g., TPLp-γPPMX). In some embodiments, the administered liposome composition comprises non-PEGylated liposomes. In some embodiments, the administered liposomes of the liposome composition comprise γPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 γ-glutamyl groups. In some embodiments, the administered liposomes of the liposome composition comprise γPPMX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form. In some embodiments, the administered liposomes of the liposome composition comprise γPPMX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPPMX containing 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D-form. In some embodiments, the liposomes of the administered liposome composition comprise gamma-pentaglutamated pemetrexed. In other embodiments, the liposomes of the administered liposome composition comprise gamma-hexaglutamated pemetrexed.In some embodiments, the liposome composition is administered to treat 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 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.

[0044] 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 gamma polyglutamated pemetrexed (Lp-γPPMX) to a subject undergoing or who has undergone cancer therapy. In some embodiments, the administered liposomal composition is PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX, or TPLp-γPPMX. In some embodiments, the liposomes of the administered liposomal composition comprise PEGylated liposomes (e.g., PLp-γPPMX, NTPLp-γPPMX, or TPLp-γPPMX). In some embodiments, the administered liposomal composition comprises targeted liposomes (e.g., TLp-γPPMX or TPLp-γPPMX). In some embodiments, the administered liposomal composition comprises PEGylated liposomes comprising a targeting moiety (e.g., TPLp-γPPMX). In some embodiments, the liposomes of the administered liposome composition comprise gamma polyglutamated pemetrexed containing 4, 5, 6, 2-10, 4-6, or more than 5 gamma glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise γPPMX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma glutamyl groups in the D-form. In some embodiments, the liposomes of the administered liposome composition comprise γPPMX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPPMX containing 2, 3, 4, 5, or more than 5 gamma glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 gamma glutamyl groups in the D-form. In some embodiments, the liposomes of the administered liposome composition comprise gamma-tetraglutamated pemetrexed. In some embodiments, the liposomes of the administered liposome composition comprise gamma-pentaglutamated pemetrexed. In other embodiments, the liposomes of the administered liposome composition comprise gamma-hexaglutamated pemetrexed.

[0045] In further embodiments, the present disclosure provides methods for treating an immune system disorder, the method comprising administering an effective amount of a liposome composition comprising liposomes comprising gamma polyglutamylated pemetrexed (e.g., Lp-γPPMX, PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX, or TPLp-γPPMX) to a subject having or at risk of having an immune system disorder. In some embodiments, the liposome composition is administered to treat an autoimmune disease. In a further embodiment, the liposome composition is administered to treat rheumatoid arthritis. In another embodiment, the liposome composition is administered to treat inflammation. In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., PLp-γPPMX, NTPLp-γPPMX, or TPLp-γPPMX). In some embodiments, the administered liposome composition comprises a targeted liposome (e.g., TLp-γPPMX or TPLp-γPPMX) containing a targeting moiety with specific affinity for a surface antigen on a target cell (e.g., an immune cell) of interest. In further embodiments, the administered liposome composition comprises a liposome (e.g., TPLp-γPPMX) that is pegylated and contains a targeting moiety. In some embodiments, the administered liposome comprises gamma-pentaglutamated pemetrexed containing 4, 5, 6, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the administered liposome comprises γPPMX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form. In some embodiments, the liposomes of the administered liposome composition comprise γPPMX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the liposomes of the administered liposome composition comprise γPPMX containing 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups in the D-form. In some embodiments, the liposomes of the administered liposome composition comprise gamma-tetraglutamated pemetrexed.In some embodiments, the liposomes of the administered liposome composition comprise gamma-pentaglutamated pemetrexed. In other embodiments, the liposomes of the administered liposome composition comprise gamma-hexaglutamated pemetrexed.

[0046] The present disclosure also provides a method for delivering gamma polyglutamated pemetrexed to tumor and / or cancer cells, comprising administering to a subject having a tumor a composition comprising gamma polyglutamated pemetrexed (L-γPPMX) 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-γPPMX). In some embodiments, the administered composition comprises gamma 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 gamma tetraglutamated pemetrexed. In some embodiments, the composition administered comprises gamma-pentaglutamated pemetrexed, hi other embodiments, the composition administered comprises gamma-hexaglutamated pemetrexed.

[0047] In further embodiments, the present disclosure provides methods for making liposomal compositions containing liposomal gamma-polyglutamated pemetrexed (γPPMX) compositions, the methods comprising: forming a mixture containing liposome components and γ-polyglutamated pemetrexed in a solution; homogenizing the mixture in the solution to form liposomes; and treating the mixture to form liposomes containing polyglutamated pemetrexed. In some embodiments, the gamma-polyglutamated pemetrexed contains 4, 5, 6, 2 to 10, 4 to 6, or more than 5 γ-glutamyl groups. In some embodiments, the γPPMX composition contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the D-form. In some embodiments, the γPPMX composition contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups in the L-form. In some embodiments, the γPPMX composition comprises 2, 3, 4, 5, or more than 5 gamma glutamyl groups in the L-form and 1, 2, 3, 4, 5, or more than 5 gamma glutamyl groups in the D-form. In some embodiments, the γPPMX composition comprises gamma pentaglutamated pemetrexed. In some embodiments, the γPPMX composition comprises gamma tetraglutamated pemetrexed. In other embodiments, the γPPMX composition comprises gamma hexaglutamated pemetrexed.

[0048] In one embodiment, the present disclosure provides a kit comprising a gamma polyglutamated pemetrexed composition described herein and / or a γPPMX delivery vehicle, such as a liposome, comprising γPPMX and a γPPMX immunoconjugate (e.g., an ADC). [Brief explanation of the drawings]

[0049] [Figure 1-1]The chemical formulas of pemetrexed (Figure 1A), representative gamma-pemetrexed polyglutamates, pemetrexed diglutamate (Figure 1B), pemetrexed triglutamate (Figures 1C and 1D), pemetrexed tetraglutamate (Figures 1E and 1F), pemetrexed pentaglutamate (Figures 1G and 1H), pemetrexed hexaglutamate (Figures 1I and 1J), pemetrexed heptaglutamate (Figures 1K and 1L), pemetrexed octaglutamate (Figures 1M and 1N), representative gamma-pemetrexed polyglutamates (Figure 1O), and representative pemetrexed analogs (Figures 1P and 1Q) are shown. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 2] An example of the dose-response relationship, expressed as the percentage of surviving cells after 48 hours of treatment, is shown for free pemetrexed L-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6), pemetrexed, and the folate receptor alpha-targeted antibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6-FR1Ab) in NCI H2342 non-small cell lung cancer (NSCLC), adenocarcinoma subtype. [Figure 3] FIG. 1 shows an example of the dose-response relationship of free pemetrexed L-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha-targeting antibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6-FR1Ab) in HT-29 (colon cancer) at 48 hours. [Figure 4]Figure 1 shows the effects of free pemetrexed L-gamma hexaglutamate (Hexa-gG6) and liposomal pemetrexed L-gamma hexaglutamate (Liposomal Hexa-gG6) on the growth of colon cancer SW260 cells after 48 hours of exposure to 256 nM of the corresponding drug. Both non-targeted and targeted liposomal pemetrexed hexa-gG6 can enter cells more efficiently than free pemetrexed hexa-gG6 to inhibit the proliferation of colon cancer SW260 cells. [Figure 5] Figure 1 shows the relative efficacy of liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6) and its enantiomer liposomal pemetrexed gamma D-hexaglutamate (liposomal gDG6) 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 6] 1 shows the therapeutic effect on HCC1806 triple-negative breast cancer cells after 48 hours of exposure to liposomal pemetrexed gamma-L hexaglutamate (Lps Hexa gG6), liposomal pemetrexed gamma-D hexaglutamate (Lps Hexa gDG6), and pemetrexed. [Figure 7] 1 shows the therapeutic effect on OAW28 ovarian cancer cells after exposure to liposomal pemetrexed gamma-L hexaglutamate (Lps Hexa gG6), liposomal pemetrexed gamma-D hexaglutamate (Lps Hexa gDG6) compared to pemetrexed over a 48 hour period. [Figure 8] 1 shows the therapeutic effect on H292 non-small cell lung cancer cells after 48 hours of exposure to liposomal pemetrexed gamma-L hexaglutamate (Lps Hexa gG6), liposomal pemetrexed gamma-D hexaglutamate (Lps Hexa gDG6), and pemetrexed. [Figure 9]This figure shows the therapeutic effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed at various dose levels ranging from 16 to 128 nM on H292 non-small cell lung cancer cells after 48 hours of exposure. Within each tested dose range, the liposomal pemetrexed gG6 formulation exhibited superior inhibition of H292 non-small cell lung cancer cells compared to pemetrexed. [Figure 10] This figure shows the therapeutic efficacy of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed at various dose levels ranging from 16 to 128 nM against HCC1806 triple-negative breast cancer cells after 48 hours of exposure. Within each tested dose range, the liposomal pemetrexed gG6 formulation was superior to pemetrexed in inhibiting HCC1806 triple-negative breast cancer cells. [Figure 11] Figure 1 shows the therapeutic effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal gamma-D hexaglutamate (liposomal gDG6), 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 gG6 liposomal formulation, but at doses of 32 nM and 64 nM, the liposomal formulation has superior therapeutic effects to pemetrexed. At 16 nM, the therapeutic effect of liposomal pemetrexed gG6 is similar to that of pemetrexed. [Figure 12] Figure 1 shows the toxicity of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed at 64 nM, 128 nM, and 264 nM toward differentiated human neutrophils. The figure shows that liposomal pemetrexed gG6 is significantly less toxic than pemetrexed toward differentiated human neutrophils. [Figure 13]Shown are the effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal gamma-D hexaglutamate (liposomal gDG6), and the corresponding pemetrexed agents at various dose levels ranging from 16 to 128 nM on neutrophils (differentiated from CD34+ cells) after 48 hours of exposure. [Figure 14] This figure shows the effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed at 16 nM, 32 nM, 64 nM, and 128 nM of the corresponding drug on AML12 liver cells after 48 hours of exposure. Remarkably, none of the liposomal drug tested at the dose levels appeared to be toxic to AML12 liver cells after treatment with liposomal pemetrexed gG6. In contrast, pemetrexed treatment resulted in a reduction in AML12 liver cell counts by approximately 40% at all doses examined. [Figure 15]

[0023] Figure 1 shows the effects of liposomal pemetrexed gamma-L hexaglutamate (liposomal gG6), liposomal pemetrexed gamma-D hexaglutamate (liposomal gDG6), and pemetrexed at 16 nM, 32 nM, 64 nM, and 128 nM of the corresponding drug on CCD841 colonic epithelial cells after 48 hours of exposure. At all concentrations tested, pemetrexed results in a reduction of CCD841 colonic epithelial cell counts of about 50% or more, compared to a reduction of about 20% or less after treatment with each of the liposomal compositions tested. [Figure 16] The structures of the polyglutamate antifolate, cisplatin (CDDP), and two possible gG6-cisplatin complexes are shown. The pH-dependent formation of inter- and / or intra-chain coordination between the carboxyl groups of the polyglutamate antifolate and cisplatin may lead to its degradation into separate molecules of gG6 and cisplatin upon encountering the acidic pH of the lysosome (pH 3-5) and in the presence of intracellular chloride ions. [Figure 17]Hematological parameters: Effect of liposomal aG6 treatment in mice at 40 mg / kg and 80 mg / kg once weekly for 4 weeks on white blood cell (WBC) count, neutrophil count, and platelet count. No significant decrease in mean neutrophil, mean white blood cell, or mean platelet count was observed. [Figure 18] This figure shows the effect of liposomal aG6 treatment in mice at 40 mg / kg and 80 mg / kg weekly doses for 4 weeks on hemoglobin and reticulocyte count indices. There is a minimal decrease in mean hemoglobin concentration at higher dose levels. Concomitantly, there is a slight increase in mean reticulocyte count indices. [Figure 19] The effects of liposomal aG6 treatment in mice at 40 mg / kg and 80 mg / kg once weekly for 4 weeks on liver markers, including serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT), in addition to serum albumin, are shown. No apparent increase in liver aminotransferase mean AST or ALT levels was observed, and furthermore, no changes in mean albumin levels were observed. [Figure 20] Relative tumor volumes in immunocompromised female Nu / J mice (6-8 weeks old) inoculated with NCI-H292 (non-small cell lung cancer) cells and treated intravenously with control, pemetrexed, or liposomal gG6 at 167 mg / kg once every three weeks are shown. These preliminary data show that liposomal gG6 results in reduced tumor control compared to pemetrexed. [Figure 21]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 (liposomal aG3), liposomal pemetrexed alpha-L-octaglutamate (liposomal aG5), liposomal pemetrexed alpha-L-hexaglutamate (liposomal aG7), and the combination of liposomal pemetrexed alpha-L-hexaglutamate (liposomal gG6) and alpha-L-dodecaglutamate (liposomal gG6 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 liposomal carrier and empty liposome control. DETAILED DESCRIPTION OF THE INVENTION

[0050] Generally, the present disclosure relates to gamma polyglutamated pemetrexed compositions. The compositions offer an advancement over existing treatments for hyperproliferative diseases, such as cancer. Methods for producing, delivering, and using the gamma polyglutamated pemetrexed compositions are also provided. The gamma polyglutamated compositions have uses including, but not limited to, the treatment or prevention of hyperproliferative diseases, such as cancer, immune system disorders, such as inflammation and rheumatoid arthritis, and infectious diseases, such as HIV and malaria.

[0051] 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.

[0052] 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, the transitional phrase "consisting of" is considered more exclusive, and "consisting essentially of" is considered intermediate).

[0053] 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.

[0054] 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.

[0055] 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 subheading may necessarily be used together in some embodiments.

[0056] 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. + , N.H. 4+ , and / or Ca 2+ In certain embodiments, the salt is a pharmaceutically acceptable salt. In further particular embodiments, the PMX salt is Na + Pemetrexed may also be referred to by its 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, as ALIMTA®, LY231514, MTA. Pemetrexed contains one L-gamma glutamyl group and is therefore considered monoglutamated for purposes of this disclosure.

[0057] The terms "polyglutamylated pemetrexed," "polyglutamylated PMX," "PMX-PG," "PPMX," and variations thereof are used interchangeably herein to refer to pemetrexed compositions containing at least one glutamyl group in addition to the glutamyl groups of pemetrexed (i.e., PMX-PGn, n≧1). References to the number of glutamyl groups in γPPMX (PMX-PG) herein include the glutamyl groups of pemetrexed. For example, a PMX-PG composition containing five glutamyl residues in addition to the glutamyl groups of PMX is referred to herein as hexaglutamylated pemetrexed or pemetrexed hexaglutamate. The polyglutamate chain contains an N-terminal glutamyl group and one or more C-terminal glutamyl groups. The N-terminal glutamyl group of the polyglutamate chain is not bound to another glutamyl group through its amino group, but is bound to one or more glutamyl groups through its carboxylic acid group. In some embodiments, the N-terminal glutamyl group of polyglutamylated pemetrexed is the glutamyl group of pemetrexed. The C-terminal glutamyl group(s) of the polyglutamate chain is / are bound to another glutamyl group through their amino group, but is / are not bound to another glutamyl group through their carboxylic acid group.

[0058] The terms "gamma glutamyl group," "gamma glutamyl group," and "gamma bond," when referring to a glutamyl group bond, refer to a glutamyl group containing a gamma carboxyl group bond. The gamma bond can be a bond between a glutamyl group and a glutamyl group of pemetrexed, or between a glutamyl group and a second glutamyl group not present in pemetrexed (e.g., a glutamyl group in a polyglutamate chain bound to pemetrexed). In some embodiments, the gamma bond is an amide bond between the gamma carboxyl group of one glutamyl group and a second glutamyl group. In some embodiments, the gamma bond refers to an amide bond of a glutamyl group in pemetrexed. In some embodiments, the gamma bond is an amide bond between the gamma carboxyl group of one glutamyl group and a second glutamyl group. Reference to gamma bonds includes gamma bonds of glutamyl groups in pemetrexed unless otherwise specified or unless the context clearly indicates otherwise. In some embodiments, the gamma glutamyl group is in the L-form. In some embodiments, the gamma glutamyl group is in the D-form. As discussed herein, during pemetrexed therapy, pemetrexed enters cells and is polyglutamated by the enzyme folylpoly-gamma-glutamate synthase (FPGS), which tandemly adds L-glutamyl groups to the gamma carboxyl groups of glutamic acid within the pemetrexed L-glutamyl group of pemetrexed. As a result, D-gamma polyglutamylated pemetrexed compositions are not formed intracellularly during pemetrexed therapy.

[0059] The terms "gamma polyglutamated pemetrexed," "gamma polyglutamated pemetrexed," "γPPMX," "gamma polyglutamated pemetrexed," "polyglutamated PMX," "γPMX-PG," and repeats thereof are used interchangeably herein to refer to pemetrexed compositions containing at least one gamma glutamyl group with a gamma carboxyl group bond in addition to the gamma glutamyl group of pemetrexed (e.g., PMX-PGn, where n≧1 gamma glutamyl group). References herein to the number of glutamyl groups in γPPMX (γPMX-PG) count the gamma glutamyl groups of pemetrexed. For example, a γPMX-PG composition containing five gamma glutamyl groups in addition to the glutamyl groups of PMX may be referred to herein as gamma hexaglutamated pemetrexed or gamma pemetrexed hexaglutamate.

[0060] The terms "alpha glutamyl group," "α glutamyl group," and "alpha linkage," when referring to a glutamyl group linkage, refer to a glutamyl group that includes an alpha carboxyl group linkage.

[0061] As used herein, the term "isolated" refers to a composition in a form not found in nature. Isolated gamma polyglutamated compositions include those that have been purified to the extent that they are no longer in the form found in nature. In some embodiments, isolated gamma polyglutamated pemetrexed is substantially pure. Isolated compositions are free or substantially free of naturally incorporated substances, such as proteins and other cellular components, such as nucleic acids, that may potentially be found in nature or in the environment in which they are produced (e.g., cell culture). Gamma polyglutamated compositions can be formulated with a diluent or adjuvant and further isolated for practical purposes—for example, when used as a diagnostic or therapeutic agent, gamma polyglutamated compositions are typically mixed with a pharmaceutically acceptable carrier or diluent. In some embodiments, isolated gamma polyglutamated compositions (e.g., gamma polyglutamates and delivery vehicles such as liposomes comprising gamma polyglutamates) contain less than 1% or less than 0.1% undesired DNA or protein content. In some embodiments, gamma polyglutamate compositions (e.g., gamma polyglutamates and delivery vehicles such as liposomes comprising gamma polyglutamates) are "isolated."

[0062] 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 entities. Targeting moieties include natural molecules, or recombinant or synthetic molecules. In some embodiments, the targeting moiety is an antibody, antigen-binding antibody fragment, bispecific antibody, or other antibody-based molecule or compound. In some embodiments, the targeting moiety is an aptamer, avimer, receptor-binding ligand, nucleic acid, biotin-avidin binding pair, peptide, protein, carbohydrate, lipid, vitamin, toxin, microbial component, hormone, receptor ligand, or any derivative thereof. Other targeting moieties are known in the art and are encompassed by the present disclosure.

[0063] The terms "specific affinity," "specifically binds," and "enhanced affinity" mean that a targeting moiety, such as an antibody or antigen-binding antibody fragment, reacts with or binds to an epitope, protein, or target molecule more frequently, more rapidly, for a longer period of time, with greater affinity, or some combination thereof, than to another substance, including a protein unrelated to the antigen containing the target epitope. Due to sequence identity between homologous proteins in different species, a particular affinity, in some embodiments, includes binding substances that recognize epitopes on proteins and / or target molecules in two or more species. Similarly, due to homology within specific regions of the polypeptide sequences of different proteins, the terms "specific affinity" or "specific binding" can include binding substances that recognize epitopes present on two or more proteins and / or target molecules. It is understood that in certain embodiments, a targeting moiety that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific affinity" does not necessarily require (although it can include) exclusive binding, e.g., binding to an epitope on only one target. Thus, a targeting moiety may, in certain embodiments, specifically bind to an epitope present on more than one target. In certain embodiments, multiple targets may be bound by the same targeting moiety that specifically binds to an epitope present on multiple targets.

[0064] 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.

[0065] Expressions known in the art, such as "binding affinity to target", "binding to target", "enhanced affinity" and similar expressions, refer to the affinity constant, a property of targeting moiety that can be directly measured by determining, for example, the amount of 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 a 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).

[0066] The term "delivery vehicle" generally refers to any composition that acts to support, promote, or facilitate the entry of gamma 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 the group consisting of a macromolecule, a protein, a peptide, a monoclonal antibody, or a fatty acid lipid.

[0067] "Subject" means a human or vertebrate mammal, including, but not limited to, a dog, a cat, a horse, a goat, and a primate, such as a monkey. Thus, the present invention can also be used to treat a disease or condition in a non-human subject. For example, cancer is one of the leading causes of death in companion animals (e.g., cats and dogs). In some embodiments of the present invention, the subject is a human. In this disclosure, the terms "subject" and "patient" are used interchangeably and have the same meaning. Generally, it is preferred to use a maximum dose, i.e., the maximum safe dose according to sound medical judgment.

[0068] 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 particular condition being treated or prevented, the age and health of the subject being treated, the severity of the condition, the duration of treatment, the nature of concurrent or concomitant therapy (if any), the specific route of administration, and similar factors within the knowledge and professional opinion of a health practitioner. An "effective amount" can 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.

[0069] 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.

[0070] "Cancer," "tumor," or "malignant tumor" are used interchangeably and refer to any of a number of cell types or diseases characterized by uncontrolled, abnormal proliferation of cells, spread of infected cells locally or to other parts of the body via the bloodstream and lymphatic system (metastasis), and / or any of the characteristic structural and / or molecular features known to be associated with these cell types or diseases. As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. A "cancerous tumor," or "malignant cell," is understood to be a cell that has specific structural characteristics, lacks differentiation, and is capable of invasion and metastasis. Cancers that can be treated using the γPPMX compositions provided herein include, but are not limited to, non-hematological tumors, including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors, such as leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or cachexia. Other types of cancers and tumors that can be treated using γPPMX 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.

[0071] Terms such as "treating," "treatment," or "to treat" refer to both (a) therapeutic measures that cure, slow, lessen the symptoms, and / or halt the progression of a diagnosed condition or disorder, and (b) prophylactic or preventative measures that prevent and / or delay the onset of the targeted disease or condition. Thus, subjects in need of treatment include those already with the cancer, disorder, or disease, those at risk of developing the cancer or condition, and those in whom the infection or condition is to be prevented. The subject has been identified, using well-known medical and diagnostic techniques, as being "at risk of having" cancer, an infectious disease, an immune system disorder, a hyperproliferative disease, or another disease or disorder referred to herein. In certain embodiments, a subject has been successfully "treated" by the methods provided herein if, for example, the subject exhibits total, partial, or temporary remission or elimination of symptoms associated with the disease or condition (e.g., cancer, inflammation, and rheumatoid arthritis). In certain embodiments, the terms "treating" or "treatment" or "to 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 γPPMX compositions alone or in combination with additional therapeutic agents.

[0072] "Subject," "patient," and "animal" are used interchangeably and refer to mammals, such as human patients and non-human primates, as well as laboratory animals, such as rabbits, rats, mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as chickens, amphibians, and reptiles. As used herein, "mammal" refers to any member of the class Mammalia, including, but not limited to, humans and non-human primates, e.g., chimpanzees and other apes and monkey species; livestock animals, such as cows, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, such as rodents, e.g., mice, rats, guinea pigs, and other members of the class Mammalia. In certain embodiments, the subject is a human.

[0073] 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.

[0074] As used herein, the term "autoimmune disease" is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate or excessive response to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, inflammation and rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, leukoplakia, myxedema, pernicious anemia, and ulcerative colitis.

[0075] As used herein, the term "therapeutic agent" refers to a drug or derivative thereof or prodrug thereof that can interact with hyperproliferative cells, such as cancer cells or immune cells, thereby reducing the proliferative state of the cells and / or killing the cells. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, cytotoxic 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.

[0076] 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.

[0077] 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 gamma 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 the group consisting of fludarabine, cytarabine, gemcitabine, decitabine, and azacitidine or derivatives thereof. In certain embodiments, the antimetabolite is 5-fluorouracil.

[0078] 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.

[0079] The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than an active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, carriers, excipients, stabilizers, diluents, or preservatives. Pharmaceutically acceptable carriers include, for example, one or more compatible solid or liquid fillers, diluents, or encapsulating substances that are suitable for administration to humans or other subjects.

[0080] The present disclosure relates generally to gamma 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.

[0081] In some embodiments, the present disclosure provides: [1] A composition comprising gamma polyglutamated pemetrexed; [2] The composition according to item [1], wherein the gamma polyglutamylated pemetrexed contains 1 to 10 glutamyl groups having gamma carboxyl group bonds; [3] The composition according to item [1] or [2], wherein the gamma polyglutamated pemetrexed contains 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups with gamma carboxyl group bonds; [4] The composition according to any one of items [1] to [3], wherein the gamma polyglutamated pemetrexed is gamma tetraglutamated pemetrexed; [5] The composition according to any one of items [1] to [3], wherein the gamma polyglutamated pemetrexed is gamma pentaglutamated pemetrexed; [6] The composition according to any one of items [1] to [3], wherein the gamma polyglutamated pemetrexed is gamma hexaglutamated pemetrexed; [7] The composition according to any one of items [1] to [6], which is the following composition: (a) gamma polyglutamated pemetrexed contains two or more L-glutamyl groups with gamma carboxyl linkages; (b) each of the glutamyl groups of the gamma polyglutamated pemetrexed is in the L-configuration and has a gamma carboxyl group bond; (c) at least one glutamyl group of the gamma polyglutamated pemetrexed is in the D-form and has a gamma carboxyl group bond; (d) each glutamyl group of gamma-polyglutamated pemetrexed other than the glutamyl group of pemetrexed is D-type and has a gamma carboxyl group bond; or (e) Gamma polyglutamated pemetrexed contains two or more L-glutamyl groups and at least one D-glutamyl group with a gamma carboxyl group bond. [8] The composition according to item [4], (a) each glutamyl group is in the L-configuration and has a gamma carboxyl linkage, or (b) each glutamyl group other than the glutamyl group of pemetrexed is in the D-configuration and each glutamyl group has a gamma carboxyl linkage. [9] The composition according to item [5], wherein (a) each glutamyl group is in the L-configuration and has a gamma carboxyl group bond, or (b) each glutamyl group other than the glutamyl group of pemetrexed is in the D-configuration and each glutamyl group has a gamma carboxyl group bond;

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

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

[10] , wherein the gamma polyglutamylated aminopterin can be polyglutamylated by FGPS under physiological conditions, and / or the polyglutamylated PMX has a lower uptake rate (less than 30%) by hepatocytes than PMX;

[12] A liposomal composition containing gamma polyglutamylated pemetrexed (Lp-γPPMX) according to any one of items [1] to

[11] ;

[13] The Lp-γPPMX composition according to item

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

[14] The Lp-γPPMX composition according to item

[12] or

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

[15] The Lp-γPPMX composition according to item

[12] or

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

[28] The Lp-γPPMX composition according to item

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

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

[27] to

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

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

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

[33] , wherein the liposome is cationic;

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

[12] to

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

[40] The Lp-γPPMX 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-γPPMX composition according to item

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

[42] The Lp-γPPMX composition according to item

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

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

[39] to

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

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

[39] to

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

[45] The Lp-γPPMX 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 a similar substance at a concentration of 1 to 200 mM and a pH of 2 to 8;

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[50] or

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

[53] The Lp-γPPMX 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-γPPMX 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 an Lp-γPPMX composition that binds to surface antigens with an equilibrium dissociation constant (Kd) in the range of

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

[50] to

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

[56] The Lp-γPPMX composition according to any one of Items

[50] to

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

[57] The Lp-γPPMX 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-γPPMX composition according to any one of Items

[39] to

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

[59] The Lp-γPPMX 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-γPPMX composition according to item

[58] or

[59] , wherein the immunostimulant is fluorescein, fluorescein isothiocyanate (FITC), DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan, resolvin (e.g., D n-6DPA Or D n-3DPA an Lp-γPPMX composition, wherein the at least one selected from the group consisting of a toll-like receptor (TLR) modulator, such as resolvin D, resolvin E, or T-series resolvin (e.g., OXPAC, PGPC), and an eritoran lipid (e.g., E5564);

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

[58] to

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

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

[58] to

[61] , further comprising a hapten;

[63] The Lp-γPPMX composition according to item

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

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

[12] to

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

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

[64] ;

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

[49] ;

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

[12] to

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

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

[12] to

[67] .

[69] A pharmaceutical composition comprising the gamma 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 of such treatment or prevention, 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 of such treatment or prevention, the method comprising administering to the subject the liposomal gamma polyglutamated pemetrexed composition according to any one of

[12] to

[69] ;

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

[69] ;

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

[12] to

[69] ;

[76] The method according to item

[74] or

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

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

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

[78] A method for treating cancer, comprising administering an effective amount of the liposomal gamma polyglutamated pemetrexed composition according to any one of items

[12] to

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

[79] The method according to item

[77] or

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

[80] The method according to item

[77] or

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

[81] The method according to item

[77] or

[78] , wherein the cancer is 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-γPPMX 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 gamma polyglutamated pemetrexed composition according to any one of items

[12] 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 gamma polyglutamated pemetrexed composition according to any one of items [8] 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 gamma polyglutamated pemetrexed composition according to any one of items

[12] to

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

[90] A method for delivering gamma polyglutamated pemetrexed to a tumor expressing a folate receptor on its surface, comprising administering the Lp-γPPMX composition according to any one of items [1] to

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

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

[12] to

[69] , comprising the steps of: forming a mixture containing liposome components and an alpha polyglutamated antifolate 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

[12] 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 to form liposomes in the solution;

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

[50] to

[69] , comprising the steps of: forming a mixture in a solution containing liposome components and alpha-polyglutamated pemetrexed; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes that entrap and / or encapsulate 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-γ), 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 entrap and / or encapsulate 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-γ), folate receptor beta (FR-β), and folate receptor delta (FR-δ);

[96] The method according to item

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

[97] The method according to any of items

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

[98] The method according to any one of items

[95] to

[97] , wherein the processing step comprises one or more steps of changing 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 starting material of alpha polyglutamated pemetrexed is encapsulated or entrapped in liposomes.

[0082] II. Gamma Polyglutamated Pemetrexed (γPPMX) Generally, the present disclosure relates to gamma polyglutamylated pemetrexed (γPPMX) compositions. γPPMX compositions contain at least one glutamyl group with a gamma carboxyl linkage. These are structurally distinct from the L-gamma polyglutamylated form of pemetrexed (Lγ1PPMX), which is generated in cells by the enzyme folylpolygamma glutamate synthase (FPGS) during pemetrexed therapy.

[0083] In some embodiments, the γPPMX composition contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl group in pemetrexed). In some embodiments, each glutamyl group in γPPMX other than the glutamyl group in pemetrexed has a gamma linkage. In some embodiments, two or more glutamyl groups in γPPMX have a gamma linkage. In some embodiments, each glutamyl group in γPPMX is in the L-form. In some embodiments, each glutamyl group in γPPMX other than the glutamyl group in pemetrexed is in the D-form. In some embodiments, γPPMX contains two or more glutamyl groups in the L-form and one or more glutamyl groups in the D-form.

[0084] In some embodiments, the gamma-polyglutamated pemetrexed is diglutamated. That is, the gamma-polyglutamated pemetrexed contains one additional glutamyl group in addition to the glutamyl groups of pemetrexed (γPMX-PG1), and the additional glutamyl group is linked to the glutamyl group in pemetrexed via a gamma bond. In some embodiments, each glutamyl group in gamma-diglutamated pemetrexed is in the L-form. In other embodiments, the gamma-diglutamated PMX contains a glutamyl group in the D-form.

[0085] In some embodiments, gamma-polyglutamated pemetrexed is triglutamylated. That is, gamma-polyglutamated pemetrexed contains two additional glutamyl groups in addition to the glutamyl group of pemetrexed (γPMX-PG2). In some embodiments, each glutamyl group of gamma-triglutamated pemetrexed is in the L-form. In other embodiments, gamma-triglutamated PMX contains a glutamyl group in the D-form. In further embodiments, each glutamyl group of gamma-triglutamated pemetrexed, other than the glutamyl group of pemetrexed, is in the D-form. In still further embodiments, gamma-triglutamated PMX contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

[0086] In some embodiments, gamma-polyglutamated pemetrexed is tetraglutamylated and thus contains three additional glutamyl groups in addition to the glutamyl groups in pemetrexed (γPMX-PG3). In some embodiments, gamma-tetraglutamylated PMX contains two or more gamma-glutamyl groups in the L-form. In further embodiments, each gamma-glutamyl group in gamma-tetraglutamylated pemetrexed is in the L-form. In other embodiments, gamma-tetraglutamylated PMX contains a gamma-glutamyl group in the D-form. In some embodiments, gamma-tetraglutamylated PMX contains two gamma-glutamyl groups in the D-form. In some embodiments, each glutamyl group in gamma-tetraglutamylated pemetrexed other than the glutamyl group in pemetrexed is in the D-form. In further embodiments, tetraglutamylated PMX contains a gamma-glutamyl group in the D-form and two or more gamma-glutamyl groups in the L-form.

[0087] In some embodiments, gamma polyglutamated pemetrexed is pentaglutamated (γPMX-PG4), comprising a chain of four additional glutamyl groups attached to the glutamyl groups of pemetrexed. In some embodiments, gamma pentaglutamated PMX comprises two or more gamma glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma pentaglutamated pemetrexed is in the L-form. In other embodiments, gamma pentaglutamated PMX comprises a glutamyl group in the D-form. In some embodiments, gamma tetraglutamated PMX comprises two or three gamma glutamyl groups in the D-form. In further embodiments, each gamma glutamyl group of gamma pentaglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In further embodiments, pentaglutamated PMX comprises a gamma glutamyl group in the D-form and two or more gamma glutamyl groups in the L-form.

[0088] In some embodiments, gamma polyglutamated pemetrexed is hexaglutamated (γPMX-PG5), comprising a chain of five additional glutamyl groups attached to the glutamyl groups of pemetrexed. In some embodiments, gamma hexaglutamated PMX comprises two or more γ-glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma hexaglutamated pemetrexed is in the L-form. In other embodiments, gamma hexaglutamated PMX comprises a γ-glutamyl group in the D-form. In some embodiments, gamma tetraglutamated PMX comprises two, three, four, or five γ-glutamyl groups in the D-form. In further embodiments, each glutamyl group of gamma hexaglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In a further embodiment, the hexaglutamylated PMX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0089] In some embodiments, gamma polyglutamated pemetrexed is heptaglutamated (γPMX-PG6), thus comprising a chain of six additional glutamyl groups attached to the glutamyl groups of pemetrexed. In some embodiments, gamma heptaglutamated PMX comprises two or more gamma glutamyl groups in the L-form. In further embodiments, each gamma glutamyl group of gamma heptaglutamated pemetrexed is in the L-form. In other embodiments, gamma heptaglutamated PMX comprises a gamma glutamyl group in the D-form. In some embodiments, gamma tetraglutamated PMX comprises 2, 3, 4, 5, or 6 gamma glutamyl groups in the D-form. In further embodiments, each gamma glutamyl group of gamma heptaglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In a further embodiment, the heptaglutamylated PMX comprises a D-glutamyl group and two or more L-glutamyl groups.

[0090] In some embodiments, gamma-polyglutamated pemetrexed is octaglutamated (γPMX-PG7), thus comprising a chain of seven additional glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma-octaglutamated PMX comprises two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma-octaglutamated pemetrexed is in the L-form. In other embodiments, gamma-octaglutamated PMX comprises a glutamyl group in the D-form. In some embodiments, gamma-octaglutamated PMX comprises 2, 3, 4, 5, 6, or 7 gamma-glutamyl groups in the D-form. In further embodiments, each glutamyl group of gamma-octaglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In further embodiments, octaglutamated PMX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

[0091] In some embodiments, gamma polyglutamated pemetrexed is nonaglutamated (γPMX-PG8), comprising a chain of eight additional glutamyl groups attached to the glutamyl groups of pemetrexed. In some embodiments, gamma nonaglutamated PMX comprises two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma nonaglutamated pemetrexed is in the L-form. In other embodiments, gamma nonaglutamated PMX comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group of gamma nonaglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In still further embodiments, nonaglutamated PMX comprises a gamma glutamyl group in the D-form and two or more gamma glutamyl groups in the L-form.

[0092] In some embodiments, gamma polyglutamated pemetrexed is deca-glutamated (γPMX-PG9), comprising a chain of nine additional glutamyl groups attached to the glutamyl groups of pemetrexed. In some embodiments, gamma deca-glutamated PMX comprises two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma deca-glutamated pemetrexed is in the L-form. In other embodiments, gamma deca-glutamated PMX comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group of gamma deca-glutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In still further embodiments, deca-glutamated PMX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

[0093] In some embodiments, gamma polyglutamated pemetrexed is undecaglutamated (γPMX-PG 10), which comprises a chain of 10 gamma glutamyl groups linked to the glutamyl group of pemetrexed. In some embodiments, gamma undecaglutamated PMX comprises two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma undecaglutamated pemetrexed is in the L-form. In other embodiments, gamma undecaglutamated PMX comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group of gamma undecaglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In still further embodiments, undecaglutamated PMX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

[0094] In some embodiments, gamma polyglutamated pemetrexed is dodecaglutamated (γPMX-PG 11 ), which comprises a chain of 11 gamma glutamyl groups linked to the glutamyl group of pemetrexed. In some embodiments, gamma dodecaglutamated PMX comprises two or more glutamyl groups in the L form. In further embodiments, each glutamyl group of gamma dodecaglutamated pemetrexed is in the L form. In other embodiments, gamma dodecaglutamated PMX comprises a glutamyl group in the D form. In further embodiments, each glutamyl group of gamma dodecaglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D form. In still further embodiments, dodecaglutamated PMX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form.

[0095] In some embodiments, gamma polyglutamated pemetrexed is triskite decaglutamated (γPMX-PG 12), which contains a chain of 12 gamma glutamyl groups linked to the glutamyl group of pemetrexed. In some embodiments, gamma-triskai deca-glutamated PMX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma-triskai deca-glutamated pemetrexed is in the L-form. In other embodiments, gamma-triskai deca-glutamated PMX contains a glutamyl group in the D-form. In further embodiments, each glutamyl group of gamma-triskai deca-glutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In still further embodiments, triskai deca-glutamated PMX contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

[0096] In some embodiments, gamma polyglutamated pemetrexed is tetradecaglutamated (γPMX-PG 13 ), which comprises a chain of 13 gamma glutamyl groups linked to the glutamyl group of pemetrexed. In some embodiments, gamma-tetradecaglutamated PMX comprises two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma-tetradecaglutamated pemetrexed is in the L-form. In other embodiments, gamma-tetradecaglutamated PMX comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group of gamma-tetradecaglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In still further embodiments, tetradecaglutamated PMX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

[0097] In some embodiments, gamma polyglutamated pemetrexed is pentadecaglutamated (γPMX-PG 14), which comprises a chain of 14 gamma glutamyl groups linked to the glutamyl groups of pemetrexed. In some embodiments, gamma pentadecaglutamated PMX comprises two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma pentadecaglutamated pemetrexed is in the L-form. In other embodiments, gamma pentadecaglutamated PMX comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group of gamma pentadecaglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In still further embodiments, pentadecaglutamated PMX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

[0098] In some embodiments, gamma polyglutamated pemetrexed is hexadecaglutamated (γPMX-PG 15 ) and comprises a chain of 15 gamma glutamyl groups linked to the glutamyl group of pemetrexed. In some embodiments, gamma hexadecaglutamated PMX comprises two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma hexadecaglutamated pemetrexed is in the L-form. In other embodiments, gamma hexadecaglutamated PMX comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group of gamma hexadecaglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In still further embodiments, hexadecaglutamated PMX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

[0099] In another embodiment, gamma polyglutamated pemetrexed is heptadecaglutamated (γPMX-PG 16) comprises a chain of 16 gamma glutamyl groups linked to the glutamyl group of pemetrexed. In some embodiments, gamma heptadecaglutamated PMX comprises two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma heptadecaglutamated pemetrexed is in the L-form. In other embodiments, gamma heptadecaglutamated PMX comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group of gamma heptadecaglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In still further embodiments, heptadecaglutamated PMX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

[0100] In some embodiments, gamma polyglutamated pemetrexed is octadeca-glutamated (γPMX-PG 17 ), which contains a chain of 17 gamma glutamyl groups linked to the glutamyl group of pemetrexed. In some embodiments, gamma octadeca-glutamated PMX contains two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma octadeca-glutamated pemetrexed is in the L-form. In other embodiments, gamma octadeca-glutamated PMX contains a glutamyl group in the D-form. In further embodiments, each glutamyl group of gamma octadeca-glutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In still further embodiments, octadeca-glutamated PMX contains a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

[0101] In some embodiments, gamma polyglutamated pemetrexed is eniadecaglutamated (γPMX-PG 18) comprises a chain of 18 gamma glutamyl groups linked to a glutamyl group of pemetrexed. In some embodiments, gamma eniadecaglutamated PMX comprises two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma eniadecaglutamated pemetrexed is in the L-form. In other embodiments, gamma eniadecaglutamated PMX comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group of gamma eniadecaglutamated pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In still further embodiments, eniadecaglutamated PMX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

[0102] In some embodiments, gamma polyglutamated pemetrexed is icosaglutamated (γPMX-PG 19 ) contains a chain of 19 gamma glutamyl groups linked to the glutamyl group of pemetrexed. In some embodiments, gamma mycosamine glutamate PMX contains two or more L-glutamyl groups. In further embodiments, each glutamyl group of gamma mycosamine glutamate pemetrexed is in the L-form. In other embodiments, gamma mycosamine glutamate PMX contains a D-glutamyl group. In further embodiments, each glutamyl group of gamma mycosamine glutamate pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In still further embodiments, gamma mycosamine glutamate PMX contains a D-glutamyl group and two or more L-glutamyl groups.

[0103] In some embodiments, gamma polyglutamated pemetrexed is henicosaglutamated (γPMX-PG 20) comprises a chain of 20 gamma glutamyl groups linked to the glutamyl groups of pemetrexed. In some embodiments, gamma heminecosamine glutamate-modified PMX comprises two or more glutamyl groups in the L-form. In further embodiments, each glutamyl group of gamma heminecosamine glutamate-modified pemetrexed is in the L-form. In other embodiments, gamma heminecosamine glutamate-modified PMX comprises a glutamyl group in the D-form. In further embodiments, each glutamyl group of gamma heminecosamine glutamate-modified pemetrexed other than the glutamyl group of pemetrexed is in the D-form. In still further embodiments, heminecosamine glutamate-modified PMX comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.

[0104] In some embodiments, gamma polyglutamated pemetrexed comprises a chain of 4 to 7 glutamyl groups linked to pemetrexed (i.e., γPMX-PGn, n=4 to 7), each of the 4 to 7 linked glutamyl groups having a gamma linkage. In some embodiments, each of the 4 to 7 linked glutamyl groups is in the L-form. In other embodiments, each of the 4 to 7 linked glutamyl groups is in the D-form. In other embodiments, the 4 to 7 linked glutamyl groups are in both the L- and D-forms.

[0105] In one embodiment, the gamma polyglutamated pemetrexed is tetraglutamated, and each of the three glutamyl groups in the polyglutamate chain bound to pemetrexed contains a gamma linkage. In some embodiments, each of the four glutamyl groups is L-type. In some embodiments, each glutamyl group in gamma tetraglutamated pemetrexed other than the glutamyl group in pemetrexed is D-type. In other embodiments, at least two glutamyl groups in gamma tetraglutamated pemetrexed are L-type and at least one glutamyl group is D-type.

[0106] In one embodiment, the gamma polyglutamated pemetrexed is pentaglutamated, and each of the four glutamyl groups in the polyglutamate chain bound to pemetrexed contains a gamma linkage. In some embodiments, each of the four glutamyl groups is in the L-form. In some embodiments, each glutamyl group in the gamma pentaglutamated pemetrexed other than the glutamyl group in pemetrexed is in the D-form. In other embodiments, at least two glutamyl groups in the gamma pentaglutamated pemetrexed are in the L-form and at least one glutamyl group is in the D-form.

[0107] In one embodiment, the gamma-polyglutamated pemetrexed is hexaglutamated. In some embodiments, each of the five glutamyl groups is in the L-form. In some embodiments, each glutamyl group in the gamma-hexaglutamated pemetrexed other than the glutamyl group in pemetrexed is in the D-form. In other embodiments, at least two glutamyl groups in the gamma-hexaglutamated pemetrexed are in the L-form and at least one glutamyl group is in the D-form.

[0108] In another embodiment, the gamma polyglutamated pemetrexed is heptaglutamated. In some embodiments, each of the six glutamyl groups is in the L-form. In some embodiments, each glutamyl group in the gamma heptaglutamated pemetrexed other than the glutamyl group in pemetrexed is in the D-form. In other embodiments, at least two glutamyl groups in the gamma heptaglutamated pemetrexed are in the L-form and at least one glutamyl group is in the D-form.

[0109] In some embodiments, gamma polyglutamylated pemetrexed (γPPMX) 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 those in pemetrexed, or any range therebetween. In some embodiments, each glutamyl group in γPPMX other than the glutamyl groups in pemetrexed has a gamma linkage. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in γPPMX have a gamma linkage. In some embodiments, γPPMX contains both L- and D-glutamyl groups. In some embodiments, each glutamyl group in the polyglutamate structure of polyglutamylated pemetrexed is in the L-configuration. In some embodiments, each glutamyl group in γPPMX other than the glutamyl group in pemetrexed is in the D form. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the glutamyl groups in γPPMX are in the L form. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in γPPMX are in the D form.

[0110] In further embodiments, the gamma polyglutamated pemetrexed contains 20 to 100, 20 to 75, 20 to 50, 20 to 40, 20 to 30, 20 to 25, or more than 100 gamma glutamyl groups, or any range therebetween. In some embodiments, each glutamyl group in the gamma polyglutamated pemetrexed is in the L-form. In other embodiments, each glutamyl group in the gamma 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 gamma polyglutamated pemetrexed are in the L-form and at least one of the glutamyl groups in the gamma polyglutamated pemetrexed is in the D-form.

[0111] In further embodiments, provided compositions comprise gamma polyglutamated pemetrexed comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups with gamma linkages. In some embodiments, the gamma polyglutamated pemetrexed comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 L-glutamyl groups. In some embodiments, the gamma polyglutamated pemetrexed comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 D-glutamyl groups. In some embodiments, the gamma polyglutamated pemetrexed contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 L-glutamyl groups and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 D-glutamyl groups.

[0112] In some embodiments, the gamma 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 routinely performed.

[0113] In some embodiments, naked gamma-PPMX compositions disclosed herein (e.g., gamma-PPMX not conjugated to a delivery vehicle) are taken up by liver cells at a rate that is significantly reduced compared to the uptake rate of pemetrexed under the same physiological conditions. In some embodiments, the rate of liver cell uptake of naked gamma-PPMX compositions is less than 30%, 20%, 15%, or 10% of the rate of pemetrexed. In further embodiments, the rate of efflux (transport) of naked gamma-PPMX compositions disclosed herein from liver cells occurs at a rate that is significantly slower (e.g., less than 30%, 20%, 15%, or 10%) than that of pemetrexed.

[0114] In some embodiments, the gamma polyglutamated pemetrexed compositions provided herein are more cytotoxic to 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 gamma polyglutamated pemetrexed is hexaglutamated pemetrexed.

[0115] In some embodiments, the gamma polyglutamated pemetrexed compositions provided herein have fewer toxic side effects than pemetrexed. In some embodiments, the gamma polyglutamated pemetrexed compositions provided herein are less toxic than pemetrexed to non-hyperproliferative cells. In some embodiments, the gamma 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 gamma polyglutamated pemetrexed is hexaglutamated pemetrexed.

[0116] In some embodiments, the gamma polyglutamated pemetrexed compositions provided herein have fewer toxic side effects than pemetrexed. In some embodiments, the gamma polyglutamated pemetrexed compositions provided herein cause fewer 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 gamma polyglutamated pemetrexed compositions provided herein cause fewer 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 a gamma polyglutamated pemetrexed composition once a week for four weeks. In some embodiments, the gamma polyglutamated pemetrexed is hexaglutamated pemetrexed.

[0117] In some embodiments, treatment with the gamma polyglutamated pemetrexed compositions provided herein does not induce significant hematological or liver toxic side effects in an in vivo mouse model. In some embodiments, hematological side effects are assessed by mean neutrophil, mean white blood cell, or mean platelet counts. In some embodiments, liver toxic side effects are assessed by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the gamma polyglutamated pemetrexed compositions provided herein do not significantly reduce mean neutrophil, mean white blood cell, or mean platelet counts. In some embodiments, the gamma polyglutamated pemetrexed compositions provided herein do not significantly increase serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. In some embodiments, the gamma 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 a gamma polyglutamated pemetrexed composition once a week for four weeks. In some embodiments, the gamma polyglutamated pemetrexed is hexaglutamated pemetrexed.

[0118] In some embodiments, the gamma polyglutamated pemetrexed composition does not contain fluorine atoms. In some embodiments, the gamma polyglutamated pemetrexed composition does not contain 4-fluoroglutamyl groups.

[0119] Gamma polyglutamated pemetrexed (αPPMX) compositions and their uses are further disclosed in U.S. Application Nos. 62 / 374,458, 15 / 675,695, 15 / 675,701, and 62 / 583,432; International Application Nos. PCT / US2017 / 046666 and PCT / US2017 / 046667, the contents of each of which are incorporated herein by reference in their entirety.

[0120] Polyglutamated Pemetrexed Analogs and Derivatives The present disclosure also encompasses gamma 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, compositions of polyglutamated pemetrexed analogs or derivatives prepared and used according to the disclosed compositions and methods are shown in Figures 1I-1J. In some embodiments, the analogs correspond to modified forms 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 analogs are variants of pemetrexed, in which the glutamyl group of pemetrexed is in the D-form. In some embodiments, the polyglutamated forms of pemetrexed or polyglutamated pemetrexed analogs or derivatives are non-fluorinated.

[0121] In further embodiments, the gamma 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.

[0122] A.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; 5,416,211; 5,344,932; U.S. Patent Publication No. 2013 / 0165654; ​​European Patents EP 0905128, EP 2882753, EP 0905128, and EP 2409978; International Patent Publications WO 2014 / 024164, WO 2012 / 056285, WO 2008 / 021410, and WO 2001 / 14379, as well as those described in 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).

[0123] The addition of glutamyl residues to the glutamyl residues of pemetrexed can be carried out using synthetic methods known in the art. In some embodiments, glutamyl residues are added sequentially to the glutamyl residues of pemetrexed. In further embodiments, polyglutamates are added to the glutamyl residues of pemetrexed using "click chemistry" methods or other bioconjugate chemistries known to those skilled in the art.

[0124] B. Pemetrexed-PG complex The inventors have made the surprising discovery that polyglutamylated pemetrexed (γPPMX) can be complexed with other compositions, including therapeutic agents, including cytotoxic compounds such as platinum-based compounds. Accordingly, in some embodiments, the present disclosure provides complexes of γPPMX (e.g., the γPPMX disclosed herein) with a therapeutic agent, or a salt or acid thereof.

[0125] In some embodiments, the γPPMX / complex comprises γPPMX and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound such as a chemotherapeutic agent. In further embodiments, the γPPMX / complex comprises a platinum-based drug such as a platinum-based chemotherapeutic agent (e.g., cisplatin, carboplatin, and oxaliplatin). In other embodiments, the γPPMX / complex comprises a taxane-based chemotherapeutic agent (e.g., paclitaxel and docetaxel). In other embodiments, the γPPMX / complex comprises a cyclodextrin. In further embodiments, the γPPMX / complex is encapsulated in a liposome.

[0126] In some embodiments, the present disclosure provides compositions comprising a complex of γPPMX and a therapeutic agent, or a salt or acid thereof. In further embodiments, the γPPMX / therapeutic agent complex comprises one or more γPPMXs 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 γPPMX / therapeutic agent complex comprises one or more γPPMXs containing 3-10, 3-9, 3-8, or 3-7 glutamyl groups, or any range therebetween. In other embodiments, the γPPMX / therapeutic agent complex comprises one or more γPPMXs 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 γPPMXs containing 3-10 glutamyl groups. In further embodiments, the γPPMX / therapeutic agent conjugate comprises one or more γPPMX containing 3-7 glutamyl groups. In further embodiments, the γPPMX / therapeutic agent conjugate comprises one or more γPPMX containing 5 glutamyl groups. In another embodiment, the γPPMX / therapeutic agent conjugate comprises one or more γPPMX 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 γPPMX / therapeutic agent in the conjugate ranges from 1 to 10:1. In some embodiments, the molar ratio of γPPMX / therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / therapeutic agent in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween.In some embodiments, the molar ratio of γPPMX / therapeutic agent 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 γPPMX / therapeutic agent complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0127] In alternative embodiments, the γPPMX complex comprises γPPMX and cyclodextrin. In some embodiments, the molar ratio of γPPMX (e.g., γPPMX 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 γPPMX 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 γPPMX 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 γPPMX / 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 γPPMX / 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 γPPMX / cyclodextrin is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the γPPMX / cyclodextrin complex is encapsulated in a liposome (e.g., as described herein or known in the art).

[0128] In some embodiments, the present disclosure provides compositions comprising a γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / platinum-based drug in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / platinum-based drug in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPPMX / platinum-based drug is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the γPPMX / platinum-based drug complex is encapsulated in a liposome (e.g., as described herein or known in the art).

[0129] In further embodiments, the γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / platinum-based analog in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / platinum-based analog 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 γPPMX / platinum-based analog is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPPMX / platinum-based analog complex is encapsulated in a liposome (e.g., as described herein or known in the art).

[0130] In further embodiments, the present disclosure provides a complex comprising γPPMX and cisplatin, or a salt or acid thereof. In some embodiments, the molar ratio of γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / cisplatin (or cisplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / cisplatin (or cisplatin salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPPMX / cisplatin (or a salt or acid of cisplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPPMX / cisplatin (or a salt or acid of cisplatin) complex is encapsulated in a liposome (e.g., as described herein or known in the art).

[0131] In another embodiment, the present disclosure provides a conjugate comprising γPPMX and carboplatin, or a salt or acid thereof. In some embodiments, the molar ratio of γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / carboplatin (or carboplatin salt or acid) in the conjugate 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 γPPMX / carboplatin in the conjugate 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 γPPMX / carboplatin (or a carboplatin salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPPMX / carboplatin (or a carboplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or known in the art).

[0132] In another embodiment, the present disclosure provides a complex comprising γPPMX and oxaliplatin, or a salt or acid thereof. In some embodiments, the molar ratio of γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / oxaliplatin (or oxaliplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / oxaliplatin (or oxaliplatin salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPPMX / oxaliplatin (or oxaliplatin salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPPMX / oxaliplatin (or oxaliplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or known in the art).

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

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

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

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

[0137] In further embodiments, the present disclosure provides a complex comprising γPPMX and larotaxel (LTX) or a salt or acid thereof. In other embodiments, the γPPMX / taxane chemotherapeutic agent complex comprises larotaxel (LTX) or a salt or acid thereof. In some embodiments, the molar ratio of γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / larotaxel (or larotaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / larotaxel (or larotaxel salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPPMX / larotaxel (or larotaxel salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPPMX / larotaxel (or larotaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or known in the art).

[0138] In further embodiments, the present disclosure provides a conjugate comprising γPPMX and cabazitaxel (CTX) or a salt or acid thereof. In other embodiments, the γPPMX / taxane chemotherapeutic agent conjugate comprises an analog of cabazitaxel (CTX), or a salt or acid thereof. In some embodiments, the molar ratio of γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / cabazitaxel (or cabazitaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / cabazitaxel (or cabazitaxel salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of γPPMX / cabazitaxel (or cabazitaxel salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPPMX / cabazitaxel (or cabazitaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or known in the art).

[0139] In further embodiments, the present disclosure provides a complex comprising γPPMX and another anti-metabolite, or a salt or acid thereof. An anti-metabolite 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 cellular functions, such as cell division. In some embodiments, the present disclosure provides a complex comprising γPPMX and pemetrexed (PMX), or a salt or acid thereof. In some embodiments, the present disclosure provides a complex comprising γPPMX and an anti-metabolite selected from the group consisting of gemcitabine, fluorouracil, capecitabine, antifolates (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 (one or more), or derivatives of any of these. In some embodiments, the molar ratio of γPPMX 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 γPPMX to antimetabolite (or antimetabolite salt or acid, or prodrug) in the complex is in the range of 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPPMX 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 γPPMX to antimetabolite (or antimetabolite salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPPMX to antimetabolite (or antimetabolite salt or acid) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween.In some embodiments, the molar ratio of γPPMX / antimetabolite (or antimetabolite salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPPMX / antimetabolite (or antimetabolite salt or acid) complex is encapsulated in a liposome (e.g., as described herein or known in the art).

[0140] In a further embodiment, the present disclosure provides a complex of γPPMX (e.g., the γPPMX 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.

[0141] 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.

[0142] As used herein, there are no particular limitations on the cyclodextrin component of a γPPMX / cyclodextrin complex, so long as the cyclodextrin is capable of forming a complex with γPPMX. 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 γPPMX and / or the liposome encapsulation.

[0143] Modification of hydroxyl groups of cyclodextrin, 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 γPPMX / 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 one or more hydroxyl groups substituted with one or more of its charged moieties. Such moieties may themselves be charged groups or may include an organic moiety (e.g., a C-C alkyl or C-C alkyl ether moiety) substituted with one or more charged moieties.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] In some embodiments, the "ionizable" or "charged" derivatized cyclodextrins, whether polyionic, amphiphilic, or otherwise, are generally weakly ionizable (e.g., having a pKai of about 4.0-8.5, 4.5-8.0, 5.0-7.5, 5.5-7.5, 6.0-6.5, and any range therebetween, inclusive).

[0148] Any one, some, or all of the hydroxyl groups of any one, some, or all of the γ-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.

[0149] The aggregate substitution occurring in a mixture of cyclodextrin derivatives is described by a term called the degree of substitution. For example, 6-ethylenediamino-β-cyclodextrin having a degree of substitution of 7 will be composed of a distribution of 6-ethylenediamino-β-cyclodextrin isomers in which the number of ethylenediamino groups per 6-ethylenediamino-β-cyclodextrin molecule is 7. The degree of substitution of a mixture of cyclodextrin derivatives can be routinely determined using mass spectrometry or nuclear magnetic resonance spectroscopy.

[0150] 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 gamma-D-glucopyranoside unit of the C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and all three C2-C3-C6 hydroxyls are substituted with an ionizable chemical group. Any such hydroxyl combination can be combined with any of the degrees of substitution described herein, as well as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and up to all gamma-D-glucopyranoside units in the modified cyclodextrin. One such derivative is sulfoalkyl ether cyclodextrin (SAE-CD). The sulfobutyl ether derivative of beta-cyclodextrin (SBE-β-CD) has been shown to have significantly improved aqueous solubility compared to the parent cyclodextrin.

[0151] Additional cyclodextrin derivatives that can be complexed with therapeutic agents in the disclosed liposomal compositions include sugammadex or Org-25969, in which the 6-hydroxy group on γ-CD has been replaced with a carboxythioacetate ether linkage, and hydroxybutenyl-β-CD. Alternative forms of cyclodextrin include 2,6-di-O-methyl-β-CD (DIMEB), 2-hydroxypropyl-3-cyclodextrin (HP-β-CD), randommethylated-β-cyclodextrin (RAMEB), sulfobutyl ether Examples of suitable cyclodextrins include β-cyclodextrin (SBE-β-CD), sulfobutylether-γ-cyclodextrin (SBEγCD), sulfobutylated-β-cyclodextrin sodium salt, (2-hydroxypropyl)-gamma cyclodextrin, (2-hydroxypropyl)-beta-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, 2,6-di-O-methyl)-β-cyclodextrin (DIMEB-50 heptakis), 2,3,6-tri-O-methyl)-β-cyclodextrin (TRIMEB heptakis), methyl-β-cyclodextrin, octakis(6-deoxy-6-iodo)-γ-cyclodextrin, and octakis(6-deoxy-6-bromo)-γ-cyclodextrin.

[0152] In some embodiments, the cyclodextrin(s) have high solubility in water to facilitate entrapment of a larger amount of cyclodextrin in the liposome internal phase. In some embodiments, the solubility of the cyclodextrin in water is at least 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, or more. In some embodiments, the water solubility of the cyclodextrin is within the range of 10-150 mg / mL, 20-100 mg / mL, 20-75 mg / mL, and any range therebetween (inclusive).

[0153] In some embodiments, a large binding constant between the cyclodextrin and γPPMX and / or other therapeutic agents complexed with the cyclodextrin is preferred and can be obtained by selecting the number of glucose units in the cyclodextrin based on the size of the therapeutic agent (e.g., Albers et al., Crit. Rev. Therap. Drug Carrier Syst. 12:311-337 (1995); Stella et al., al., Toxicol. Pathol. 36:30-42 (2008). If the binding constant is pH dependent, a cyclodextrin can be selected that has a high binding constant at the pH of the liposome internal phase. As a result, the solubility (nominal solubility) of the therapeutic agent in the presence of the cyclodextrin can be further improved. In some embodiments, the binding constant between the cyclodextrin and the therapeutic agent is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or greater. In some embodiments, the binding constant between the cyclodextrin and the therapeutic agent is 100-1,200, 200-1,000, 300-750, and any range therebetween.

[0154] In some embodiments, the cyclodextrin of the γPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is not underivatized.

[0155] In some embodiments, the cyclodextrin of the γPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is derivatized. In further embodiments, the cyclodextrin derivative of the complex has Formula I: [ka] wherein n is 4, 5, or 6; R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently -H, a linear or branched C1-C8 alkylene group, or an optionally substituted linear or branched C1-C6 group, and at least one of R1, R2, R3, R4, R5, R6, R7, R8, and R9 is a linear or branched C1-C8 alkylene (e.g., C1-C8-(alkylene)-SO3 - (base).

[0156] In some embodiments, the cyclodextrin derivative of the γPPMX / 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 the group consisting of -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.

[0157] In some embodiments, the cyclodextrin derivative of the γPPMX / 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.

[0158] In some embodiments, the cyclodextrin derivative of the γPPMX / 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.

[0159] In some embodiments, the cyclodextrin derivative of the γPPMX / 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.

[0160] In a further embodiment, the γPPMX / cyclodextrin complex and / or cyclodextrin / therapeutic agent complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0161] III. γPPMX Delivery Carrier In alternative embodiments, the present disclosure provides γPPMX delivery systems and their use for delivering a γPPMX payload to a cell(s) in vitro or in vivo. In some embodiments, γPPMX 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-γPPMX 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.

[0162] A. Liposomes In some embodiments, the present disclosure provides liposome compositions comprising liposomes encapsulating (i.e., loaded with) gamma polyglutamated pemetrexed (e.g., γPPMX as disclosed herein). In some embodiments, the liposomes in the liposome composition comprise γPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups (including the glutamyl groups in pemetrexed). In some embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains two or more L-glutamyl groups. In other embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains D-glutamyl groups. In further embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains D-glutamyl groups and two or more L-glutamyl groups. In further embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains two or more glutamyl groups with gamma carboxyl bonds. In some embodiments, the liposome composition comprises liposomes containing gamma pentaglutamated PMX. In further embodiments, the liposomes comprise L-γ pentaglutamated PMX, D-γ pentaglutamated PMX, or L- and D-γ pentaglutamated PMX. In some embodiments, the liposome composition comprises liposomes containing gamma hexaglutamated PMX (Lp-γPPMX). In further embodiments, the liposomes comprise L-γ hexaglutamated PMX, D-γ hexaglutamated PMX, or L- and D-γ 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-γPPMX composition is non-PEGylated. In some embodiments, the Lp-γPPMX composition is non-targeted (NTLp-γPPMX). In other embodiments, the Lp-γPPMX composition is targeted (TLp-γPPMX). In some embodiments, the liposome composition comprises liposomes having a diameter of 20 nm to 500 nm, or any range therebetween.In some embodiments, the liposome composition comprises liposomes having a diameter of 20 nm to 400 nm, or any range therebetween. In some embodiments, the liposome composition comprises liposomes having a diameter of 20 nm to 300 nm, or any range therebetween. In some embodiments, the liposome composition comprises liposomes having a diameter of 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposome composition comprises liposomes having a diameter of 20 nm to 150 nm, or any range therebetween. In further embodiments, the liposome composition comprises liposomes having a diameter of 80 nm to 120 nm, or any range therebetween. In further embodiments, 30 to 70%, 30 to 60%, or 30 to 50% w / w of gamma polyglutamated pemetrexed, or any range therebetween, is encapsulated (entrapped) in Lp-γPPMX during the liposome preparation process. In some embodiments, the Lp-γPPMX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w of gamma polyglutamated 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% w / w of gamma polyglutamated pemetrexed is encapsulated in Lp-γPPMX during the liposome production process.

[0163] In some embodiments, provided liposomes further comprise an immunostimulatory agent, a detectable marker, or both, disposed on the exterior surface of the liposome. The immunostimulatory agent or detectable marker can be ionically or covalently bound to the exterior surface of the liposome, which may optionally include binding to a steric stabilizing component of the liposome.

[0164] The term "immunostimulatory agent," also known as "immunostimulant" and "immunostimulator," refers to a substance that stimulates immunity (including a pre-existing immune response) by inducing activation or increased activity of any component of the immune system. These immunostimulatory agents include one or more of haptens, adjuvants, protein immunostimulators, nucleic acid immunostimulators, and chemical immunostimulators. Many adjuvants include substances designed to stimulate the immune response, such as lipid A, proteins derived from Bordetella pertussis, or Mycobacterium tuberculosis. Certain adjuvants are commercially available, for example, Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron, or zinc; insoluble suspensions of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A; IFN-alpha, IFN-gamma, FLT3 ligand; and immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3). Cytokines such as GM-CSF, interleukins 2, 7, 12, and 15, and other similar growth factors can also be used as adjuvants. In a preferred embodiment, the immunostimulant may be at least one selected from the group consisting of fluorescein, DNP, beta-glucan, beta-1,3-glucan, and beta-1,6-glucan. In a further preferred embodiment, the immunostimulant is a toll-like receptor (TLR) modulator. In a further embodiment, the toll-like receptor (TLR) modulator is one or more of oxidized low-density lipoprotein (e.g., OXPC, PGPC), erythrocyanide (e.g., E5564), and resolvin.In some embodiments, the liposomes contain fluorescein isothiocyanate (FITC), which, based on the inventors' experiments, surprisingly functions as both an immunostimulant and a detectable marker.

[0165] 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.

[0166] 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.

[0167] In some embodiments, the liposome further comprises an agent that increases uptake of the liposome into a desired intracellular compartment, including the cytosol.

[0168] 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 (e.g., RQIKIWFQNRRMKWKKRKKRRQR RR (SEQ ID NO: 1), RKKRRXR RRGC, where X is any natural or unnatural amino acid (SEQ ID NO: 2), CCGCCAAGAAGCG (SEQ ID NO: 3), GCGTGCACACGCGCGTAGACTTCCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTGAGCGGCGTGGCGCGGGGGCGTCAT (SEQ ID NO: 4), ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTC ATCCGTCAGCTC (SEQ ID NO: 5), or ACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTG (SEQ ID NO: 6), or a mitochondrial permeability fragment thereof.

[0169] In some embodiments, the liposomes in the provided liposome compositions comprise a mitochondrial penetrant selected from the group consisting of 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).

[0170] 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.

[0171] In some embodiments, the liposomes in the provided liposome compositions comprise 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 permeabilizing agent 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), or a combination thereof. 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), Sequence number 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) , GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 37), GRKKRRQRRR (SEQ ID NO: 38), RRRRRRR (SEQ ID NO: 39), RRRRRRRR (SEQ ID NO: 40), RRRRRRRRR (SEQ ID NO: 41), RRRRRRRRRR (SEQ ID NO: 42), RRRRRRRRRRR (SEQ ID NO: 43), and YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG(R)n (wherein n=2-15R in L- and / or D-form) (SEQ ID NO: 44), or a cell-permeable fragment thereof.

[0172] As discussed above, liposomes may contain a steric stabilizer that can extend their lifespan in the circulation. For those embodiments incorporating a steric stabilizer, the steric stabilizer may be at least one member selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidylpolyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymers; oligoglycerin, polyethylene glycol and polypropylene oxide-containing copolymers, poloxamer 188, and polyvinyl alcohol. In some embodiments, the steric stabilizer or stabilizers is PEG. In one embodiment, the steric stabilizer is PEG. In a further embodiment, the PEG has a number-average molecular weight (Mn) of 200 to 5,000 daltons. These PEGs can be of any structure, such as linear, branched, star or comb structures, and are commercially available.

[0173] In some embodiments, the liposome composition comprises PEGylated liposomes (PLp-γPPMX). In some embodiments, the PEGylated liposomes in the liposome composition comprise γPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains two or more L-glutamyl groups. In other embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains D-glutamyl groups. In further embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains D-glutamyl groups and two or more L-glutamyl groups. In some embodiments, the liposome composition comprises PEGylated liposomes containing gamma pentaglutamated PMX. In further embodiments, the liposomes comprise L-γ-pentaglutamated PMX, D-γ-pentaglutamated PMX, or L- and D-γ-pentaglutamated PMX. In some embodiments, the liposome composition comprises PEGylated liposomes comprising gamma-hexaglutamated PMX. In further embodiments, the liposomes comprise L-γ-hexaglutamated PMX, D-γ-hexaglutamated PMX, or L- and D-γ-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-γPPMX composition is non-targeted (NTPLp-γPPMX). In other embodiments, the PLp-γPPMX composition is targeted (TPLp-γPPMX). In some embodiments, the liposome composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w / w) gamma polyglutamated pemetrexed.In some embodiments, the liposome composition comprises PEGylated liposomes containing at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w / w) gamma polyglutamated pemetrexed, which is encapsulated in PLp-γPPMX during liposome preparation. 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 400 nm. In some embodiments, the liposome composition comprises PEGylated liposomes having a diameter ranging from 20 nm to 300 nm. In some embodiments, the liposome composition comprises PEGylated liposomes having a diameter ranging from 20 nm to 200 nm. In a further embodiment, the liposome composition comprises PEGylated liposomes having diameters ranging from 80 nm to 120 nm.

[0174] In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated pemetrexed in the provided liposome compositions is pentaglutamated. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated pemetrexed in the provided compositions is hexaglutamated. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated pemetrexed in the compositions has 4 to 10, 4 to 6, or more than 5 gamma glutamyl groups.

[0175] In some embodiments, the gamma polyglutamated pemetrexed composition (e.g., a delivery vehicle such as gamma polyglutamate and polyglutamate-containing liposomes) is in an aqueous solution. In some embodiments, the γPPMK composition is administered as a liposomal composition at a dose of 1000 mg / mL per square meter of body surface area (m 2In a further embodiment, the γPPMX composition is administered as a liposomal composition at a dose of 0.1 to 1000 mg of γPPMX per square meter of body surface area, or any range therebetween.

[0176] (1) Liposome composition Lipids and other components of liposomes contained in the liposome composition can be any lipid, combination of lipids and ratios, or combination of lipids and other liposome components and their respective ratios known in the art. However, those skilled in the art will understand that liposomal encapsulation of any particular drug, such as, but not limited to, gamma polyglutamated 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.

[0177] The liposome composition formulations provided herein can be in 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 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 cyrodessication). 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.

[0178] 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)), and 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 steric 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.

[0179] 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.

[0180] The properties of liposomes are influenced by the nature of the lipids used to prepare the liposomes. A wide variety of lipids have been used to prepare liposomes. These include cationic, anionic, and neutral lipids. In some embodiments, liposomes containing gamma polyglutamated 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.

[0181] 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.

[0182] 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 gamma antibodies, folate receptor beta antibodies, and / or folate receptor delta antibodies.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] In some embodiments, the γPPMX compositions provided herein are formulated in liposomes comprising a cationic lipid. In one embodiment, the cationic lipid is a lipid selected from the group consisting of, but not limited to, the cationic lipids 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 / 0 Nos. 44638, 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 No. 20100036115, and U.S. Patent Application Publication No. 20120202871. Each of these patent documents is incorporated herein by reference in its entirety. In another embodiment, the cationic lipid may be selected from, but is not limited to, Formula A as described in WO 2012 / 040184, WO 2011 / 153120, WO 201 / 1149733, WO 2011 / 090965, WO 2011 / 043913, WO 2011 / 022460, WO 2012 / 061259, WO 2012 / 054365, and WO 2012 / 044638, each of which is incorporated herein by reference in its entirety. In yet another embodiment, the cationic lipid may be selected from, but is not limited to, formulas CLI-CLXXIX of WO 2008103276, 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-dimemyl-hexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylhenicosa-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-dimethyltricosa-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 Amine, (18Z)-N,N-dimethylheptacos-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 amine, (15Z)-N,N-dimethyleptacos-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]heneicosan-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]hexadecan-8-amine N,N-dimethyl-1-[(1S,2R)-octylcyclopropyl]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)propan-2-amine, S-N,N-dimethyl 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}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 Pan-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,1 4Z)-Icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-Icosa-11,14-dien-1-yloxy]-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)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-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 N,N-dimethyl-1-{[-(2-octylcyclopropyl)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.

[0189] 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.

[0190] 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.

[0191] 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 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.

[0192] In some embodiments, the γPPMX 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 the cationic peptides described in International Publication No. WO 2012 / 013326, which is incorporated herein by reference in its entirety. In another embodiment, the γPPMX composition is formulated in a lipid-polycation complex, which further includes a neutral lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0193] The liposome components can include any molecule (e.g., chemical / reagent / protein) bound thereto, and in some embodiments, the liposome components provided include at least a member selected from the group consisting of 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.

[0194] 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.

[0195] 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.

[0196] In some embodiments, the liposomal gamma polyglutamated pemetrexed composition is PEGylated (i.e., PEGylated liposomal gamma polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate (PLp-γPPMX or TPLp-γPPMX)). In some embodiments, the PLp-γPPMX or TPLp-γPPMX is water-soluble. That is, the PLp-γPPMX or TPLp-γPPMX is in the form of an aqueous solution.

[0197] In some embodiments, the liposomes of the disclosed liposome compositions comprise a lipid selected from the group consisting of 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.

[0198] In some embodiments, the pH of the solution containing the liposome composition is pH 5-8, or pH 2-6, or pH 2-8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is pH 5-8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is pH 6-7, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is pH 6-7.5, 6.5-7.5, 6.7-7.5, or 6.3-7.0, or any range therebetween.

[0199] 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).

[0200] 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.

[0201] 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).

[0202] (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 gamma polyglutamated pemetrexed provided herein. In further embodiments, the interior space of the liposome comprises a tonicity agent. In some embodiments: In some embodiments, the concentration (wt%) of the tonicity agent is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 1-50%, or any range therebetween. In some embodiments, the interior space of the liposome comprises a sugar (e.g., trehalose, maltose, sucrose, lactose, mannose, mannitol, glycerol, dextrose, fructose, etc.). In further embodiments, the concentration (wt%) of the sugar is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 1-50%, or any range therebetween. In some embodiments, the pH of the liposome interior space is between 2 and 8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is between 5 and 8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is between 6 and 7, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is between 6 and 7.5, 6.5 and 7.5, 6.7 and 7.5, or 6.3 and 7.0, or any range therebetween. In some embodiments, the interior space contains a buffer. In further embodiments, the buffer is a buffer selected from the group consisting of 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 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.

[0203] In some embodiments, the interior space of the liposome contains trehalose. In further embodiments, the concentration (wt%) of trehalose is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 5-20%, or any range therebetween. In 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 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 solution. In some embodiments, the buffer is selected from the group consisting of 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 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.

[0204] In some embodiments, the interior space of the liposome contains dextrose. In further embodiments, the concentration (wt%) of dextrose is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 5-20%, or any range therebetween. In still further embodiments, the concentration (wt%) of dextrose is 1-15%, or any range therebetween. In additional embodiments, the dextrose is present at a concentration of about 5%-20% (wt%) dextrose, or any combination of one or more lyoprotectants or cryoprotectants is present at a total concentration of 5%-20%. In some embodiments, the pH of the solution containing the liposome composition is 6-7.5, 6.5-7.5, 6.7-7.5, or 6.3-7.0, or any range therebetween. In some embodiments, the interior space contains a buffer. In some embodiments, the buffer is selected from the group consisting of 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.

[0205] In further embodiments, the present disclosure provides liposome compositions comprising liposomes encapsulating (loaded with) gamma polyglutamated pemetrexed (e.g., γPPMX as disclosed herein). In some embodiments, the liposomes in the liposome composition comprise γPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 gamma glutamyl groups (including the glutamyl groups of pemetrexed). In some embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains two or more L-glutamyl groups. In other embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains D-glutamyl groups. In further embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains a D-glutamyl group and two or more L-glutamyl groups. In further embodiments, the gamma polyglutamated pemetrexed in Lp-γPPMX contains two or more glutamyl groups with gamma carboxyl bonds. In some embodiments, the liposome composition comprises liposomes containing gamma pentaglutamated PMX. In further embodiments, the liposomes comprise L-γ pentaglutamated PMX, D-γ pentaglutamated PMX, or L- and D-γ pentaglutamated PMX. In some embodiments, the liposome composition comprises liposomes containing gamma hexaglutamated PMX (Lp-γPPMX). In further embodiments, the liposomes comprise L-γ hexaglutamated PMX, D-γ hexaglutamated PMX, or L- and D-γ hexaglutamated PMX.

[0206] In some embodiments, the targeted PEGylated liposomal gamma-polyglutamated (e.g., pentaglutamated or hexaglutamated) pemetrexed comprises a vehicle comprising a liposome comprising an interior space; aqueous gamma-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.

[0207] In some embodiments, gamma polyglutamated pemetrexed-encapsulated liposomes (i.e., Lp-γPPMX, including PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) have an interior space containing fewer than 500,000 or fewer than 200,000 gamma polyglutamated pemetrexed molecules. In some embodiments, the liposome interior space contains 10 to 100,000 gamma polyglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposome interior space contains 10,000 to 100,000 gamma 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 gamma polyglutamated pemetrexed molecules. In some embodiments, the liposomes are non-PEGylated and the interior space of the liposomes contains 10 to 100,000 gamma 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 gamma polyglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposomes are targeted, non-PEGylated (TLp-γPPMX), and have an interior space containing less than 500,000 or less than 200,000 gamma polyglutamated pemetrexed molecules. In some embodiments, the liposomes are targeted, non-PEGylated, and the interior space of the liposomes contains 10 to 100,000 gamma polyglutamated 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 gamma polyglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposomes are non-targeted and non-PEGylated (NTLp-γPPMX) and have an interior space containing less than 500,000 or less than 200,000 gamma 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 gamma 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 gamma polyglutamated pemetrexed molecules, or any range therebetween.

[0208] In some embodiments, the liposome encapsulates gamma polyglutamated pemetrexed containing 2 to 10 glutamyl groups (i.e., Lp-γPPMX, including PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) and has an interior space containing fewer than 500,000 or fewer than 200,000 gamma polyglutamated pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome interior space contains 10 to 100,000 gamma 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 gamma 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 gamma polyglutamated pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposomes are not PEGylated and the interior space of the liposomes contains 10 to 100,000 gamma polyglutamated pemetrexed molecules containing 2 to 10 glutamyl groups, or any range therebetween. In further embodiments, the liposomes are not PEGylated and the interior space of the liposomes contains 10,000 to 100,000 gamma polyglutamated pemetrexed molecules containing 2 to 10 glutamyl groups, or any range therebetween. In some embodiments, the liposomes are targeted and non-PEGylated (TLp-γPPMX) and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma 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 gamma 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, or any range therebetween, gamma polyglutamated pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposomes are non-targeted and non-PEGylated (NTLp-γPPMX), and have an interior space containing fewer than 500,000 or fewer than 200,000, gamma 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, or any range therebetween, gamma polyglutamated pemetrexed molecules containing 2 to 10 glutamyl groups. In a further embodiment, the liposome is non-targeted and non-PEGylated, and the interior space of the liposome contains 10,000 to 100,000 gamma polyglutamated pemetrexed molecules containing 2 to 10 glutamyl groups, or any range therebetween.

[0209] In some embodiments, the liposomes encapsulate gamma-tetraglutamated pemetrexed (i.e., Lp-γPPMX, including PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma-tetraglutamated pemetrexed molecules. In some embodiments, the liposome interior space contains 10 to 100,000 gamma-tetraglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposome interior space contains 10,000 to 100,000 gamma-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 gamma-tetraglutamated pemetrexed molecules. In some embodiments, the liposomes are non-PEGylated and the interior space of the liposomes contains 10 to 100,000 gamma-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 gamma-tetraglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposomes are targeted, non-PEGylated (TLp-γPPMX), and have an interior space containing less than 500,000 or less than 200,000 gamma-tetraglutamated pemetrexed molecules. In some embodiments, the liposomes are targeted, non-PEGylated, and the interior space of the liposomes contains 10 to 100,000 gamma-tetraglutamated 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 gamma-tetraglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposomes are non-targeted and non-PEGylated (NTLp-γPPMX), and have an interior space containing less than 500,000 or less than 200,000 gamma-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 gamma-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 gamma-tetraglutamated pemetrexed molecules, or any range therebetween.

[0210] In some embodiments, the liposomes encapsulate gamma pentaglutamated pemetrexed (i.e., Lp-γPPMX, including PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma pentaglutamated pemetrexed molecules. In some embodiments, the liposome interior space contains 10 to 100,000 gamma pentaglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposome interior space contains 10,000 to 100,000 gamma pentaglutamated 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 gamma-pentaglutamated pemetrexed molecules. In some embodiments, the liposomes are non-PEGylated and have an interior space containing 10 to 100,000 gamma-pentaglutamated 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 gamma-pentaglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposomes are targeted, non-PEGylated (TLp-γPPMX), and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma-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 gamma-pentaglutamated 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 gamma-pentaglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposomes are non-targeted and non-PEGylated (NTLp-γPPMX) and have an interior space containing less than 500,000 or less than 200,000 gamma-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 gamma-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 gamma-pentaglutamated pemetrexed molecules, or any range therebetween.

[0211] In some embodiments, the liposomes encapsulate gamma hexaglutamated pemetrexed (i.e., Lp-γPPMX, including PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma hexaglutamated pemetrexed molecules. In some embodiments, the liposome interior space contains 10 to 100,000 gamma hexaglutamated pemetrexed molecules, or any range therebetween. In further embodiments, the liposome interior space contains 10,000 to 100,000 gamma hexaglutamated 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 gamma hexaglutamated pemetrexed molecules. In some embodiments, the liposomes are non-PEGylated and have an interior space containing 10 to 100,000 gamma hexaglutamated 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 gamma hexaglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposomes are targeted, non-PEGylated (TLp-γPPMX), and have an interior space containing fewer than 500,000 or fewer than 200,000 gamma hexaglutamated pemetrexed molecules. In some embodiments, the liposomes are targeted and non-PEGylated, and the interior space of the liposomes contains 10 to 100,000 gamma hexaglutamated 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 gamma hexaglutamated pemetrexed molecules, or any range therebetween. In some embodiments, the liposomes are non-targeted and non-PEGylated (NTLp-γPPMX), and have an interior space containing less than 500,000 or less than 200,000 gamma hexaglutamated 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 gamma hexaglutamated 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 gamma hexaglutamated pemetrexed molecules, or any range therebetween.

[0212] In some embodiments, the present disclosure provides liposomal gamma polyglutamated pemetrexed compositions, wherein liposomes encapsulate gamma 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 5% to 20% trehalose by weight. 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.

[0213] A. Non-polyglutamylated polyglutamylated antifolates In some embodiments, liposomal gamma polyglutamated pemetrexed (e.g., Lp-γPPMX, including PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) compositions comprise gamma polyglutamated pemetrexed (e.g., γPPMX disclosed herein) and one or more non-polyglutamylatable polyglutamate antifolate compositions.

[0214] In some embodiments, Lp-γPPMX (e.g., PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) comprises gamma polyglutamated pemetrexed (e.g., the γPPMX disclosed herein) and pemetrexed (PMX). In some embodiments, Lp-γPPMX (i.e., liposomal gamma polyglutamated pemetrexed) comprises gamma 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-γPPMX comprises gamma-polyglutamated pemetrexed and lometrexol. In some embodiments, Lp-γPPMX comprises gamma-polyglutamated pemetrexed and pemetrexed. In some embodiments, Lp-γPPMX comprises gamma-polyglutamated pemetrexed and leucovorin. In some embodiments, Lp-γPPMX comprises gamma-polyglutamated pemetrexed and a triazine antifolate derivative (e.g., a sulfonyl fluoride triazine such as NSC127755). In some embodiments, Lp-γPPMX comprises gamma-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.

[0215] B. Non-polyglutamylatable antifolates In some embodiments, Lp-γPPMX (e.g., PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) comprises gamma-polyglutamated pemetrexed (e.g., γPPMX disclosed herein) and a so-called "non-polyglutamylatable" antifolate. In some embodiments, the liposome comprises gamma-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 the group consisting of thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide (GAR) transformylase, and aminoimidazolecarboxamide ribonucleotide (AICAR) transformylase. In some embodiments, the liposomes comprise gamma-polyglutamated pemetrexed and a non-polyglutamate-resistant antifolate that inhibits DHFR. In some embodiments, the liposomes comprise gamma-polyglutamated pemetrexed and a non-polyglutamate-resistant antifolate that inhibits TS. In some embodiments, the liposomes comprise gamma-polyglutamated pemetrexed and a non-polyglutamate-resistant antifolate that inhibits GAR or AICAR transformylase. In further embodiments, the non-polyglutamate-resistant 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), and BGC945 (ONX0801).

[0216] C platinum In some embodiments, the liposomes comprise gamma polyglutamated pemetrexed (e.g., Lp-γPPMX, such as PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX), including gamma polyglutamated pemetrexed (e.g., γPPMX disclosed herein) and a platinum-based chemotherapeutic agent, or a salt or acid thereof. In some embodiments, the liposomes comprise a gamma polyglutamated pemetrexed / platinum-based drug conjugate (e.g., as described in Section IIB).

[0217] In some embodiments, the Lp-γPPMX comprises a platinum-based chemotherapeutic agent selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In some embodiments, the Lp-γPPMX comprises an analog of a platinum-based chemotherapeutic agent selected from the group consisting of cisplatin, carboplatin, or oxaliplatin, or salts or acids thereof.

[0218] In some embodiments, Lp-γPPMX comprises gamma polyglutamated pemetrexed and cisplatin, or a salt or acid thereof. In some embodiments, Lp-γPPMX comprises gamma polyglutamated pemetrexed and a cisplatin analog, or a salt or acid thereof.

[0219] In some embodiments, Lp-γPPMX comprises gamma polyglutamated pemetrexed and carboplatin, or a salt or acid thereof. In some embodiments, the liposome comprises gamma polyglutamated pemetrexed and a carboplatin analog, or a salt or acid thereof.

[0220] In some embodiments, Lp-γPPMX comprises gamma polyglutamated pemetrexed and oxaliplatin, or a salt or acid thereof. In some embodiments, the liposome comprises gamma polyglutamated pemetrexed and an oxaliplatin analog, or a salt or acid thereof.

[0221] In some embodiments, the liposome comprises gamma polyglutamated pemetrexed (e.g., γPPMX disclosed herein) and a platinum-based chemotherapeutic agent selected from the group consisting of nedaplatin, heptaplatin, and lobaplatin, or salts or acids thereof. In some embodiments, Lp-γPPMX comprises gamma polyglutamated pemetrexed and an analog of a platinum-based chemotherapeutic agent selected from the group consisting of nedaplatin, heptaplatin, and lobaplatin, or salts or acids thereof.

[0222] In some embodiments, the Lp-γPPMX comprises gamma polyglutamated pemetrexed and a platinum-based chemotherapeutic agent selected from the group consisting of stratoplatin, paraplatin, platinol, cycloplatin, dexorumaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamines, satraplatin, enloplatin, JM216, 254-S, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or a salt or acid thereof. In some embodiments, the Lp-γPPMX comprises gamma polyglutamated pemetrexed and a platinum-based chemotherapeutic agent selected from the group consisting of stratoplatin, paraplatin, platinol, cycloplatin, dexorumaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamines, satraplatin, enloplatin, JM216, 254-S, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or a salt or acid thereof.

[0223] In some embodiments, the liposome composition comprises a liposome further comprising one or more of an immunostimulatory agent, a detectable marker, and a maleimide disposed on at least one of the PEG or exterior surface of the liposome.

[0224] D Cyclodextrin In further embodiments, the γPPMX liposomes comprise γPPMX (eg, a γPPMX disclosed herein) and a cyclodextrin (eg, a cyclodextrin in Section IIB herein).

[0225] In some embodiments, the γPPMX liposome comprises a complex formed by a cyclodextrin and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound, or a salt or acid thereof. In further embodiments, the therapeutic agent is a chemotherapeutic agent, or a salt or acid thereof. In another embodiment, the chemotherapeutic agent is a platinum-based drug. In another embodiment, the chemotherapeutic agent is a taxane drug. In further embodiments, the therapeutic agent of the cyclodextrin / therapeutic agent complex is a member selected from the group consisting of gemcitabine, a gemcitabine-based therapeutic agent, doxorubicin, an antifolate, an antifolate-based chemotherapeutic agent, or a salt or acid, acid form, or free base form thereof. In further embodiments, the molar ratio of cyclodextrin to therapeutic agent in the complex is in the range of 1-10:1. In some embodiments, the molar ratio of cyclodextrin to therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to therapeutic agent in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to therapeutic agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50.

[0226] In some embodiments, the γPPMX liposome comprises γPPMX and a cyclodextrin / platinum-based chemotherapeutic agent complex. In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or a salt or acid thereof. In other embodiments, the cyclodextrin / platinum-based chemotherapeutic agent complex comprises an analog of cisplatin, carboplatin, or oxaliplatin, or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin to platinum-based agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to platinum-based drug in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to platinum-based drug in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to therapeutic agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50.

[0227] In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the cyclodextrin / platinum-based chemotherapeutic agent complex comprises an analog of cisplatin, carboplatin, or oxaliplatin, or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin to platinum-based agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to platinum-based agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to platinum-based drug in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to platinum-based drug is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the cyclodextrin / platinum-based drug complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0228] In further embodiments, the present disclosure provides a complex comprising a cyclodextrin and cisplatin, or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin to cisplatin (or a cisplatin salt or acid) in the complex is in the range of 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to cisplatin (or a cisplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to cisplatin (or cisplatin salt or acid) in the complex ranges from 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to cisplatin (or cisplatin salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the cyclodextrin / cisplatin (or cisplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

[0229] In another embodiment, the present disclosure provides a complex comprising a cyclodextrin and carboplatin, or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin to carboplatin (or a carboplatin salt or acid) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to carboplatin (or a carboplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to carboplatin (or carboplatin salt or acid) in the complex is 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to carboplatin (or carboplatin salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In a further embodiment, the cyclodextrin / carboplatin (or carboplatin salt or acid) complex is encapsulated in a liposome (eg, as described herein or otherwise known in the art).

[0230] In another embodiment, the present disclosure provides a complex comprising a cyclodextrin and oxaliplatin, or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin to oxaliplatin (or oxaliplatin salt or acid) in the complex is in the range of 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to oxaliplatin (or oxaliplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to oxaliplatin (or oxaliplatin salt or acid) in the complex is 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to oxaliplatin (or oxaliplatin salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In a further embodiment, the cyclodextrin / oxaliplatin (or oxaliplatin salt or acid) complex is encapsulated in a liposome (eg, as described herein or otherwise known in the art).

[0231] In a further embodiment, the present disclosure provides a conjugate comprising a cyclodextrin and a platinum-based chemotherapeutic agent selected from the group consisting of nedaplatin, heptaplatin, lobaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexorumaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamines, satraplatin, enloplatin, JM216, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or a salt or acid thereof. In other embodiments, the cyclodextrin / platinum-based chemotherapeutic agent complex comprises nedaplatin, heptaplatin, lobaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexorumaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enloplatin, JM216, NK121, CI973, DWA2114R, NDDP, or an analog of nedaplatin, or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin to oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to platinum-based chemotherapeutic agent (or salt or acid or analog thereof) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to platinum-based chemotherapeutic agent (or salt or acid or analog thereof) in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range therebetween.In some embodiments, the molar ratio of cyclodextrin / platinum-based chemotherapeutic agent (or salt or acid or analog thereof) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the cyclodextrin / platinum-based chemotherapeutic agent (or salt or acid or analog thereof) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

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

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

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

[0235] In further embodiments, the present disclosure provides a complex comprising a cyclodextrin and larotaxel (LTX) or a salt or acid thereof. In other embodiments, the cyclodextrin / taxane chemotherapeutic agent complex comprises an analog of larotaxel (LTX), or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin to larotaxel (or a salt or acid of larotaxel) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin to larotaxel (or larotaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin to larotaxel (or larotaxel salt or acid) in the complex is 1:1-20, 1:1-10, or 1:2-8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin to larotaxel (or larotaxel salt or acid) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the cyclodextrin / larotaxel (or larotaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).

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

[0237] The cyclodextrin of the cyclodextrin / therapeutic agent complex can be derivatized or underivatized. In some embodiments, the cyclodextrin is derivatized. In further embodiments, the cyclodextrin is a derivatized beta cyclodextrin (e.g., hydroxypropyl beta cyclodextrin (HP-beta-CD) and sulfobutyl ether beta-CD ((SBE)-beta-cyclodextrin)). In some embodiments, the cyclodextrin of the cyclodextrin / therapeutic agent complex is a derivatized beta cyclodextrin that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 2-hydroxylpropyl-3-group substitutions of hydroxy groups, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfoalkyl ether group substitutions of hydroxy groups. In further embodiments, the cyclodextrin of the cyclodextrin / therapeutic agent complex is a derivatized beta cyclodextrin that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more sulfobutyl ether group substitutions of hydroxy groups.

[0238] In some embodiments, the cyclodextrin of the cyclodextrin / therapeutic agent complex included in the γPPMX liposome composition 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 straight-chain or branched C1-C8 alkylene group, a 2-hydroxylpropyl-3- group; or an optionally substituted straight-chain or branched C1-C6 group, and at least one of R1, R2, R3, R4, R5, R6, R7, R8, and R9 is a straight-chain or branched C1-C8 alkylene group or a 2-hydroxylpropyl-3- group.

[0239] In some embodiments, the cyclodextrin of the cyclodextrin / therapeutic agent complex included in the γPPMX liposome composition has Formula II: [ka] wherein n is 4, 5, or 6; R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently -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 -H or H or 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 amine cations such as ammonium ions and cations of (C1-C6)-alkylamines, piperidines, pyrazines, (C1-C6)-alkanolamines and (C4-C8)-cycloalkanolamines.

[0240] In some embodiments, the γPPMX liposomes contain between 100 and 100,000 cyclodextrin / therapeutic agent complexes.

[0241] In some embodiments, the cyclodextrin derivative of the γPPMX / 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.

[0242] In some embodiments, the cyclodextrin derivative of the cyclodextrin / cyclodextrin 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.

[0243] In some embodiments, the cyclodextrin / therapeutic agent complex and / or cyclodextrin derivative has Formula III: [ka] wherein R is (e)(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; (f)(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; (g)(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 (h)(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.

[0244] Additional cyclodextrins and cyclodextrin / platinum-based therapeutic complexes that can be included in γPPMX liposomes and used according to the disclosed methods are disclosed in U.S. Patent Application Serial No. 62 / 583,543, the contents of which are incorporated herein by reference in their entirety.

[0245] In some embodiments, the γPPMX liposomes comprise a complex of a cyclodextrin and a platinum-based chemotherapeutic agent, or a salt thereof. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin or a cisplatin analog. In some embodiments,...

Claims

1. A liposome composition comprising a liposome encapsulating gamma polyglutamylated pemetrexed, the gamma polyglutamylated pemetrexed comprising 2 to 10 glutamyl groups having gamma carboxyl group bonds; The liposome is pegylated and contains a targeting moiety that has specific affinity for a surface antigen of a target cell; A liposome composition, wherein the liposomes encapsulate one or more non-polyglutamylatable antifolates, or alternatively, encapsulate one or more non-polyglutamylatable antifolates.

2. The liposome composition of claim 1 , wherein the gamma polyglutamated pemetrexed is gamma tetraglutamated pemetrexed or gamma pentaglutamated pemetrexed.

3. The liposome composition of claim 1 , wherein the gamma polyglutamated pemetrexed is gamma hexaglutamated pemetrexed.

4. (a) the gamma polyglutamylated pemetrexed contains two or more L-type glutamyl groups with gamma carboxyl linkages; (b) each of the glutamyl groups of the gamma polyglutamylated pemetrexed is in the L-configuration and has a gamma carboxyl group linkage; (c) at least one glutamyl group of the gamma polyglutamylated pemetrexed is in the D-form and has a gamma carboxyl group bond; (d) each glutamyl group of said gamma polyglutamylated pemetrexed other than the glutamyl group of pemetrexed is in the D-configuration and has a gamma carboxyl group linkage; or (e) the gamma polyglutamylated pemetrexed comprises two or more L-glutamyl groups and at least one D-glutamyl group having a gamma carboxyl group bond; The liposome composition of claim 1.

5. 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.

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

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

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

9. 2. The liposome composition of claim 1, wherein the liposomes are formed from liposome components, the liposome components comprising 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, The liposome components may comprise at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC, and one or more of the liposome components may further comprise a steric stabilizer, the steric stabilizer being 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-phenylindole) (PVP ... poly(2-ethyl-2-oxazoline); phosphatidylpolyglycerol; poly N-(2-hydroxypropyl) methacrylamide; amphiphilic poly-N-vinylpyrrolidone; L-amino acid based polymers; oligoglycerin, copolymers comprising polyethylene glycol and polypropylene oxide, poloxamer 188, and polyvinyl alcohol, wherein the steric stabilizer is PEG and the PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons. Liposomal compositions.

10. 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 composition of claim 1, wherein the liposome has a zeta potential of -30 to -50 mV.

11. 2. The liposome composition of claim 1, wherein the liposome has an interior space containing gamma polyglutamylated pemetrexed and an aqueous pharma- ceutically acceptable carrier, The pharma- ceutically acceptable carrier may comprise a tonicity agent such as dextrose, mannitol, glycerol, potassium chloride, sodium chloride at a concentration greater than 1%, 1%-50% trehalose, 1%-50% dextrose, 5% dextrose suspended in HEPES buffer, or sodium acetate and calcium acetate at a combined concentration of 50 mM-500 mM; The interior space of the liposome may have a pH of 5 to 8 or a pH of 6 to 7 or any range therebetween; The liposome may contain less than 500,000 or less than 200,000 gamma polyglutamated pemetrexed molecules, or between 10 and 100,000 gamma polyglutamated pemetrexed molecules or any range therebetween. Liposomal compositions.

12. 10. The liposome composition of claim 1, further comprising 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., DNP ... ｎ－６ＤＰＡ Or D ｎ－３ＤＰＡ resolvin D, resolvin E, or T series resolvins such as erythropoietin, oxidized low density lipoprotein (e.g., OXPAC, PGPC), and toll-like receptor (TLR) modulators such as erythropoietin lipids (e.g., E5564); may further comprise at least one cryoprotectant selected from the group consisting of mannitol, trehalose, sorbitol, and sucrose, or may further comprise carboplatin and / or pembrolizumab; Liposomal compositions.

13. A pharmaceutical composition comprising the liposome composition of claim 1.

14. A composition for treating cancer, immune system disorders, or infectious diseases, comprising the liposome composition according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 13.

15. 15. The composition of claim 14 for treating cancer, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, colon cancer, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, ovarian cancer, cervical cancer, mesothelioma or non-small cell lung cancer (NSCLC), and sarcoma such as osteosarcoma.

16. 13. A method of making a gamma polyglutamated pemetrexed composition comprising the liposome composition of any one of claims 1 to 12, the method comprising: forming a mixture in a solution comprising liposome components and gamma polyglutamated pemetrexed; homogenizing the mixture in the solution to form liposomes; and treating the mixture to form liposomes comprising gamma polyglutamated pemetrexed. The processing steps may include one or more of the following steps: thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw technique, reverse phase evaporation, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, freeze-dried double emulsion, 3D printing, membrane contacting, and stirring, or the processing steps may include one or more steps of modifying the size of the liposomes by one or more of the following steps: extrusion, high pressure microfluidization, and / or sonication; method.

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