Polyglutamated antifolates and uses thereof

Abstract

To provide compositions to treat diseases including hyperproliferative diseases such as cancer, disorders of the immune system such as rheumatoid arthritis and infectious disease such as HIV.SOLUTION: Provided is a composition comprising polyglutamated antifolates.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Cases This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 62 / 374,458, filed Aug. 12, 2016, in the U.S. Patent and Trademark Office, which is incorporated herein by reference in its entirety. This application also claims priority to U.S. Application No. 15 / 675,695, filed Aug. 11, 2017, and U.S. Application No. 15 / 675,701, filed Aug. 11, 2017, the contents of each of which are incorporated herein by reference in their entirety. All references, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety. [Background technology]

[0002] The present disclosure relates generally to L-polyglutamated antifolate compositions comprising delivery vehicles, such as targeted or non-targeted liposomes, loaded with polyglutamated antifolates, and methods of making and using the compositions to treat diseases, including hyperproliferative diseases such as cancer, disorders of the immune system such as rheumatoid arthritis, and infectious diseases such as HIV.

[0003] Folate is essential for cell proliferation and tissue regeneration. Mammalian cells do not synthesize folates de novo but rely on extracellular folates taken up by the three major folate uptake systems: the reduced folate carrier (RFC) system, the folate receptor (FR) α and β systems, and the proteolytic folate cotransporter (PCFT) system. Antifolates that target folate-dependent biosynthetic pathways function as antiproliferative agents. Naturally occurring folates are present in cells as polyglutamates through the action of folylpolyglutamate synthetase (FPGS), which adds up to six glutamyl groups in the γ-peptide bond to the folate substrate. Polyglutamation serves at least three main purposes: (1) It promotes the accumulation of intracellular folates in a vast excess of monoglutamate pools that are freely mobile in and out of cells; (2) it allows selective intracellular retention of these relatively large anionic molecules; and (3) it greatly enhances folate cofactor affinity for several folate-dependent enzymes, including thymidylate synthase and AICAR transformylase (see, e.g., Figure 2).

[0004] Antifolates are a class of antiproliferative agents that were first developed over 70 years ago as "folate mimetic molecule" cytotoxic agents. The rationale was to design a molecular class that would exploit the physiological folate transport mechanisms and their facilitatory intracellular mode of action on DNA replication during cell division to counter the effects of folate in rapidly replicating cells, such as cancer cells. Specifically, antifolates were designed to mimic folate in its systemic transport, physiological cellular uptake (e.g., via reduced folate carriers (RFCs) and proton-coupled folate transporters (PCFTs)) and intracellular processing. Antifolates specifically act during DNA and RNA synthesis and exert their cytotoxic effects during the S phase of the cell cycle. As a result, they have a highly toxic effect on rapidly dividing cells, such as malignant and myeloid cells.

[0005] Antifolates are widely recognized for their inhibition of folate metabolism. The main antifolate enzyme targets and examples of antifolates that target these enzymes include (a) dihydrofolate reductase (DHFR) [e.g., methotrexate (MTX) and pralatrexate], (b) thymidylate synthase (TS) [e.g., raltitrexed (RTX), GW1843U, and pemetrexed (PMX)], (c) β-glycinamide ribonucleotide formyltransferase (GARFTase) [e.g., lometrexol (LMX), PMX], and (d) 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) [e.g., PMX]. Inhibition of the above enzymes suppresses new nucleotide biosynthesis, causing an imbalance of purine and pyrimidine precursors, preventing cells from performing accurate DNA replication, and ultimately leading to cell death. It is therefore not surprising that inhibitors of the folate metabolic pathway would play an important role in treating hyperproliferative diseases, including hematological malignancies and solid tumors, as well as disorders of the immune system, such as rheumatoid arthritis.

[0006] Each of the above antifolates is transported by the reduced folate carrier (RFC), the major transport system at physiological pH. RFC is widely expressed in normal and diseased cells, and as a result these agents have dose-limiting toxicities that are a major obstacle in cancer chemotherapy.

[0007] Pemetrexed (marketed under the trade name ALIMTA®) is an antifolate containing a 6-5 condensed pyrrolo[2,3,-d]pyrimidine nucleus that inhibits thymidylate synthase (TS), glycinamide ribonucleotide formyltransferase (GARFT), and dihydrofolate reductase (DHFR), folate-dependent enzymes involved in the synthesis of thymidine and purine nucleotides. Similar to methotrexate, pemetrexed is transported into cells by RFC and membrane folate-binding protein, where it is polyglutamylated by folylpoly-γ-glutamate synthetase. The polyglutamylated form of pemetrexed has higher intracellular retention and higher affinity for TS and GARFT compared to pemetrexed monoglutamate. Pemetrexed is approved for the treatment of mesothelioma and non-small cell lung cancer (NSCLC). Myelosuppression is commonly a dose-limiting toxicity of pemetrexed therapy, limiting the clinical application of this agent. Pretreatment with folic acid and vitamin B is currently used to ameliorate the most frequent side effects, including myelosuppression, fatigue, and skin rash.

[0008] One of the challenges in cancer treatment is to deliver cytotoxic agents to cancer cells while minimizing and / or reducing the effect of such agents on normal healthy cells.To address the toxicity of antifolates in normal cells, WO2016 / 25882 reports, for example, liposomal formulations of antifolates that are targeted to cancer cells using antibodies with specific affinity to folate receptors expressed by many cancer cells.This formulation can reduce and / or minimize the effect of antifolates on healthy cells, meaning that patients may experience fewer side effects.

[0009] Despite advances in the treatment of cancer and other hyperproliferative disorders with antifolates, there is a need for additional compositions and methods to improve efficacy and reduce dose-limiting toxicities associated with antifolate therapy. The present disclosure provides compositions and methods that address these needs. Summary of the Invention

[0010] The present disclosure generally relates to polyglutamated antifolate compositions comprising a delivery vehicle such as a liposome loaded with a polyglutamated antifolate, and methods of making and using the compositions to treat diseases including hyperproliferative diseases such as cancer, disorders of the immune system such as rheumatoid arthritis, and infectious diseases such as HIV. The present disclosure further relates to pentaglutamated and hexaglutamated antifolate compositions comprising a delivery vehicle such as a liposome loaded with a pentaglutamated and hexaglutamated antifolate, and methods of making and using the compositions to treat diseases including hyperproliferative diseases, disorders of the immune system, and infectious diseases.

[0011] By way of example and not limitation, the present disclosure describes liposomal compositions comprising polyglutamylated (e.g., pentaglutamylated and hexaglutamylated) forms of antifolates, such as MTX, PMX, LTX, AG2034, RTX, piritrexim, pralatrexate, AG2034, GW1843, aminoprelin, and LY309887. These compositions provide improvements to the efficacy and safety of delivering antifolates to cancer cells by providing preferential delivery of a higher cytotoxic payload (e.g., polyglutamylated antifolates) compared to the cytotoxicity of the antifolates in their monoglutamylated state to which they are administered. The present disclosure also provides targeted liposomal compositions comprising targeting moieties that have specific affinity for epitopes (antigens) expressed on the surface of target cells of interest. The targeted liposome compositions provide further improvements in the efficacy and safety of delivering antifolates to cancer cells by specifically delivering polyglutamylated (e.g., pentaglutamylated or hexaglutamylated) antifolates to target cells.

[0012] In some embodiments, the disclosure provides a composition comprising a polyglutamated antifolate (e.g., pentaglutamated or hexaglutamated). In further embodiments, the composition comprises a pentaglutamated antifolate. In further embodiments, the composition comprises a hexaglutamated antifolate. According to some embodiments, the polyglutamated antifolate is one or more members selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In a further embodiment, the polyglutamylated member is pentaglutamylated. In a further embodiment, the polyglutamylated member is hexaglutamylated.

[0013] In one embodiment, the composition comprises polyglutamylated PMX, MTX, RTX, and / or LTX. In a further embodiment, the composition comprises pentaglutamylated PMX, MTX, RTX, and / or LTX. In a further embodiment, the composition comprises hexaglutamylated PMX, MTX, RTX, or LTX.

[0014] In one embodiment the composition comprises polyglutamylated PMX. In a further embodiment the composition comprises pentaglutamylated PMX. In a further embodiment the composition comprises hexaglutamylated PMX.

[0015] In another embodiment, the composition comprises polyglutamylated MTX. In a further embodiment, the composition comprises pentaglutamylated MTX. In a further embodiment, the composition comprises hexaglutamylated MTX.

[0016] In another embodiment, the composition comprises polyglutamylated RTX. In a further embodiment, the composition comprises pentaglutamylated RTX. In a further embodiment, the composition comprises hexaglutamylated RTX.

[0017] In additional embodiments, the composition comprises polyglutamylated LTX. In further embodiments, the composition comprises pentaglutamylated LTX. In further embodiments, the composition comprises hexaglutamylated LTX.

[0018] In additional embodiments, the disclosure provides liposomal polyglutamated antifolate (LPA) compositions comprising a polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate. For example, the polyglutamated antifolate can be in a HEPES buffered solution within the liposome. In further embodiments, the LPA composition is PEGylated (PLPA). In some embodiments, the PLPA composition comprises a pentaglutamated antifolate. In some embodiments, the PLPA composition comprises a hexaglutamated antifolate. In some embodiments, the PLPA liposomes are anionic or neutral. In other embodiments, the PLPA liposomes are cationic. In some embodiments, the PLPA composition comprises at least 10%, at least 20%, or at least 30% liposomes with entrapped polyglutamated antifolate. In some embodiments, the PLPA liposomes have a diameter in the range of 20-200 nm, 30-175 nm, or 50-150 nm. In some embodiments, the PLPA liposomes have a diameter in the range of 30 to 175 nm or 50 to 150 nm, hi further embodiments, the PLPA liposomes have a diameter in the range of 80 to 120 nm.

[0019] In some embodiments, the PLPA composition comprises a polyglutamated antifolate selected from the group consisting of polyglutamated MTX, polyglutamated PMX, polyglutamated LTX, polyglutamated AG2034, polyglutamated RTX, polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In further embodiments, the polyglutamated antifolate is pentaglutamated. In further embodiments, the polyglutamated antifolate is pentaglutamated. In one embodiment, the PLPA composition comprises polyglutamated PMX, MTX, RTX, or LTX. In further embodiments, the PLPA composition comprises pentaglutamated PMX, MTX, RTX, or LTX. In a further embodiment, the PLPA composition comprises hexaglutamylated PMX, MTX, RTX, or LTX. In one embodiment, the PLPA composition comprises polyglutamylated PMX. In a further embodiment, the PLPA composition comprises pentaglutamylated PMX. In a further embodiment, the PLPA composition comprises hexaglutamylated PMX.

[0020] In another embodiment, the PLPA composition comprises polyglutamylated MTX. In a further embodiment, the PLPA composition comprises pentaglutamylated MTX. In a further embodiment, the PLPA composition comprises hexaglutamylated MTX.

[0021] In another embodiment, the PLPA composition comprises polyglutamylated RTX. In a further embodiment, the PLPA composition comprises pentaglutamylated RTX. In a further embodiment, the PLPA composition comprises hexaglutamylated RTX.

[0022] In additional embodiments, the PLPA composition comprises polyglutamylated LTX. In further embodiments, the PLPA composition comprises pentaglutamylated LTX. In further embodiments, the PLPA composition comprises hexaglutamylated LTX.

[0023] In some embodiments, the present disclosure provides liposomal polyglutamated antifolate compositions, wherein the liposome comprises a PEGylated and polyglutamated antifolate and a targeting moiety attached to one or both of the PEG and the exterior of the liposome, wherein the targeting moiety has a specific affinity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is an antibody or a fragment of an antibody. In additional 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 multivalent antibody. In additional embodiments, the targeting moiety has a specific affinity for an epitope on a tumor cell surface antigen that is present on tumor cells but not present or accessible on non-tumor cells. In some embodiments, the targeting moiety-PLPA further comprises one or more of an immunostimulant, a detectable marker, and a maleimide disposed on at least one of the PEG and the exterior of the liposome. In some embodiments, the targeting moiety-PLPA liposome is anionic or neutral. In other embodiments, the targeting moiety-PLPA liposomes are cationic. In some embodiments, the targeting moiety-PLPA compositions comprise at least 10% liposome-entrapped polyhexaglutamated antifolate. In additional embodiments, the targeting moiety-PLPA liposomes have a diameter in the range of 20-200 nm. In further embodiments, the liposomes have a diameter in the range of 80-120 nm.

[0024] In some embodiments, the targeting moiety-PLPA comprises a polypeptide targeting moiety, such as an antibody or antibody fragment, and the targeting moiety has an affinity for at least 0.5×10 as measured using Biacore analysis. -10 ~10×10 -6In further embodiments, the targeting moiety comprises a polypeptide that specifically binds a folate receptor. In some embodiments, the folate receptor bound by the targeting moiety is one or more folate receptors selected from the group consisting of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ).

[0025] The present disclosure also provides a method of killing hyperproliferative cells, comprising contacting the hyperproliferative cells with a PLPA and / or LPA composition. In some embodiments, the hyperproliferative cells are cancer cells. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vitro. In further embodiments, the cancer cells are primary cells or cell line-derived cells obtained or derived from a cancer selected from the group consisting of lung cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colon cancer, esophageal cancer, cervical cancer, renal cancer, bile duct cancer, gallbladder cancer, and hematological malignancies.

[0026] In additional embodiments, the disclosure provides methods for treating cancer comprising administering an effective amount of a liposomal polyglutamylated (e.g., pentaglutamylated or hexaglutamylated) antifolate (LPA) composition and / or a pegylated LPA (PLPA) composition to a subject having or at risk of having cancer. In some embodiments, the method is administered to treat a cancer selected from the group consisting of lung cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colon cancer, esophageal cancer, cervical cancer, kidney cancer, bile duct cancer, gallbladder cancer, and hematological malignancies. In some embodiments, the polyglutamated antifolate administered is one or more members selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In further embodiments, the administered composition comprises a pentaglutamated antifolate. In further embodiments, the administered composition comprises a hexaglutamated antifolate. In some embodiments, the administered composition comprises a polyglutamated form of PMX, MTX, RTX, and / or LTX. In a further embodiment, the composition administered comprises pentaglutamylated forms of PMX, MTX, RTX, and / or LTX. In a further embodiment, the composition administered comprises hexaglutamylated forms of PMX, MTX, RTX, and / or LTX. In one embodiment, the composition administered comprises pentaglutamylated forms of PMX. In one embodiment, the composition administered comprises hexaglutamylated forms of PMX. In another embodiment, the composition administered comprises pentaglutamylated forms of MTX. In another embodiment, the composition administered comprises hexaglutamylated forms of MTX. In another embodiment, the composition administered comprises pentaglutamylated forms of RTX.In another embodiment, the composition administered comprises a hexaglutamylated form of RTX. In a further embodiment, the composition administered comprises a pentaglutamylated form of LTX. In a further embodiment, the composition administered comprises a hexaglutamylated form of LTX.

[0027] In additional embodiments, the disclosure provides a method for treating cancer comprising administering to a subject having or at risk of having cancer an effective amount of an optionally PEGylated liposome. In some embodiments, the method is administered to treat a cancer selected from the group consisting of lung cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colon cancer, esophageal cancer, cervical cancer, kidney cancer, bile duct cancer, gallbladder cancer, and hematological malignancies. In some embodiments, the polyglutamated antifolate administered is one or more members selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In further embodiments, the administered composition comprises a pentaglutamated antifolate. In further embodiments, the administered composition comprises a hexaglutamated antifolate. In some embodiments, the administered composition comprises a polyglutamated form of PMX, MTX, RTX, and / or LTX. In some embodiments, the administered composition comprises pentaglutamylated forms of PMX, MTX, RTX, and / or LTX. In some embodiments, the administered composition comprises hexaglutamylated forms of PMX, MTX, RTX, and / or LTX. In a further embodiment, the administered composition comprises pentaglutamylated forms of PMX. In a further embodiment, the administered composition comprises hexaglutamylated forms of PMX. In another further embodiment, the administered composition comprises pentaglutamylated forms of MTX. In another further embodiment, the administered composition comprises pentaglutamylated forms of MTX. In another further embodiment, the administered composition comprises pentaglutamylated forms of RTX. In another further embodiment, the administered composition comprises hexaglutamylated forms of RTX.In yet another embodiment, the composition administered comprises a pentaglutamylated form of LTX. In yet another embodiment, the composition administered comprises a hexaglutamylated form of LTX.

[0028] In some embodiments, the disclosure provides a method for treating cancer comprising administering an effective amount of an LPA composition and / or a pegylated LPA (PLPA) composition to a subject having or at risk of having cancer, wherein the PLPA and / or LPA composition further comprises a targeting moiety having specific affinity for a surface antigen (epitope) of the cancer. In further embodiments, the disclosure provides a method for treating cancer comprising administering a liposomal polyglutamated antifolate composition comprising a polyglutamate antifolate and a targeting moiety having specific binding affinity for the folate receptor to a subject having or at risk of having cancer that expresses a folate receptor on its surface that is bound by the targeting moiety. In further embodiments, the targeting moiety has specific binding affinity for the folate receptor alpha (FR-alpha), folate receptor beta (FR-beta), and / or folate receptor delta (FR-delta).

[0029] In further embodiments, the method is administered to treat a cancer selected from the group consisting of lung cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colon cancer, esophageal cancer, cervical cancer, kidney cancer, bile duct cancer, gallbladder cancer, and hematological malignancies. In some embodiments, the polyglutamated antifolate administered is one or more members selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In a further embodiment, the composition administered comprises a pentaglutamated antifolate. In a further embodiment, the composition administered comprises a hexaglutamated antifolate. In some embodiments, the composition administered comprises polyglutamated forms of PMX, MTX, RTX, and / or LTX. In some embodiments, the composition administered comprises pentaglutamated forms of PMX, MTX, RTX, and / or LTX. In some embodiments, the composition administered comprises hexaglutamated forms of PMX, MTX, RTX, and / or LTX. In a further embodiment, the composition administered comprises pentaglutamated forms of PMX. In a further embodiment, the composition administered comprises hexaglutamated forms of PMX. In another further embodiment, the composition administered comprises pentaglutamated forms of MTX. In another further embodiment, the composition administered comprises hexaglutamated forms of MTX. In another further embodiment, the composition administered comprises pentaglutamated forms of RTX. In another further embodiment, the composition administered comprises hexaglutamated forms of RTX. In yet another embodiment, the composition administered comprises a pentaglutamylated form of LTX. In yet another embodiment, the composition administered comprises a hexaglutamylated form of LTX.

[0030] In additional embodiments, the present disclosure provides a method for cancer maintenance therapy comprising administering an effective amount of an LPA composition and / or a PLPA composition to a subject undergoing or who has undergone cancer therapy.

[0031] In additional embodiments, the disclosure provides methods for treating an immune system disorder comprising administering to a subject having or at risk of having an immune system disorder an effective amount of a liposomal polyglutamylated (e.g., pentaglutamylated or hexaglutamylated) antifolate composition comprising a liposomal polyglutamylated (e.g., pentaglutamylated or hexaglutamylated) antifolate comprising a targeting moiety having specific affinity for a surface antigen on an immune cell of interest. In some embodiments, the method is administered to treat an autoimmune disease. In further embodiments, the method is administered to treat rheumatoid arthritis. In some embodiments, the polyglutamate antifolate administered is one or more members selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In further embodiments, the administered composition comprises a pentaglutamate antifolate. In further embodiments, the administered composition comprises a hexaglutamate antifolate. In some embodiments, the administered composition comprises a polyglutamate form of PMX, MTX, RTX, and / or LTX. In some embodiments, the composition administered comprises pentaglutamylated forms of PMX, MTX, RTX, and / or LTX. In some embodiments, the composition administered comprises pentaglutamylated forms of PMX, MTX, RTX, and / or LTX. In another further embodiment, the composition administered comprises hexaglutamylated forms of MTX. In a further embodiment, the composition administered comprises pentaglutamylated forms of PMX. In a further embodiment, the composition administered comprises hexaglutamylated forms of PMX. In another further embodiment, the composition administered comprises pentaglutamylated forms of RTX.In another further embodiment, the composition administered comprises a hexaglutamylated form of RTX. In an additional further embodiment, the composition administered comprises a pentaglutamylated form of LTX. In an additional further embodiment, the composition administered comprises a hexaglutamylated form of LTX.

[0032] The present disclosure also provides a method of delivering a polyglutamic antifolate to a tumor, comprising administering to a subject having a tumor a liposomal polyglutamylated (e.g., pentaglutamylated and / or hexaglutamylated) antifolate (LPA) composition and / or pegylated LPA (PLPA) comprising a targeting moiety having specific binding affinity for a surface antigen on the tumor, wherein the targeted PLPA and / or targeted LPA composition is delivered to the tumor in a therapeutically effective dose.

[0033] In an additional embodiment, the disclosure provides a method of preparing a composition comprising a liposomal polyglutamated antifolate composition, the method comprising forming a mixture comprising liposome components, a polyglutamated antifolate in solution, homogenizing the mixture to form liposomes in the solution, and treating the mixture to form liposomes comprising the polyglutamated antifolate.

[0034] Pharmaceutical compositions are also provided that include liposomal polyglutamylated (e.g., pentaglutamylated and / or hexaglutamylated) antifolate (LPA) compositions and / or pegylated LPA (PLPA) compositions, optionally further including a targeting moiety having specific affinity for a surface antigen on the surface of a target cell of interest. [Brief description of the drawings]

[0035] [Figure 1]Illustrates folate homeostasis and cellular accumulation of antifolates. Influx and efflux (anti)folate transporters. Once inside the cell, (anti)folates are polyglutamylated in the cytosol and mitochondria, whereas hydrolysis occurs in liposomes, a competing process. Figure 1 is adapted from Gonen et al., Drug Resistance Updates 15:183-210 (2012). [Diagram 2] Cellular folate metabolism and its compartmentalization in the cytosol and mitochondria. Figure 2 is adapted from Gonen et al., Drug Resistance Updates 15:183-210 (2012). [Figure 3A-B] 3A shows the disruption of cell polarity and disorganization that characterize advanced epithelial tumors. As shown in FIG. 3A, normal simple epithelium contains a single layer of individual cells that show clear apical-basal polarity. The cells are tightly packed and connected to each other by apical junctional complexes that separate the apical and basement membrane regions. In normal cells where polarity is preserved, FR-α is bound at the apical surface of the cells, which is distant from and does not directly contact folate in the blood circulation. As shown in FIG. 3B, high-grade epithelial tumors show loss of apical-basal polarity and overall disorganization, bringing FR-α into direct contact with folate in the blood circulation. [Figure 4] Molecular mechanisms underlying antifolate resistance in cancer. Figure 4 is adapted from Gonen et al., Drug Resistance Updates 15:183-210 (2012). [Diagram 5] FIG. 1 shows the chemical formula of L-alpha hexaglutamylated pemetrexed. [Figure 6] FIG. 1 shows the chemical formula of an example L-gamma polyglutamated antioxidant composition encompassed by the present disclosure. [Figure 7]FIG. 7 shows 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 adenocarcinoma subtype NCI H2342 non-small cell lung cancer (NSCLC). Results are cell viability measured by luciferase luminescence. As summarized in FIG. 7, free pemetrexed gG6 appears to be the least potent in inhibiting cell viability measured by IC50. Both liposomal pemetrexed gG6 and liposomal pemetrexed gG6-FR1Ab are 7-fold and 40-fold more potent than free pemetrexed, respectively. [Figure 8] FIG. 1 shows the dose-response relationship of free pemetrexed L-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha targeted antibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6-FR1Ab) in adenocarcinoma subtype NCI H2342 non-small cell lung cancer (NSCLC) as percentage of viable cells 48 hours after treatment. [Figure 9] FIG. 1 shows an example of dose-response relationship of free pemetrexed L-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha targeted antibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6-FR1Ab) in adenocarcinoma subtype NCI H2342 non-small cell lung cancer (NSCLC) as percentage of viable cells 48 hours after treatment. [Figure 10]Figure 1 shows 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. Free pemetrexed gG6 appears to be the least potent. Furthermore, liposomal pemetrexed gG6 is twice as potent as pemetrexed, and liposomal pemetrexed gG6-FR1Ab is 5 times more potent than free pemetrexed. [Figure 11] FIG. 1 shows examples of dose-response relationships at 48 hours in HT-29 (colon cancer) for free pemetrexed L-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha targeted antibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6-FR1Ab). [Figure 12] Figure 1 shows the effect of pemetrexed, free pemetrexed L-gamma hexaglutamate (hexa gG6), liposomal pemetrexed L-gamma hexaglutamate (liposomal hexa gG6), and free pemetrexed on the proliferation of ovarian cancer OAW28 cells after 48 hours of exposure to 256 nM of the corresponding drugs. Liposomal pemetrexed hexa gG6 can enter cells more efficiently and inhibit the proliferation of ovarian cancer OAW28 cells than pemetrexed hexa gG6. [Figure 13] Figure 1 shows the effect of free pemetrexed L-gamma hexaglutamate (hexa gG6) and liposomal pemetrexed L-gamma hexaglutamate (liposomal hexa gG6) on the proliferation of colon cancer SW260 cells after 48 hours of exposure to 256 nM of the corresponding drug. Non-targeted and targeted liposomal pemetrexed hexa gG6 can enter cells and inhibit the proliferation of colon cancer SW260 cells more efficiently than free pemetrexed hexa gG6. [Figure 14]FIG. 1 shows the relative potency of liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6) compared to pemetrexed after 48 hour exposure to 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 15] FIG. 1 shows the therapeutic effect of liposomal pemetrexed L-gamma hexaglutamate (Lps hexa gG6) compared to pemetrexed on HCC1806 triple-negative breast cancer cells after 48 hours of exposure. [Figure 16] FIG. 1 shows the therapeutic effect of liposomal pemetrexed L-gamma hexaglutamate (Lps hexa gG6) compared to pemetrexed on H292 non-small cell lung cancer cells after 48 hours of exposure. [Figure 17] FIG. 1 shows the therapeutic effect of liposomal pemetrexed L-gamma hexaglutamate (Lps hexa gG6) compared to pemetrexed on OAW28 ovarian cancer cells after 48 hours of exposure. [Figure 18] Figure 1 shows the therapeutic effect on H292 non-small cell lung cancer cells after 48 hours of exposure to different doses of liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6) and pemetrexed ranging from 16 to 128 nM. At each of the dose ranges tested, the liposomal pemetrexed gG6 formulation is superior to pemetrexed in inhibiting H292 non-small cell lung cancer cells. [Figure 19] Figure 1 shows the therapeutic effect of liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6) and pemetrexed at 16 nM, 32 nM, 64 nM, and 128 nM on HCC1806 triple-negative breast cancer cells after 48 hours of exposure. At each of the doses tested, the liposomal pemetrexed gG6 formulation is superior to pemetrexed in inhibiting HCC1806 triple-negative breast cancer cells. [Figure 20]Figure 1 shows the therapeutic effect of liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6) and pemetrexed at a range of concentrations on OAW28 ovarian cancer cells after exposure for 48 hours. At a dose of 128 nM, pemetrexed appears to inhibit cell proliferation more efficiently than the liposomal pemetrexed gG6 liposomal formulation, while at doses of 32 nM and 64 nM, the liposomal formulation has a superior therapeutic effect to pemetrexed, and at 16 nM, the therapeutic effect of liposomal pemetrexed gG6 is comparable to pemetrexed. [Figure 21] 1 shows the toxicity of liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6) and pemetrexed at 16 nM, 32 nM, 64 nM, and 128 nM to differentiating human neutrophils. The figure shows that liposomal pemetrexed gG6 is significantly less toxic to differentiating human neutrophils than pemetrexed. [Figure 22] Figure 1 shows the effect of liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6) and the equivalent of pemetrexed at 16nM, 32nM, 64nM and 128nM on AML12 hepatocytes after 48 hours of exposure.Strikingly, there does not appear to be any toxicity to AML12 hepatocytes after treatment with liposomal pemetrexed gG6 and liposomal drugs at any dose tested.In contrast, pemetrexed treatment causes a reduction in AML12 hepatocyte count by about 40% at all doses tested. [Figure 23] Figure 1 shows the effect of liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6) and its pemetrexed counterpart at 16 nM, 32 nM, 64 nM, and 128 nM on CCD841 colonic epithelial cells after 48 hours of exposure. At all concentrations tested, pemetrexed caused a reduction in CCD841 colonic epithelial cell numbers of about 50% or more, compared to a reduction in cell numbers of about 20% or less after treatment with liposomal gG6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] This application describes advances over conventional cancer treatments and methods for delivering antifolates to cancer cells. In particular, the present disclosure relates to a previously unrecognized antifolate system.

[0037] More specifically, the present application describes encapsulated polyglutamated antifolates, including, but not limited to, pemetrexed polyglutamate and lometrexol polyglutamate. In a non-limiting example, the encapsulated polyglutamated antifolate (e.g., pemetrexed polyglutamate) is targeted with a targeting moiety that has specific affinity for one or more of the folate receptors α, β, and δ. In another non-limiting example, the encapsulated polyglutamated antifolate is not targeted.

[0038] Folic acid is a water-soluble B vitamin. Its main role in the body / cell is as a cofactor for various methyltransferases involved in serine, methionine, thymidine and purine biosynthesis, a crucial process involved in cell division. Folic acid's main function is to mediate the transfer of one-carbon units involved in nucleotide biosynthesis, homocysteine ​​(Hcy) remethylation and biological methylation reactions. As an essential cofactor for de novo biosynthesis of nucleotides, folic acid plays a key role in DNA synthesis, stability and integrity, and repair. Folic acid also provides the main methyl group donor for DNA methylation (which regulates gene expression), protein methylation (an important post-translational modification), and lipid methylation (important in their synthesis, e.g. phosphatidylcholine). As described in more detail below, for this reason, antifolates have been developed as cancer treatments with the aim of blocking the action of folic acid, thereby inhibiting cell division, DNA / RNA synthesis and repair, and protein synthesis.

[0039] In nature, folic acid occurs in polyglutamic acid form in animal products and leafy vegetables. Polyglutamic acid is not suitable for physiological transport. Natural polyglutamic acid is degraded mainly to monoglutamic acid in the jejunum before physiological transport and uptake by cells. Dietary folic acid in polyglutamic acid form is then cleaved to monoglutamic acid in the jejunum where it is absorbed. Once absorbed by enterocytes, folic acid is either polyglutamated and retained intracellularly or released into the portal circulation for various compartments for metabolism, storage, or enterohepatic recirculation. Folic acid enters the plasma and is rapidly eliminated by entering hepatocytes and other cells.

[0040] The liver takes up most of the folate that enters the portal circulation, while the remaining folate passes through the liver, enters the portal circulation, and is taken up by other tissues where it is converted to its polyglutamate form for intracellular storage. The liver is the major storage site for folate. Surgical biliary drainage can result in a fall in serum folate within 6 hours, whereas dietary restriction does not produce a comparable fall for 3 weeks, probably because total body stores of folate are estimated to be 500-20,000 mcg. This observation indicates that there is a large enterohepatic circulation of folate.

[0041] Monoglutamate is the only circulating form of folate in the blood and the only form of folate transported across cell membranes. Once absorbed by enterocytes, folate is either polyglutamated and retained intracellularly or released into the portal circulation for various compartments for metabolism, storage, or enterohepatic recycling. Once monoglutamate is taken up into cells, intracellular folate exists primarily as polyglutamate, a form that acts biologically as a cofactor for various methyltransferases involved in serine, methionine, thymidine, and purine biosynthesis, a crucial process involved in cell division.

[0042] Polyglutamylation involves the addition of a glutamic acid group in a gamma linkage to the terminal carboxyl group of a vicinal folylglutamate by the enzyme folylpolyglutamate synthetase (FPGS), which uses ATP as its energy source.

[0043] Polyglutamation refers to the addition of glutamic acid residues to a molecule (such as an antifolate) such that the resulting molecule after polyglutamation has more than one glutamic acid residue. Several names are used in the literature to refer to glutamic acid residues, including glutamic acid, glutamyl group, and glutamyl radical. Each of the glutamic acid residues (glutamyl groups) can independently be in the L- or D-form.

[0044] For example, pemetrexed, whose chemical name is "N-[4-2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-l-glutamic acid, already has one glutamyl group (monoglutamylated). The addition of glutamic acid residues to pemetrexed as described herein results in polyglutamylated pemetrexed. For example, the addition of more than five glutamic acid residues to pemetrexed results in a total of six glutamic acid groups (one from pemetrexed and five additional glutamyl groups added), referred to herein as pemetrexed hexaglutamate or hexaglutamated pemetrexed. In the literature, this material has also been referred to as pemetrexed pentaglutamate or pentaglutamated pemetrexed.

[0045] Gamma polyglutamation refers to the addition of glutamic acid residues to a molecule as previously described, where a peptide bond is between the amino group of glutamic acid and the carboxyl group at the gamma carbon of the glutamic acid side chain. Alpha polyglutamation refers to the addition of glutamic acid residues to a molecule as previously described, where a peptide bond is between the amino group of glutamic acid and the carboxyl group at the alpha carbon of the glutamic acid side chain.

[0046] A single molecule may be formed by gamma polyglutamylation alone, alpha polyglutamylation alone, or a combination of gamma and alpha polyglutamylation.

[0047] Antifolates were developed over 70 years ago as "folate mimetic molecule" cytotoxic agents. The rationale was to design a molecular family that would exploit the physiological folate transport mechanism and their facilitatory intracellular mechanism of action on DNA replication during cell division to counter the effects of folate in rapidly replicating cells, such as cancer cells. Specifically, antifolates were designed to mimic folate in its systemic transport, physiological cellular uptake (e.g., via reduced folate carriers (RFCs) and proton-coupled folate transporters (PCFTs)) and intracellular processing. Antifolates specifically act during DNA and RNA synthesis and exert their cytotoxic effects during the S phase of the cell cycle. As a result, they have a high toxic effect on rapidly dividing cells, such as malignant and myeloid cells.

[0048] Generally, there are four types of transporters for folates and antifolates in the human body: reduced folate carrier (RFC), folate receptor (FR), proton-coupled folate transporter (PCFT), and ATP-binding cassette transporter. As mentioned above, none of these transport mechanisms is effective for transporting free polyglutamic acid across the cell membrane.

[0049] RFCs are saturable anion-dependent cell membrane carriers with high affinity for reduced folates and hydrophilic antifolates such as MTX or PMX, and a twofold higher affinity for PMX than for MTX. They are members of a superfamily of solute carriers and exploit high transmembrane anion gradients, particularly the organophosphate gradient, which is regulated by the energy state of the cell, to achieve the difficult transport of folate into cells.

[0050] The delivery of cytotoxic antifolates by RFC is not tumor-specific: because RFC is widely expressed in normal tissues and is highly active at neutral pH, characteristic of most normal tissues, antifolates delivered via RFC have been shown to cause toxicity in limited types of normal tissues.

[0051] Folate receptors α, β, and δ (FR-α, FR-β, and FR-δ) are high-affinity folate-binding proteins that are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor region. FRs have a particularly high affinity for FAs. They transport folates into cells via an endocytic mechanism. In the cytoplasm, upon acidification of vesicles to a pH of 6.0–6.5, folate is released from the receptor and exported from endosomes by a mechanism proposed to be partially mediated by PCFT. Although FR-α has a higher affinity for its preferred substrate than RFC, its folate transport into cells requires a series of steps including binding, invagination, vesicle formation and translocation, acidification, and export of the substrate from the vesicle into the cytoplasm. Due to these multiple steps required for transport into cells, the rate of FR-mediated folate transport is 1% of that mediated by RFC.

[0052] Proton-coupled folate transporters (PCFTs) are folate-H+ symporters that function most efficiently in acidic extracellular environments, which are characteristic of hypoxic environments in particular. A critical difference between RFC and PCFT is their optimum pH, which affects their affinity for transport substrates. At pH 7.4, RFC activity is optimal and PCFT activity is minimized, so that antifolate transport is primarily mediated by RFC. Alternatively, PCFT activity is more prominent when the pH is lowered. In addition to mediating intestinal folate absorption and uptake into tissues with acidic extracellular environments, such as solid tumors, or, less efficiently, transport into tissues in neutral pH environments, PCFT may play a role in folate receptor-mediated endocytosis.

[0053] Several ATP-binding cassette transporters are low affinity, high capacity ATP-dependent efflux pumps for folates and antifolates. These include the multidrug resistance associated proteins MRP1-MRP5 and the breast cancer resistance protein BCRP. All of these drug resistance efflux pumps have been shown to be involved in pumping antifolates out of cells as well as other classes of drugs.

[0054] Intracellular folates are converted to polyglutamates by FPGS, while GGH removes the terminal glutamate, thereby facilitating the export of folates out of the cell and back into the extracellular circulation by ATP-binding cassette transporters, often referred to as folate efflux pumps. Without wishing to be bound by theory, it is believed that polyglutamylated folates are better retained in cells because they are poor substrates for ATP-binding cassette transporters. Furthermore, without wishing to be bound by theory, it is believed that polyglutamylated folates are better substrates for intracellular folate-dependent enzymes compared to monoglutamates.

[0055] As in the case of folate, antifolates such as MTX, PMX and RTX are believed to be retained in tumor and normal cells by FPGS-induced polyglutamation, which is exported from the cell after hydrolysis to monoglutamate by GGH. As with physiological folates, polyglutamated antifolates are retained longer in the cell, thereby increasing their cytotoxicity by extending the period of exposure. Polyglutamated antifolates generally have a higher affinity for, and consequently inhibit, their target folate-dependent enzymes in thymidylate and purine biosynthesis to a greater extent than the monoglutamate forms. Thus, FPGS and GGH are believed to be important enzymes for maintaining intracellular homeostasis of folate and antifolates for optimal folate-dependent one-carbon transfer reactions and antifolate-induced cytotoxic effects.

[0056] Once inside the cell, folates and antifolates (such as, but not limited to, pemetrexed and pralatrexate) are polyglutamylated to polyglutamates by FPGS. This process is necessary for biological activity. For example, polyglutamylation promotes retention (increasing intracellular concentration) and increases affinity for folate-dependent enzymes (including thymidylate synthase). The ability of cells to generate polyglutamates also enhances the cytotoxic effects of antifolates, making them highly effective cytotoxic agents.

[0057] The applicant set out to study the cytotoxic effects of polyglutamylated forms of antifolates to evaluate their effectiveness in treating cancerous cells. Because polyglutamylated forms of antifolates are not taken up by either normal or cancer cells (as described in more detail below), the applicant hypothesized that direct delivery of such polyglutamylated forms to cancer cells could ameliorate the toxicity of antifolates by reducing undesirable toxicity to normal cells that may result from the excretion of excess antifolates in cancer cells.

[0058] In particular, the excess amount of antifolate present in cancer cells can be returned to the circulation via the high capacity ATP-dependent efflux pump of folic acid and antifolate.This is true even when antifolate is delivered using the targeted liposome method reported in WO2016 / 25882, the entire contents of which are incorporated herein by reference in their entirety.In other words, the technology of WO2016 / 25882 can deliver antifolate to cancer cells while mostly avoiding normal cells, but the excess antifolate delivered can flow back to the circulation and be taken up by normal cells, resulting in undesirable toxicity.Although these toxicities are significantly reduced compared to the toxicity associated with the conventional administration of free antifolate in the patient's circulation, they are still undesirable.The antifolate and delivery system described herein can further reduce the significantly reduced but toxic effect on normal cells achieved by WO2016 / 25882, resulting in even greater reduction in toxicity to normal cells.

[0059] The present inventors have realized the potential benefits of using polyglutamylated antifolates for cancer treatment. For example, polyglutamates, especially pentaglutamate, can allow cells to retain their folate pool more efficiently. However, due to their high negative charge and other properties, pentaglutamate does not currently have a known substrate for transport across cell membranes.

[0060] This disclosure describes an advanced and improved technology to deliver various classes of antifolates (e.g., polyglutamic acid) to cancer cells that could not be taken up by cancer cells in the past. Due to lack of transport across cell membranes, polyglutamic acid delivered to cancer cells is effectively retained within the cancer cells. This reduces and / or eliminates the toxicity associated with non-polyglutamic acid antifolates, such as monoglutamic acid antifolates, because polyglutamic acid is not returned to the circulation. In fact, even if they were returned, they would not be taken up by healthy cells.

[0061] The therapeutic benefits afforded by polyglutamated antifolate compositions and their use are numerous and surprising.

[0062] First, the active form of many cytotoxic agents, especially antifolates, is polyglutamylated, and therefore delivery of a conventionally undeliverable polyglutamylated form of an antifolate means that the agent is immediately active. Furthermore, some solid tumors are known to have low or no FPGS activity and polyglutamylation. This means that tumors with low FPGS activity are more resistant to chemotherapy mediated by non-polyglutamylated agents, because the agent still relies on low endogenous FPGS activity for activation after it is taken up into the cell.

[0063] Secondly, retention of drugs in their non-polyglutamated state is also problematic. Part of the problem in treating tumors, especially those with low endogenous FPGS activity, is at least twofold. In the previous paragraph, we discuss the problem of low activity of drugs due to low levels of FPGS. In addition to this drawback, non-polyglutamated drugs, such as non-polyglutamated pemetrexed, have limited retention in cells because they can be actively transported out of the cell. Transport of non-polyglutamated drugs (e.g., non-polyglutamated pemetrexed) is a two-fold problem in itself. First, if a non-polyglutamated drug is transported out of the cell, it can no longer act in the cancer cell for treatment. Also, cytotoxic drugs, such as antifolates, located outside the cell can re-enter other cells, including normal cells, causing undesirable toxicity to normal cells. Upon entry into the cell, certain cellular processes may function to reduce the polyglutamated antifolates presented herein to monoglutamates. However, recent studies suggest that the toxicity and activity of the provided polyglutamylated antifolates may kill cells before such reduction can occur.

[0064] Third, because polyglutamylated antifolates are retained intracellularly and not transported out of cells, administration of polyglutamylated antifolates (e.g., intravenous administration) can be performed for only a limited period of time, thus further reducing the possibility of undesired toxicity to normal tissues. The shortened administration period also improves patient comfort and reduces costs, since the administration period in clinical practice can be relatively reduced. For example, retention of polyglutamylated drugs in tumor cells means that tumor cells are unlikely to recover from treatment, even after the drug is removed from the extracellular environment (e.g., intravenous administration is stopped).

[0065] Fourth, the activity of the disclosed polyglutamated antifolates, such as targeted and non-targeted pegylated liposomal polyglutamated antifolates, is not diminished in tumors, even when the tumors are FPGS-deficient, because they do not rely on FPGS to activate the agents.

[0066] For all the reasons stated above, liposomal polyglutamated antifolates have a higher therapeutic index than non-polyglutamated antifolates delivered in the same manner, i.e., we expect the drugs to have minimized / reduced toxicity and patients will be able to tolerate higher doses of the drug with fewer side effects, an effect not present with non-polyglutamated administered drugs.

[0067] A non-exhaustive list of polyglutamated antifolates encompassed by the compositions and methods of the present disclosure includes polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887.

[0068] In some embodiments, the pentaglutamated antioxidant is a member selected from the group consisting of pentaglutamated methotrexate (MTX), pentaglutamated pemetrexed (PMX), pentaglutamated lometrexol (LTX), pentaglutamated AG2034, pentaglutamated raltitrexed (RTX), pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887.

[0069] In some embodiments, the polyglutamated antifolate composition (e.g., polyglutamic acid and delivery vehicle such as liposomes that contain polyglutamic acid) components are "isolated". As used herein, the term isolated refers to a composition that is in a form not found in nature. Isolated polyglutamated antifolate compositions include those that have been purified to the extent that they are no longer in a form found in nature. In some embodiments, the polyglutamated antifolate composition components that are isolated are substantially pure. Isolated compositions are free or substantially free of materials with which they are naturally associated, such as other intracellular components such as proteins and nucleic acids that may be found in nature or in the environment in which they are prepared (e.g., cell culture). The polyglutamated antifolate compositions may be formulated with a diluent or adjuvant, and may even be isolated for practical purposes, for example, when the polyglutamated antifolate compositions are used in diagnosis or therapy, they are typically mixed with a pharma- ceutically acceptable carrier or diluent. In some embodiments, the isolated polyglutamated antifolate composition (eg, polyglutamic acid and a delivery vehicle such as a liposome that includes polyglutamic acid) contains less than 1% or less than 0.1% DNA or protein.

[0070] In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated antifolates in the provided liposomal compositions are pentaglutamated. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated antifolates in the provided liposomal compositions are pentaglutamated. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated antifolates in the provided liposomal compositions are pentaglutamated. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated antifolates in the provided liposomal compositions are hexaglutamated. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated antifolates in the liposomal compositions are hexaglutamated. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated antifolates in the liposomal compositions have 5-10 or ≧4 glutamyl groups. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated antifolates in the liposome composition have 5-10 or ≧4 glutamyl groups.

[0071] In some embodiments, the alpha (L-alpha or D-alpha) or D-gamma polyglutamated antifolate composition (e.g., polyglutamic acid and delivery vehicle such as liposomes containing polyglutamic acid) is in an aqueous solution. In some embodiments, the polyglutamated antifolate is at a concentration of 100 ng / mL to 700 mg / mL. In some embodiments, the polyglutamated antifolate is at a concentration of greater than 100 ng / mL, 250 ng / mL, 500 ng / mL, 750 ng / mL, 1 ug / mL, 100 ug / mL, 250 ug / mL, 500 ug / mL, 750 ug / mL, 1 mg / mL, 100 mg / mL, 250 mg / mL, or 500 mg / mL. In some embodiments, the concentration of the liposomes containing the polyglutamated antifolate is at a concentration of 100 ng / mL to 1 mg / mL. In some embodiments, the concentration is greater than 100 ng / mL, 250 ng / mL, 500 ng / mL, 750 ng / mL, 1 ug / mL, 100 ug / mL, 250 ug / mL, 500 ug / mL, 750 ug / mL, or 1 mg / mL.

[0072] In some embodiments, the hexaglutamated antioxidant is a member selected from the group consisting of hexaglutamated methotrexate (MTX), hexaglutamated pemetrexed (PMX), hexaglutamated lometrexol (LTX), hexaglutamated AG2034, hexaglutamated raltitrexed (RTX), hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887.

[0073] Pemetrexed, for example, is a multi-targeted antifolate that inhibits at least three enzymes involved in folate metabolism and purine and pyrimidine synthesis. These enzymes are thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) (see Figure 2). Pemetrexed is FDA approved and currently remains an important treatment for mesothelioma and non-small cell lung cancer.

[0074] In the treatment of mesothelioma and non-small cell lung cancer, pemetrexed has been approved in combination with cisplatin for the treatment of malignant mesothelioma and for the initial and maintenance treatment of patients with non-small cell lung cancer. In the second-line therapy of non-small cell lung cancer, pemetrexed has shown efficacy comparable to docetaxel, but with significantly lower toxicity. The most common and serious toxicity of pemetrexed - myelosuppression and mucositis - has been significantly improved by folic acid and vitamin B12 supplementation, but still has undesirable toxicity. In some specific embodiments, the polyglutamated antifolate is hexaglutamated pemetrexed (PMX). In further embodiments, the polyglutamated antifolate is pentaglutamated PMX. In further embodiments, the polyglutamated antifolate is hexaglutamated PMX.

[0075] These toxicities may be reduced, minimized and / or eliminated using the polyglutamated antifolates described herein. In some specific embodiments described herein, the polyglutamated antifolate is polyglutamated pemetrexed (PMX). In further embodiments, the polyglutamated antifolate is pentaglutamated PMX. In further embodiments, the polyglutamated antifolate is hexaglutamated PMX.

[0076] In some embodiments, the polyglutamated antifolate is polyglutamated raltitrixed (RTX). In further embodiments, the polyglutamated antifolate is pentaglutamated RTX. In further embodiments, the polyglutamated antifolate is hexaglutamated RTX.

[0077] Lometrexol was the first potent antifolate that inhibits purine synthesis by directly inhibiting its glycinamide ribonucleotide transferase (GARFT) activity. In a particular embodiment, the polyglutamated antifolate is polyglutamated lometrexol (LTX). In a further embodiment, the polyglutamated antifolate is pentaglutamated LTX. In a further embodiment, the polyglutamated antifolate is hexaglutamated LTX.

[0078] As discussed herein, the use of antifolates such as pemetrexed in clinical practice has led to significant clinical dilemmas. Rapidly regenerating normal tissue cells such as bone marrow, GI tract and oral mucosa cells have been shown to be sensitive to the cytotoxic effects of this drug class because they also replicate their DNA more frequently. As a result, in clinical practice, the use of antifolates has often led to severe and life-threatening hematological and non-hematological toxic side effects. Moreover, the toxicity was considered to be dose-limiting on the one hand and to hinder the ability to achieve good efficacy on the other. Because of their associated severe and life-threatening toxicity, promising antifolates have either failed during development, e.g. lometrexol, or have limited use in clinical practice, e.g. raltitrexed (TOMUDEX®) and pemetrexed (ALIMTA®).

[0079] The elements described herein are also at the core of resistance mechanisms to antifolates in general, the antitumor activity of which is enhanced by polyglutamation, such as methotrexate, pemetrexed, lometrexol, and raltitrexed.

[0080] The disclosed polyglutamated antifolates provide a strategy to overcome the pharmacological challenges associated with dose-limiting toxicity of antifolates. The methods provided deliver conventionally undeliverable (due to lack of transport mechanisms) polyglutamate forms of antifolate payload to tumor cells while (1) minimizing / reducing exposure to normal tissue cells, (2) optimizing / improving the cytotoxic effect of antifolates on cancer cells, and (3) minimizing / reducing the effects of efflux pumps.

[0081] The polyglutamylated chemicals provided herein achieve many benefits over comparable antifolates that are not delivered to cells in polyglutamylated form. For example, polyglutamylated antifolates are administered as a bolus that avoids the FPGS-induced polyglutamylated activity that is required for antifolates such as PMX, MTX, RTX, and LTX to have increased activity (via increased binding affinity to folate-dependent enzymes) and cellular retention. As reflected in the figures, disclosures, and examples herein, the retention of highly polyglutamic acids (e.g., agents containing 4, 5, or more than 5 glutamic acid groups) in cells is a direct function of their chain length. Thus, a significant portion of unbound MTX-Glu4 and most of MTX-Glu5 remain in cells for at least 24 hours after removal of extracellular agents, and continue to exert inhibitory effects on DHFR, DNA synthesis, and cell viability.

[0082] Novel chemical entities can be designed to increase and / or maximize intratumor concentrations of polyglutamylated antifolates by closely exploiting the molecular biology of cells and the folate pathway. Specific targeted forms of novel chemical entities can exploit the distinct cellular polarity between tumor and normal cells, where cancer-specific morphology has not been recognized as useful for the goal of mitigating clinical and pharmacological challenges associated with the use of antifolates for cancer therapy. Additionally, novel chemical entities minimize tumor cell resistance to therapy mediated by cellular efflux pumps.

[0083] By way of example, and without limitation, the targeted liposomal polyglutamated antifolate composition may comprise a PEGylated liposome comprising an entrapped and / or encapsulated polyglutamated antifolate; and a targeting moiety comprising an amino acid chain (e.g., an antibody or an antibody fragment), the amino acid chain comprising a plurality of amino acids, the targeting moiety having a specific affinity for at least one type of folate receptor, the targeting moiety being attached to one or both of the PEG and the exterior of the liposome. In an exemplary embodiment, the liposome-encapsulated polyglutamated antifolate (LPA) may be a pentaglutamated form of pemetrexed or any suitable pentaglutamated form of an antifolate. In an exemplary embodiment, the liposome-encapsulated polyglutamated antifolate (LPA) may be a hexaglutamated form of pemetrexed or any suitable hexaglutamated form of an antifolate.

[0084] By way of example, and without limitation, the non-targeted liposomal polyglutamated antifolate composition may comprise a PEGylated liposome comprising an entrapped and / or encapsulated polyglutamated antifolate. In an exemplary embodiment, the liposome-encapsulated polyglutamated antifolate (LPA) may be a pentaglutamated form of pemetrexed or any suitable pentaglutamated form of an antifolate. In an exemplary embodiment, the liposome-encapsulated polyglutamated antifolate (LPA) may be a hexaglutamated form of pemetrexed or any suitable hexaglutamated form of an antifolate.

[0085] In some embodiments, the targeted liposomal, optionally PEGylated, polyglutamated antifolate composition comprises a PEGylated liposome comprising an entrapped and / or encapsulated polyglutamated antifolate; and a targeting moiety comprising an amino acid chain (e.g., an antibody or antibody fragment), the amino acid chain comprising a plurality of amino acids, the targeting moiety having a specific affinity for at least one type of folate receptor. The specific affinity can be, for example, but not limited to, 0.5×10 to at least one type of folate receptor. -10 ~10×10 -6The equilibrium dissociation constant (Kd) can be determined to be in the molar [0.05 nanomolar to 10 micromolar] range, and the targeting moiety is attached to either or both of the PEG and the exterior of the liposome.

[0086] In some embodiments, the targeted liposome composition, optionally PEGylated, comprises a PEGylated liposome comprising an entrapped and / or encapsulated polyglutamated antifolate (LPA) and a targeting moiety comprising an amino acid chain (e.g., an antibody or antibody fragment), the amino acid chain comprising a plurality of amino acids, the targeting moiety having a specific affinity for at least one type of folate receptor. In some embodiments, the specific affinity of the targeting moiety is greater than or equal to 0.5×10 for at least one type of folate receptor. -10 ~10×10 -6 Equilibrium dissociation constants (Kd) in the molar range. In further embodiments, the targeting moiety is attached to one or both of the PEG and the exterior of the liposome.

[0087] There are at least three main limitations to the use of free cytotoxic agents, such as antifolates, that require polyglutamation for enhanced activity. The new chemical entities discussed and disclosed herein are designed to address these fundamental limitations. These limitations include: The first limitation is toxicity to normal tissue cells, which is due to cellular uptake of the monoglutamate form, e.g., via RFC or PCFT, and its subsequent intracellular conversion by FPGS to a gamma polyglutamated form that has enhanced cytotoxicity with prolonged cellular retention in normal tissue cells. The second limitation is the delivery of insufficient amounts of most active polyglutamated forms of the agent, due to the inability of such compounds to cross the cell membrane. Instead, their intracellular availability depends on the ability of the cell to polyglutamate the monoglutamate form of the agent that is transported from the extracellular environment. The third limitation is the retention of adequate amounts of the most cytotoxic forms, such as the pentaglutamated and hexaglutamated forms of antifolates, within tumor cells. This is due to the ability of cancer cells to downregulate FPGS resulting in decreased polyglutamylation, and in some cases, to increase the degradation of polyglutamylated forms by GGH and the upregulation of efflux pumps (ATP cassettes) capable of exporting mono- and other less highly glutamylated forms of (1-, 2-, 3-) glutamate from cells, but not the more highly (e.g., pentaglutamylated and hexaglutamylated) forms of folates and their analogues.

[0088] The polyglutamylated antifolate new chemical entities discussed and disclosed herein do not have the aforementioned drawbacks and limitations and are designed to address these and other limitations.Indeed, the new chemical entities reduce / minimize the pharmacological challenges associated with systemic transport, cellular uptake and intratumoral accumulation of active drugs.The novel chemical entities of the present disclosure preferentially target tumor cells for exposure to pentaglutamylated and hexaglutamylated antifolates and / or minimize exposure of such antifolate cocktails to normal cells, particularly high turnover (e.g., rapid replicating) normal cells in the epithelial lining of bone marrow and gastrointestinal tract.

[0089] In some embodiments, the disclosure provides a composition comprising a polyglutamated antifolate. In further embodiments, the composition comprises a pentaglutamated antifolate. In further embodiments, the composition comprises a hexaglutamated antifolate. According to some embodiments, the polyglutamated antifolate is a member selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In further embodiments, the polyglutamated member is pentaglutamated. In a further embodiment, the polyglutamylated member is hexaglutamylated.

[0090] In one embodiment, the composition comprises polyglutamylated PMX, MTX, RTX, and / or LTX. In a further embodiment, the composition comprises pentaglutamylated PMX, MTX, RTX, and / or LTX. In a further embodiment, the composition comprises hexaglutamylated PMX, MTX, RTX, and / or LTX.

[0091] In one embodiment the composition comprises liglutamylated PMX. In a further embodiment the composition comprises pentaglutamylated PMX. In a further embodiment the composition comprises hexaglutamylated PMX.

[0092] In another embodiment, the composition comprises polyglutamylated MTX. In a further embodiment, the composition comprises pentaglutamylated MTX. In a further embodiment, the composition comprises hexaglutamylated MTX.

[0093] In another embodiment, the composition comprises polyglutamylated RTX. In a further embodiment, the composition comprises pentaglutamylated RTX. In a further embodiment, the composition comprises hexaglutamylated RTX.

[0094] In additional embodiments, the composition comprises polyglutamylated LTX. In further embodiments, the composition comprises pentaglutamylated LTX. In further embodiments, the composition comprises hexaglutamylated LTX.

[0095] In additional embodiments, the disclosure provides liposomal polyglutamated antifolate compositions comprising a polyglutamated antifolate. In some embodiments, the liposomes are optionally PEGylated (PLPA). In some embodiments, the PLPA compositions comprise a pentaglutamated antifolate. In some embodiments, the PLPA compositions comprise a hexaglutamated antifolate. In some embodiments, the PLPA liposomes are anionic or neutral. In other embodiments, the PLPA liposomes are cationic. In some embodiments, at least 10% of the liposome-entrapped PLPA compositions comprise a polyglutamated antifolate. In some embodiments, the PLPA liposomes have a diameter in the range of 20-200 nm. In further embodiments, the liposomes have a diameter in the range of 80-120 nm.

[0096] According to some embodiments, the LPA comprises a polyglutamated antifolate selected from the group consisting of polyglutamated MTX, polyglutamated PMX, polyglutamated LTX, polyglutamated AG2034, polyglutamated RTX, polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887.In further embodiments, the polyglutamated antifolate is pentaglutamated.In further embodiments, the polyglutamated antifolate is hexaglutamated.

[0097] In one embodiment, the LPA comprises polyglutamylated PMX, MTX, RTX, and / or LTX. In a further embodiment, the LPA comprises pentaglutamylated PMX, MTX, RTX, and / or LTX. In a further embodiment, the LPA comprises hexaglutamylated PMX, MTX, RTX, and / or LTX.

[0098] In one embodiment, the LPA comprises polyglutamylated PMX. In a further embodiment, the LPA comprises pentaglutamylated PMX. In a further embodiment, the LPA comprises hexaglutamylated PMX.

[0099] In another embodiment, the LPA comprises polyglutamylated MTX. In a further embodiment, the LPA comprises pentaglutamylated MTX. In a further embodiment, the LPA comprises hexaglutamylated MTX.

[0100] In another embodiment, the LPA comprises polyglutamylated RTX. In a further embodiment, the LPA comprises pentaglutamylated RTX. In a further embodiment, the LPA comprises hexaglutamylated RTX.

[0101] In additional embodiments, the LPA comprises polyglutamylated LTX. In further embodiments, the LPA comprises pentaglutamylated LTX. In further embodiments, the LPA comprises hexaglutamylated LTX.

[0102] Liposomes In some embodiments, targeted PEGylated liposomal polyglutamated antifolate is provided.In some embodiments, targeted PEGylated liposomal polyglutamated antifolate can comprise: a liposome comprising an internal space; a polyglutamated antifolate disposed in the internal space; a PEG molecule attached to the outside of the liposome; and a targeting moiety comprising a protein (e.g., an antibody or an antibody fragment) having specific affinity for at least one antigen (e.g., folate receptor (e.g., FR-α, FR-β, and / or FR-δ)) expressed on the surface of cancer cell, and the targeting moiety is attached to at least one of the PEG and the outside of the liposome.

[0103] In other embodiments, non-targeted liposomal polyglutamated antifolates are provided.

[0104] The liposomes included in the targeted or non-targeted liposome composition may be any liposome known in the art. However, it will be understood by those skilled in the art that liposomal encapsulation of any particular agent, such as, but not limited to, the polyglutamated antifolates described herein, may involve the most routine experimentation to achieve a useful and functional liposomal formulation. In general, the liposomes provided may have any liposomal structure, such as a structure having an interior space separated from the external medium by one or more lipid biphases, or any microcapsule having a semipermeable membrane with a lipophilic core that separates the membrane from the interior. The lipid biphase may be any arrangement of amphiphilic molecules characterized by a hydrophilic portion (hydrophilic part) and a hydrophobic portion (hydrophobic part). Usually, the amphiphilic molecules in the bilayer are arranged in a two-dimensional sheet with the hydrophobic portion facing the inside of the sheet, while the hydrophilic portion faces the outside. The amphiphilic molecules forming the provided liposomes may be any known or later discovered amphiphilic molecules, such as synthetic or naturally occurring lipids or biocompatible lipids. Liposomes may also be formed by amphiphilic polymers and surfactants, such as polymersomes and niosomes. For purposes of this disclosure, but without limitation, these liposome-forming materials are also referred to as "lipids."

[0105] The liposome composition formulations provided herein may be in liquid or dry formulation form, such as dry powder or dry solid. The dry powder or dry solid may be subjected to initial drying, e.g., under lyophilization conditions, or optionally, both the dry solid or dry powder may be subjected to initial drying only, or both initial drying and secondary drying. In dry formulation form, the powder or solid may have, e.g., 1-6% moisture, e.g., 2-5% moisture, or 2-4% moisture, etc. One exemplary method of drying is lyophilization (also called freeze-drying, or cryodesication). Any of the compositions and methods of the present disclosure may 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 generally polyhydroxy compounds, such as sugars (mono-, di-, and polysaccharides), polyalcohols and their derivatives, glycerol, or polyethylene glycols, trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol, or aminoglycosides, etc. In further embodiments, the lyoprotectant or cryoprotectant comprises up to 10% or up to 20% of the solution outside the liposomes, inside the liposomes, or both outside and inside the liposomes.

[0106] In some embodiments, the liposomes contain steric stabilizers that increase their longevity in the circulation. One or more steric stabilizers, such as hydrophilic polymers (polyethylene glycol (PEG)), glycolipids (monosialoganglioside (GM1)), or others, occupy the space immediately adjacent to the liposome surface and exclude other macromolecules from this space. As a result, access and binding of plasma opsonins to the liposome surface is hindered, thus inhibiting macrophage interaction with such liposomes, or any other exclusion mechanism, and enhancing the longevity of the liposomes in the circulation. In some embodiments, the steric stabilizer or ensemble of 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 200-5000 Daltons. These PEGs can be of any structure, such as linear, branched, star-shaped, or comb-shaped structures, and are commercially available.

[0107] 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 to 150 nm (nanometers). In other embodiments, the liposomes have a diameter in the range of 40 to 70 nm.

[0108] The properties of liposomes are influenced by the nature of the lipids used to make the liposomes. A wide variety of lipids have been used to make liposomes. These include cationic, anionic, and neutral lipids. In some embodiments, the liposomes containing polyglutamated antifolates are anionic or neutral. In other embodiments, the liposomes provided are cationic. The measurement of charge (e.g., anionic, neutral, or cationic) can generally be measured 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 equal to or less than zero. In some embodiments, the zeta potential of the liposomes is in the range of 0 to -150 mV. In another embodiment, the zeta potential of the liposomes is in the range of -30 to -50 mV.

[0109] In some embodiments, cationic lipids are used to make cationic liposomes, which are commonly used as gene transfection agents.The positive charge on cationic liposomes can interact with the negative charge on cell surface.After cationic liposomes are bound to cells, liposomes are transported to the inside of cells by endocytosis.

[0110] In some embodiments, neutral to anionic liposomes are used. In one embodiment, anionic liposomes are used. For example, using a mixture of neutral lipids, such as HSPC, and anionic lipids, such as PEG-DSPE, results in the formation of anionic liposomes that are less likely to bind nonspecifically to normal cells. Specific binding to tumor cells can be achieved by using tumor targeting antibodies, such as, for example, folate receptor antibodies, including, for example, folate receptor alpha antibodies, folate receptor beta antibodies, and / or folate receptor delta antibodies.

[0111] By way of example, at least one type (or some) of the lipids are amphipathic lipids, defined as having hydrophilic and hydrophobic portions (generally a hydrophilic head and a hydrophobic tail). The hydrophobic portion generally faces into the hydrophobic phase (e.g., within the bilayer), while the hydrophilic portion generally faces into the aqueous phase (e.g., outside the bilayer). The hydrophilic portion may include polar or charged groups, such as carbohydrates, phosphates, carboxyl groups, sulfate groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other groups. The hydrophobic portion may include non-polar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and groups substituted by one or more aromatic, cyclo-aliphatic, or heterocyclic groups. Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0112] Typically, for example, the lipid is a phospholipid. Phospholipids include, but are 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 be used.

[0113] The lipids comprising the liposomes provided herein may 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 pH. At physiological pH, such lipids include, for example, dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, epharin, 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, palmitoyloleylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0114] In summary, anionic and neutral lipids are referred to herein as non-cationic lipids. Such lipids may contain specific phosphorus, but they are not so limited. Examples of non-cationic lipids include lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmito ... 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, palmitoyloleoyl-phosphatidylethanolamine (POPE), l-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, and cholesterol.

[0115] Liposomes can be assembled using any liposome assembly method using liposome components (also referred to as liposome ingredients) known in the art. Liposome components include lipids such as, for example, 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 neutral suitable for making anionic liposomes may 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-maleimide PEG-2000·Na.

[0116] The lipid derivatives, for example, at least include the attachment (preferably covalent attachment) of one or more steric stabilizers and / or functional groups to the liposome component, after which the steric stabilizers and / or functional groups are considered to be part of the liposome component. The functional groups include groups 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 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), phosphatidyl polyglycerol, poly[N-(2-hydroxypropyl)methacrylamide], amphiphilic poly-N-vinylpyrrolidone, L-amino acid-based polymers, and polyvinyl alcohol.

[0117] As the liposomal components may include any molecule (i.e. chemical / reagent / protein) that is attached thereto, in some embodiments, the liposomal 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 liposomal components provided include DSPE, DSPE-PEG, DSPE-maleimide, HSPC, HSPC-PEG, HSPC-maleimide, cholesterol, cholesterol-PEG, and cholesterol-maleimide. In exemplary embodiments, the liposomal components that make up the liposomes include DSPE, DSPE-FITC, DSPE-maleimide, cholesterol, and / or HSPC.

[0118] 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 cross-link reagents and moieties to the lipid. When a lipid is functionalized, any liposomes it forms are also functionalized. In some embodiments, the reactive group is a group that reacts with a cross-linking agent (or other moiety) to form a cross-link. The reactive group in the liposomal lipid bilayer is located anywhere on the lipid that allows it to come into contact with a cross-linking agent and be cross-linked to another moiety (e.g., a steric stabilizer or targeting moiety). In some embodiments, the reactive group is on the head group of the lipid, including, for example, a phospholipid. In some embodiments, the reactive group is a maleimide group. The maleimide groups can be cross-linked to each other in the presence of a dithiol cross-linking agent, including, but not limited to, dithiolthreitol (DTT).

[0119] It should be understood that the use of other functionalized lipids, other reactive groups, and other cross-linking agents beyond those mentioned above is also contemplated. In addition to maleimide groups, other contemplated examples include, but are not limited to, other thiol reactive groups, amino groups such as primary and secondary amines, carboxyl groups, hydroxyl groups, aldehyde groups, alkyne groups, azide groups, carbonyls, alloacetyl (e.g., iodoacetyl) groups, imidoester groups, N-hydroxysuccinimide esters, sulfhydryl groups, and pyridyl disulfide groups.

[0120] Functionalized and non-functionalized lipids are available from a number of commercial sources, including Avanti Polar 5 Lipids (Alabaster, Alabama).

[0121] In some embodiments, the provided liposomes further comprise an immunostimulatory agent, a detectable marker, or both disposed on the exterior of the liposome. The immunostimulatory agent or detectable marker can be ionically or covalently bound to the exterior of the liposome, for example, optionally to a steric stabilizer of the liposome.

[0122] The term immunostimulant, also known as immunopotentiator and immunostimulant, refers to a substance that stimulates the immune system by inducing the activation or increasing the activity of any of its components. These immunostimulants may include one or more of the following: heptenes, adjuvants, protein immunostimulants, nucleic acid immunostimulants, and chemical immunostimulants. Many adjuvants include substances designed to stimulate the immune response, such as lipid A, Bordetella pertussis or Mycobacterium tuberculosis derived proteins. Certain adjuvants are commercially available, e.g., 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, calcium, iron, or zinc salts, insoluble suspensions of acylated tyrosine, acylated sugars, cationic or anionic derivatized polysaccharides, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A, and quil A. Cytokines such as GM-CSF, interleukin-2, -7, -12, and other growth factors can also be used as adjuvants. In an exemplary embodiment, the immunostimulant may be at least one selected from the group consisting of fluorescein, DNP, β-glucan, β-1,3-glucan, β-1,6-glucan.

[0123] Detectable markers are detectable by any suitable means known in the art, such as magnetic resonance imaging (MRI), optical imaging, fluorescence / luminescence imaging, or nuclear imaging techniques, and may include, for example, at least, 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 dye.

[0124] In some embodiments, the immunostimulatory agent and / or detectable marker is bound to the exterior by co-incubating it with liposome. For example, the immunostimulatory agent and / or detectable marker can be bound to the liposome membrane by hydrophobic interaction or ionic bond such as avidin / biotin bond or metal chelation bond (e.g., Ni-NTA). Alternatively, the immunostimulatory agent or detectable marker can be covalently bound to the exterior of the liposome, for example, by covalently binding to a liposome component or to a steric stabilizer, such as PEG.

[0125] One exemplary reagent is fluorescein isothiocyanate (FITC), which, based on experiments, can act as both an immunostimulant and a detectable marker.

[0126] In further non-limiting embodiments, the liposomes provided enclose an interior space. In some embodiments, the interior space includes, but is not limited to, an aqueous solution. In some embodiments, the interior space includes a polyglutamated antifolate agent provided herein. In some embodiments, the interior space further includes a pharma- ceutically acceptable carrier, such as trehalose. In additional embodiments, the trehalose is present at about 5-20% by weight trehalose, or at a total concentration of 5-20% of one or more lyoprotectants or any combination of lyoprotectants. In some embodiments, the interior space includes a buffering agent. In further embodiments, the buffering agent is a HEPES buffer or a citrate buffer. In still further embodiments, the citrate buffer is at a concentration of 5-200 mM. In some embodiments, the interior space has a pH of 2.8-6. In additional embodiments, the interior space of the liposome includes sodium acetate and / or calcium acetate. In some embodiments, the interior space of the liposome includes a total concentration of sodium acetate and calcium acetate of 50 mM to 500 mM.

[0127] In some embodiments, the targeted PEGylated liposomal polyglutamylated (e.g., pentaglutamylated or hexaglutamylated) antifolate comprises a liposome comprising an interior space; an aqueous polyglutamylated antifolate disposed within the interior space; and a medium containing a targeting moiety comprising a protein having specific affinity for at least one folate receptor, where the targeting moiety is disposed outside the liposome. In some embodiments, the medium is an aqueous solution. In some embodiments, the interior space, the exterior space (e.g., the medium), or both the interior space and the medium comprise one or more lyoprotectants or cryoprotectants as described above. In some embodiments, the cryoprotectant is mannitol, trehalose, sorbitol, and / or sucrose.

[0128] In some embodiments, the non-targeted liposomal polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate comprises a liposome comprising an interior space; and a medium containing an aqueous polyglutamated antifolate disposed within the interior space. In some embodiments, the medium is an aqueous solution. In some embodiments, the interior space, the exterior space (e.g., the medium), or both the interior space and the medium comprise one or more lyoprotectants or cryoprotectants as described above. In some embodiments, the cryoprotectant is mannitol, trehalose, sorbitol, and / or sucrose.

[0129] As previously mentioned, liposomes may include steric stabilizers that can extend their lifetime in circulation. In 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), phosphatidyl polyglycerol, poly[N-(2-hydroxypropyl)methacrylamide], amphiphilic poly-N-vinylpyrrolidone, L-amino acid based polymers, and polyvinyl alcohol. In some embodiments, the steric stabilizer or population of steric stabilizers is PEG. In one embodiment, the steric stabilizer is PEG. In a further embodiment, the PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons. These PEGs can be of any configuration, such as linear, branched, star or comb configurations, and are commercially available.

[0130] In some embodiments, the liposomal polyglutamated antifolate (LPA or PLPA) is water soluble. That is, the liposomal polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate is in the form of an aqueous solution. In some embodiments, the LPA or PLPA comprises an interior space that contains less than 200,000 molecules of the polyglutamated antifolate. In some embodiments, the LPA or PLPA comprises 10,000 to 100,000 molecules of the polyglutamated antifolate. In further embodiments, the LPA or PLPA comprises 10,000 to 100,000 molecules of the pentaglutamated antifolate. In further embodiments, the LPA or PLPA comprises 10,000 to 100,000 molecules of the pentaglutamated antifolate.

[0131] In some embodiments, the inner space of LPA or PLPA contains less than 200,000 molecules of polyglutamated antifolate selected from the group consisting of polyglutamated MTX, polyglutamated PMX, polyglutamated LTX, polyglutamated AG2034, polyglutamated RTX, polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the liposome contains less than 200,000 molecules of polyglutamated PMX. In some embodiments, the liposome contains less than 200,000 molecules of polyglutamated MTX. In some embodiments, the liposome contains less than 200,000 molecules of polyglutamated RTX. In some embodiments, the liposomes contain less than 200,000 molecules of pentaglutamylated LTX. In some embodiments, the liposomes contain less than 200,000 molecules of hexaglutamylated LTX.

[0132] In some embodiments, the inner space of the LPA or PLPA contains 10,000 to 100,000 molecules of a polyglutamated antifolate selected from the group consisting of polyglutamated MTX, polyglutamated PMX, polyglutamated LTX, polyglutamated AG2034, polyglutamated RTX, polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the liposome contains 10,000 to 100,000 molecules of polyglutamated PMX. In some embodiments, the liposome contains 10,000 to 100,000 molecules of polyglutamated MTX. In some embodiments, the liposome contains 10,000 to 100,000 molecules of polyglutamylated RTX. In some embodiments, the liposome contains 10,000 to 100,000 molecules of pentaglutamylated LTX. In some embodiments, the liposome contains 10,000 to 100,000 molecules of hexaglutamylated LTX.

[0133] In some embodiments, the inner space of LPA or PLPA contains less than 200,000 molecules of pentaglutamated antifolate selected from the group consisting of pentaglutamated MTX, pentaglutamated PMX, pentaglutamated LTX, pentaglutamated AG2034, pentaglutamated RTX, pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887. In some embodiments, the liposome contains less than 200,000 molecules of pentaglutamated PMX. In some embodiments, the liposome contains less than 200,000 molecules of pentaglutamated MTX. In some embodiments, the liposome contains less than 200,000 molecules of pentaglutamated RTX. In some embodiments, the liposome contains less than 200,000 molecules of pentaglutamylated RTX.

[0134] In some embodiments, the inner space of the LPA or PLPA contains less than 200,000 molecules of hexaglutamated antifolate selected from the group consisting of hexaglutamated MTX, hexaglutamated PMX, hexaglutamated LTX, hexaglutamated AG2034, hexaglutamated RTX, hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887. In some embodiments, the liposome contains less than 200,000 molecules of hexaglutamated PMX. In some embodiments, the liposome contains less than 200,000 molecules of hexaglutamated MTX. In some embodiments, the liposome comprises less than 200,000 molecules of hexaglutamylated RTX.In some embodiments, the liposome comprises less than 200,000 molecules of hexaglutamylated RTX.

[0135] In some embodiments, the liposomal antifolate is pegylated (i.e., a pegylated liposomal polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate (PLPA or PLPNA)). In some embodiments, the PLPA or PLPNA is water soluble. That is, the PLPA or PLPNA is in the form of an aqueous solution. In some embodiments, the PLPA or PLPNA comprises an interior space that comprises less than 200,000 molecules of the polyglutamated antifolate. In some embodiments, the PLPA or PLPNA comprises between 10,000 and 100,000 molecules of the polyglutamated antifolate. In further embodiments, the PLPA or PLPNA comprises between 10,000 and 100,000 molecules of the pentaglutamated antifolate. In further embodiments, the PLPA or PLPNA comprises between 10,000 and 100,000 molecules of the hexaglutamated antifolate.

[0136] In some embodiments, the inner space of PLPA or PLPNA contains less than 200,000 molecules of polyglutamated antifolate selected from the group consisting of polyglutamated MTX, polyglutamated PMX, polyglutamated LTX, polyglutamated AG2034, polyglutamated RTX, polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the liposome contains less than 200,000 molecules of polyglutamated PMX. In some embodiments, PLPA or PLPNA contains less than 200,000 molecules of polyglutamated MTX. In some embodiments, PLPA or PLPNA contains less than 200,000 molecules of polyglutamated RTX. In some embodiments, the PLPA or PLPNA contains less than 200,000 molecules of pentaglutamylated LTX. In some embodiments, the PLPA or PLPNA contains less than 200,000 molecules of hexaglutamylated LTX.

[0137] In some embodiments, the inner space of the PLPA or PLPNA contains 10,000 to 100,000 molecules of a polyglutamated antifolate selected from the group consisting of polyglutamated MTX, polyglutamated PMX, polyglutamated LTX, polyglutamated AG2034, polyglutamated RTX, polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the liposome contains 10,000 to 100,000 molecules of polyglutamated PMX. In some embodiments, the PLPA or PLPNA contains 10,000 to 100,000 molecules of polyglutamated MTX. In some embodiments, the PLPA or PLPNA comprises 10,000-100,000 molecules of polyglutamylated RTX. In some embodiments, the PLPA or PLPNA comprises 10,000-100,000 molecules of pentaglutamylated LTX. In some embodiments, the PLPA or PLPNA comprises 10,000-100,000 molecules of hexaglutamylated LTX.

[0138] In further embodiments, the inner space of PLPA or PLPNA comprises less than 200,000 molecules of pentaglutamated antifolate selected from the group consisting of pentaglutamated MTX, pentaglutamated PMX, pentaglutamated LTX, pentaglutamated AG2034, pentaglutamated RTX, pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887. In some embodiments, PLPA or PLPNA comprises less than 200,000 molecules of pentaglutamated PMX. In some embodiments, PLPA or PLPNA comprises less than 200,000 molecules of pentaglutamated MTX. In some embodiments, the PLPA or PLPNA comprises less than 200,000 molecules of pentaglutamylated RTX. In some embodiments, the PLPA or PLPNA comprises less than 200,000 molecules of pentaglutamylated RTX.

[0139] In further embodiments, the interior space of the PLPA or PLPNA comprises less than 200,000 molecules of hexaglutamated antifolate selected from the group consisting of hexaglutamated MTX, hexaglutamated PMX, hexaglutamated LTX, hexaglutamated AG2034, hexaglutamated RTX, hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887. In some embodiments, the PLPA or PLPNA comprises less than 200,000 molecules of hexaglutamated PMX. In some embodiments, the PLPA or PLPNA comprises less than 200,000 molecules of hexaglutamated MTX. In some embodiments, the PLPA or PLPNA comprises less than 200,000 molecules of hexaglutamylated RTX. In some embodiments, the PLPA or PLPNA comprises less than 200,000 molecules of hexaglutamylated RTX.

[0140] In further embodiments, the internal space of the PLPA or PLPNA contains 10,000 to 100,000 molecules of a pentaglutamated antifolate selected from the group consisting of pentaglutamated MTX, pentaglutamated PMX, pentaglutamated LTX, pentaglutamated AG2034, pentaglutamated RTX, pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887. In some embodiments, the PLPA or PLPNA contains 10,000 to 100,000 molecules of polyglutamated PMX. In some embodiments, the PLPA or PLPNA contains 10,000 to 100,000 molecules of pentaglutamated MTX. In some embodiments, the PLPA or PLPNA comprises 10,000 to 100,000 molecules of pentaglutamylated RTX. In some embodiments, the PLPA or PLPNA comprises 10,000 to 100,000 molecules of pentaglutamylated RTX.

[0141] In further embodiments, the interior space of the PLPA or PLPNA contains 10,000 to 100,000 molecules of a hexaglutamated antifolate selected from the group consisting of hexaglutamated MTX, hexaglutamated PMX, hexaglutamated LTX, hexaglutamated AG2034, hexaglutamated RTX, hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887. In some embodiments, the PLPA or PLPNA contains 10,000 to 100,000 molecules of hexaglutamated PMX. In some embodiments, the PLPA or PLPNA comprises 10,000 to 100,000 molecules of hexaglutamylated MTX. In some embodiments, the PLPA or PLPNA comprises 10,000 to 100,000 molecules of hexaglutamylated RTX. In some embodiments, the PLPA or PLPNA comprises 10,000 to 100,000 molecules of hexaglutamylated RTX.

[0142] In some embodiments, the pH of the solution containing the liposome composition is pH 5-8 or pH 2-6.

[0143] Targeted Liposomes In some embodiments, the present disclosure provides liposomal polyglutamated antifolate compositions, in which liposomes are PEGylated and comprise a polyglutamated antifolate and a targeting moiety attached to one or both of the PEG and the exterior of the liposome, in which the targeting moiety has a specific affinity for a surface antigen on a target cell of interest. Such liposomes may be generally referred to herein as "targeted liposomes", e.g., the liposomes comprise one or more targeting moieties or biodistribution modifiers on the surface of the liposome or otherwise attached to the liposome. The targeting moiety of the targeted liposome may be any moiety or agent that can specifically bind a desired target (e.g., an antigen target expressed on the surface of a target cell of interest). In one embodiment, the targeted liposome specifically and preferentially binds to targets on the surface of a desired target cell into which the targeted liposome is internalized, and the encapsulated polyglutamated cytotoxic agent (e.g., polyglutamated antifolates such as pentaglutamated or hexaglutamated PMX, LTX, and MTX) exerts its cytotoxic effect. In further embodiments, the targeted cell is a cancer cell, a tumor cell, or a metastatic cell. In some embodiments, the targeted liposome is an immunoliposome.

[0144] The terms bind or bound refer to any type of bond, such as, for example, a covalent bond, an ionic bond (e.g., avidin-biotin), a bond through hydrophobic interactions, and a bond via a functional group such as maleimide or a linker such as PEG. For example, detectable markers, steric stabilizers, liposomes, liposome components, immunostimulants can be directly bound to each other by maleimide functional groups or by PEG-maleimide groups.

[0145] In some embodiments, the targeting moiety attached to the liposome is a polypeptide. In further embodiments, the targeting moiety is an antibody or a fragment of an antibody. In additional 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 multivalent antibody. In additional embodiments, the targeting moiety has a specific affinity for an epitope on a tumor cell surface antigen that is present on tumor cells but not present or accessible on non-tumor cells. In some embodiments, the targeting moiety further comprises one or more of an immunostimulant, a detectable marker, and a maleimide disposed on at least one of the PEG and the exterior of the liposome. In some embodiments, the targeting moiety of a liposomal polyglutamated antifolate (LPA) or pegylated liposomal polyglutamated antifolate (PLPA) liposome is anionic or neutral. In other embodiments, the targeting moiety of an LPA or PLPA liposome is cationic. In some embodiments, the targeting moiety of the LPA or PLPA liposome composition comprises at least 10% liposome-encapsulated polyglutamic acid antifolate. In some embodiments, the targeting moiety-LPA or targeting moiety-PLPA liposomes have a diameter in the range of 20-200 nm. In further embodiments, the liposomes have a diameter in the range of 80-120 nm.

[0146] In some embodiments, the targeting moiety-LPA or targeting moiety-PLPA comprises a polypeptide targeting moiety, such as an antibody or antibody fragment, and the targeting moiety has a molecular weight of less than 0.5×10 as measured using Biacore analysis. -10 ~10×10 -6 In a further embodiment, the targeting moiety-LPA or targeting moiety-PLPA comprises a polypeptide targeting moiety.

[0147] In some embodiments, the targeting moiety-LPA or targeting moiety-PLPA comprises a polypeptide targeting moiety, such as an antibody or antibody fragment, and the targeting moiety is selected from the group consisting of GONMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-alpha, folate receptor-beta, mucin 1, folate receptor-delta, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, EGFR, CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98.

[0148] In further embodiments, the targeting moiety comprises a polypeptide targeting moiety, such as an antibody or antibody fragment, and the targeting moiety has binding specificity for a folate receptor. In some embodiments, the targeting moiety has a binding specificity of 0.5×10 as measured using Biacore analysis. -10 ~10×10 -6 In some embodiments, the folate receptor bound by the targeting moiety is one or more folate receptors selected from the group consisting of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ).

[0149] In some embodiments, the targeting moiety is an antibody or an antigen-binding portion of an antibody that specifically binds a target of interest expressed on the surface of a target cell of interest. In some embodiments, the targeting moiety is a full-length antibody. In some embodiments, the targeting moiety is an antigen-binding portion of an antibody. In some embodiments, the targeting moiety comprises one or more complementarity determining regions (CDRs) from an antibody. Examples of suitable proteins that can act as targeting moieties for the disclosed targeted liposomes include one or more of full-length human antibodies, humanized antibodies, chimeric antibodies, antigen-binding fragments of antibodies, single-chain antibodies, single-domain antibodies, bispecific antibodies, synthetic antibodies, pegylated antibodies, and multivalent antibodies. The antibodies of the provided targeted liposomes may have a combination of the above properties. For example, a humanized antibody may be an antigen-binding fragment and may be pegylated and multiplexed.

[0150] The term "humanized antibody" refers to a non-human (e.g., murine) form of a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof that contains minimal non-human (e.g., murine) sequence. In general, a humanized antibody is a human immunoglobulin in which residues from the complementarity determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, and hamster) that has the desired specificity, affinity, and capacity (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some instances, Fv network region (FR) residues of a human immunoglobulin are replaced by the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capacity. Humanized antibodies can be further modified by substitution of additional residues within the Fv framework regions and / or the substituted non-human residues to refine and optimize antibody specificity, affinity, and / or potency. Generally, a humanized antibody contains at least one, typically two or three, variable regions, including all or almost all of the CDR regions corresponding to a non-human immunoglobulin, where all or almost all of the FR regions are of human immunoglobulin consensus sequence. A humanized antibody may also contain at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are reported in U.S. Patent Nos. 5,225,539 and 5,639,641.

[0151] As discussed herein, folate receptors (FRs) differ from reduced folate carriers (RFCs) in that they utilize different pathways to bring folate and antifolates into cells. In some embodiments, the targeting moiety specifically binds a folate receptor. In further embodiments, the targeting moiety specifically binds a folate receptor selected from folate receptor alpha, folate receptor beta, and folate receptor delta. Antibodies against folate receptor alpha can generally be made using techniques known in the art. Additionally, many anti-folate receptor antibody sequences are publicly and / or commercially available and readily available.

[0152] The murine antibody against folate receptor is an example of the antibody that is a murine antibody against folate receptor that can be used as the targeting moiety of the disclosed targeted liposome.The sequences of these antibodies are known and are reported, for example, in U.S. Patent Nos. 5,646,253, 8,388,972, 8,871,206, and 9,133,275, and International Application Nos. PCT / US2011 / 056966 and PCT / US2012 / 046672.For example, based on the sequences already disclosed in the public domain, the antibody genes are synthesized and placed in a transient expression vector, and the antibody is produced in a HEK-293 transient expression system.The antibody can be a complete antibody, a Fab, or any of the various antibody variants described herein or otherwise known in the art.

[0153] In some embodiments, the targeted liposome comprises between 30 and 500 targeting moieties (e.g., between 30 and 250 targeting moieties or between 30 and 200 targeting moieties). In some embodiments, the targeted liposomes provided comprise fewer than 220 targeting moieties, fewer than 200 targeting moieties, or fewer than 175 targeting moieties. In some embodiments, the targeting moieties are non-covalently attached to the exterior of the liposome (e.g., via ionic interactions or a GPI anchor).

[0154] In some embodiments, the external molecule of the targeted liposome comprises a lipid, a targeting moiety, a steric stabilizer (e.g., PEG), a maleimide, and cholesterol. In some embodiments, the targeting moiety is covalently attached via a maleimide functional group. In some embodiments, the targeting moiety is covalently attached to a liposome component or a steric stabilizer such as a PEG molecule. In some embodiments, all targeting moieties are attached to one component of the liposome, such as PEG. In another embodiment, the targeting moiety is attached to another component of the liposome. For example, some targeting moieties can be attached to a lipid component or cholesterol, some targeting moieties can be attached to a steric stabilizer (e.g., PEG), and still other targeting moieties can be attached to a detectable marker or another targeting moiety.

[0155] In some embodiments, the targeting moiety of the targeted liposome has affinity and specificity for (i.e., specifically binds to) an antigen expressed on the surface of a cancer cell. In some further embodiments, the targeting moiety of the targeted liposome has affinity and specificity for an antigen selected from the group consisting of folate receptor alpha, folate receptor beta, and folate receptor delta. In one embodiment, the targeting moiety has specific affinity for (i.e., specifically binds to) one or more antigens selected from the group consisting of folate receptor alpha, folate receptor beta, and folate receptor delta. In a further embodiment, the targeting moiety has specific affinity for at least two antigens selected from the group consisting of folate receptor alpha, folate receptor beta, and folate receptor delta. In another embodiment, the targeting moiety has specific affinity for three antigens, e.g., folate receptor alpha, folate receptor beta, and folate receptor delta. The targeting moiety may have affinity and specificity for one epitope of an antigen, since sometimes the targeting moiety does not bind the complete antigen, but only one epitope of many epitopes in the antigen.

[0156] The terms "epitope" or "antigenic determinant" are used interchangeably herein and refer to a portion of an antigen that can be recognized and specifically bound by a particular antibody or binding moiety. When the antigen is a polypeptide, epitopes can be formed from both contiguous amino acids and non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are generally retained upon protein denaturation, while epitopes formed by tertiary folding are generally lost upon protein denaturation. Epitopes generally include at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation.

[0157] In some embodiments, the targeting moiety has specific affinity for an epitope on a tumor cell surface antigen that is present on tumor cells but not present or accessible on non-tumor cells. For example, in some conditions, tumor antigens are present on the surface of both normal cells and malignant cancer cells, but tumor epitopes are only exposed on cancer cells. As a further example, tumor antigens may have a structural change in cancer, causing cancer cell-specific epitopes to be presented. Targeting moieties that have specific affinity for the epitopes described herein are useful and are encompassed by the disclosed compositions and methods. In some embodiments, the tumor cells that have cancer cell-specific epitopes are cancer cells. Examples of such tumor cell surface antigens include folate receptor alpha, folate receptor beta, and folate receptor delta.

[0158] In some embodiments, the liposome composition is provided as a pharmaceutical composition comprising liposomes and a carrier, e.g., a pharma- ceutical acceptable carrier. Examples of pharma- ceutical acceptable carriers included in the provided pharmaceutical compositions include physiological saline, isotonic dextrose, isotonic sucrose, Ringer's solution, and Hank's solution. In some embodiments, a buffer substance is added to maintain the optimal pH for storage stability of the pharmaceutical composition. In some embodiments, the pH of the pharmaceutical composition is 6.0-7.5. In some embodiments, the pH is 6.3-7.0. In further embodiments, the pH is 6.5. Ideally, the pH of the pharmaceutical composition allows both stability of the liposome membrane lipids and retention of the entrapped material. Examples of buffer substances are histidine, hydroxyethylpiperazine-ethylsulfonic acid (HEPES), morpholinoethylsulfonic acid (MES), succinic acid, tartaric acid, and citric acid, typically at concentrations of 2-20 mM. Other suitable carriers include, for example, water, buffered aqueous solutions, 0.4% NaCl, and 0.3% glycine. Protein, carbohydrate, or polymer stabilizers, and osmolality adjusters, such as gelatin, albumin, dextran, or polyvinylpyrrolidone, can be added. The isotonicity of the composition can be adjusted to a physiological amount of 0.25 to 0.35 mol / kg with glucose or more inert compounds such as lactose, sucrose, mannitol, or dextrin. These compositions can generally be sterilized using standard sterilization techniques known in the art (e.g., filtration). The resulting aqueous solutions can be packaged for use or filtered and lyophilized under aseptic conditions, and the lyophilized preparations are mixed with a sterile aqueous medium before administration.

[0159] The provided pharmaceutical liposome compositions may also contain other pharma- ceutical acceptable additives required to approximate physiological conditions, such as pH adjusting and buffering agents, and osmolality adjusting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride. In addition, the liposome suspension may contain lipid-protecting agents that protect lipids against free radical and lipid-peroxidative damage on storage. Lipophilic free-radical quenchers, such as α-tocopherol, and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.

[0160] The liposome concentration in the provided liquid pharmaceutical composition may vary widely as needed, for example, typically less than about 0.05% or at least about 2-10% to 30-50% by weight, and may be selected primarily by liquid volume and viscosity according to the particular mode of administration selected. For example, the concentration may be increased to reduce the fluid load associated with the treatment. This may be particularly desirable in patients with congestive heart failure or severe hypertension associated with atherosclerosis. Alternatively, liposomal pharmaceutical compositions consisting of irritating lipids may be diluted to low concentrations to reduce inflammation at the site of administration.

[0161] Some embodiments relate to methods of delivering targeted PEGylated liposomal formulations of polyglutamated antifolates to tumors expressing folate receptors on their surface. Exemplary methods include administering at least one of any of the liposome-containing compositions of the present disclosure in an amount that delivers a therapeutically effective dose of the targeted PEGylated liposomal polyglutamated antifolate to the tumor.

[0162] The amount of liposomal pharmaceutical composition administered will depend on the particular polyglutamated antifolate therapeutic agent entrapped within the liposome, the disease state being treated, the type of liposome used, and the judgment of the clinician. Generally, the amount of liposomal pharmaceutical composition administered will be sufficient to deliver a therapeutically effective dose of the particular therapeutic agent.

[0163] The amount of liposomal pharmaceutical composition required to deliver a therapeutically effective dose can be determined by routine in vitro and in vivo methods common in the prior art of drug testing. See, for example, DB Budman, AHCalvert, EK Rowinsky (eds.), Handbook of Anticancer Drug Development, LWW, 2003. Therapeutically effective dosages of various therapeutic compositions are known to those skilled in the art. In some embodiments, the therapeutic agent delivered by the pharmaceutical liposomal composition provides activity at least equal to or greater than that obtained by administering the same amount of the therapeutic agent in its routine non-liposomal formulation. In general, dosages in liposomal pharmaceutical compositions range, for example, from about 0.005 to about 500 mg of therapeutic agent per kg of body weight, and in most cases, from about 0.1 to about 100 mg of therapeutic agent per kg of body weight.

[0164] As used herein, "effective amount" refers to the dosage of an agent sufficient to produce a medically desirable result. The effective amount will vary with the desired outcome, the particular condition being treated or prevented, the age and physical condition of the subject being treated, the severity of symptoms, the duration of treatment, the nature of concurrent or concomitant therapy (if any), the particular route of administration, and factors within the knowledge and expertise of the physician. An "effective amount" can be determined empirically and routinely in relation to the stated purpose. In this disclosure, the terms "subject" and "patient" are used interchangeably and have the same meaning. It is generally preferred that a maximum dose, i.e., the highest safe dose according to sound medical judgment, be used.

[0165] For example, when a subject has a tumor, an effective amount can be the amount of agent (e.g., polyglutamylated antifolate) that reduces tumor volume or burden (e.g., measured by imaging the tumor). An effective amount can also be routinely assessed by the presence and / or frequency of cancer cells in blood or other body fluids or tissues (e.g., biopsy). When a tumor affects the normal functioning of a tissue or organ, an effective amount can be routinely assessed by measuring the normal functioning of the tissue or organ. In some examples, an effective amount is the amount required to reduce or eliminate one or more, preferably all, symptoms.

[0166] The terms "treat" or "treatment" or "to treat" and the like refer to both (a) therapeutic measures that cure, delay, reduce, and / or halt the progression of the symptoms of a diagnosed pathological condition or disorder, and (b) prophylactic or preventative measures that prevent and / or delay the onset of the target disease or condition. Thus, subjects in need of treatment include those who already have a cancer or condition, those at risk of having a cancer or condition, and those with an infection or condition that should be prevented. In certain embodiments, a subject is successfully "treated" by the methods provided herein, for example, when the subject shows all, some, or temporary improvement or disappearance of symptoms associated with a disease or condition (e.g., cancer, rheumatoid arthritis).

[0167] Also provided are pharmaceutical compositions comprising the provided polyglutamated antifolate compositions (e.g., liposomes comprising pentaglutamated or hexaglutamated antifolates). The pharmaceutical compositions are sterile compositions comprising the sample liposomes and, preferably, an antifolate, preferably in a pharma- ceutical acceptable carrier.

[0168] The term "delivery vehicle" generally refers to any composition, such as viral sequences, viral material, or lipid or liposomal formulations, that acts to aid, promote or facilitate the introduction of a polyglutamated antifolate into a cell.

[0169] The term "pharmaceutical acceptable carrier" refers, for example, to one or more compatible solid or liquid fillers, diluents, or encapsulating substances that are suitable for administration to a human or other subject.

[0170] The term "carrier" refers to an organic or inorganic material, natural or synthetic, with which the liposomal composition is combined to facilitate administration. The components of the pharmaceutical composition are mixed in a manner that precludes interactions that would significantly impair their desired pharmaceutical efficacy. Suitable buffering agents include acetic acid and salts (1-2% W / V), citric acid and salts (1-3% W / V), boric acid and salts (0.5-2.5% W / V), and phosphoric acid and salts (0.8-2% W / V). Suitable preservatives include benzalkonium chloride (0.003-0.03% W / V), chlorobutanol (0.3-0.9% W / V), and parabens (0.01-0.25% W / V).

[0171] Unless otherwise stated herein, various routes of administration are available. The particular mode selected will depend on the particular active agent selected, the particular condition being treated, and the dosage required for therapeutic efficacy. The provided methods can be carried out using any known mode of administration that is medically acceptable and in accordance with good medical practice. In some embodiments, the route of administration is injection. In further embodiments, the injection is by a parenteral route selected from intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal, intraarticular, intradural, intrathecal, intravenous, intramuscular, or intrasternal injection. In some embodiments, the route of administration is infusion. In additional embodiments, the route of administration is oral, nasal, mucosal, sublingual, intratracheal, ophthalmic, rectal, vaginal, ocular, topical, transdermal, pulmonary, or inhalation.

[0172] In some embodiments, the PLPA and / or targeted PLPA is prepared as an infusion composition, an injection composition, a parenteral composition, or a topical composition. In further embodiments, the injection comprises one or more of intraperitoneal injection, intratumoral direct injection, intraarterial injection, intravenous injection, subcutaneous injection, intramuscular injection, transdermal and intranasal delivery. In further embodiments, the PLPA and / or targeted PLPA is a liquid solution or suspension. However, solid dosage forms suitable for solution or suspension in a liquid vehicle prior to injection are also provided herein. In some embodiments, the targeted PEGylated liposomal polyglutamated antifolate is formulated as an enteric coated tablet or gel capsule according to methods known in the art.

[0173] In some embodiments, the targeted PEGylated liposomal polyglutamated antifolates are administered to tumors of the central nervous system using slow, sustained intracranial injection of liposomes directly into the tumor (e.g., convection-enhanced delivery (CED)). See Saito et al., Cancer Research 64:2572-2579 (2004); Mamot et al., J. Neuro-Oncology 68:1-9 (2004). In other embodiments, the formulation is applied directly to the tissue surface. Sustained release, pH-dependent release, and other specific chemical or environmental condition mediated release administration of PEGylated liposomal polyglutamated antifolates (e.g., depot injections and disintegrating implants) are also provided. Such release-mediated compositions are further described herein or otherwise known in the art.

[0174] For administration by inhalation, the compositions can conveniently be delivered in the form of an aerosol spray preparation from pressurized packs or a nebulizer by use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.

[0175] When it is desired to deliver the compositions to the whole body, they can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Injectable preparations can be provided in unit dosage form, e.g., in ampoules or in multi-dose containers. Parenteral preparations of pharmaceuticals include aqueous solutions of the material. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Alternatively, liposomal suspensions can be prepared as oil-based suspensions. Suitable lipophilic solvents or excipients include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides.

[0176] Alternatively, the non-targeted or targeted PEGylated liposomal polyglutamated antifolates may be in powder or lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0177] The provided compositions (e.g., polyglutamated antifolates, and polyglutamated antifolate-containing liposomes) can also be formulated into rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing standard suppository bases such as cocoa butter or other glycerides.

[0178] The provided compositions have in vivo, ex vivo and in vitro applications. In some embodiments, the compositions have in vivo applications. In vivo uses can include uses such as cell culture and tissue engineering, where selective processing of subpopulations of cells is desired. For example, during the culture of stem cells from normal patients or patients with cancer, cells can be treated with the sample composition or sample liposome discussed to address cancerous subpopulations of cells. Cancerous subpopulations can arise because donors are naturally cancerous or because cells spontaneously transform during in vitro processing.

[0179] In some embodiments, the liposome composition is provided in a kit that includes a container with the liposomes, and optionally a container with a substance (antigen) to be targeted or preferentially bound by the liposome, and instructions, such as instructions or information related to using the liposome composition in one or more applications. Such instructions can be provided in any medium, such as a hard copy paper, electronic media, or through a database or website that includes instructions.

[0180] In some embodiments, the disclosure provides a method of killing hyperproliferative cells comprising contacting the hyperproliferative cells with a delivery vehicle (e.g., a liposome) comprising a polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate. In some embodiments, the polyglutamated antifolate is a member selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the polyglutamated antioxidant comprises a pentaglutamated antioxidant selected from the group consisting of pentaglutamated MTX, pentaglutamated PMX, pentaglutamated LTX, pentaglutamated AG2034, pentaglutamated RTX, pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887. In some embodiments, the polyglutamated antifolate comprises a hexaglutamated antifolate selected from the group consisting of hexaglutamated MTX, hexaglutamated PMX, hexaglutamated LTX, hexaglutamated AG2034, hexaglutamated RTX, hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887. In some embodiments, the polyglutamated antifolate comprises pentaglutamated PMX. In some embodiments, the polyglutamated antifolate comprises hexaglutamated PMX. In some embodiments, the polyglutamated antifolate comprises pentaglutamated MTX.In some embodiments, the polyglutamated antifolate comprises hexaglutamated MTX. In some embodiments, the polyglutamated antifolate comprises pentaglutamated RTX. In some embodiments, the polyglutamated antifolate comprises hexaglutamated RTX. In some embodiments, the polyglutamated antifolate comprises pentaglutamated LTX. In some embodiments, the polyglutamated antifolate comprises hexaglutamated LTX. In some embodiments, the delivery vehicle is a liposome. In further embodiments, the liposome is PEGylated. In additional embodiments, the delivery vehicle comprises a targeting moiety on its surface that specifically binds an antigen on the surface of the hyperproliferative cell. In further embodiments, the delivery vehicle is selected from the group consisting of GONMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-alpha, folate receptor-beta, mucin 1, folate receptor-delta, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6 ), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, EGFR, CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98.In a further embodiment, the delivery vehicle is a liposome, and the liposome is capable of expressing any of the following proteins: GONMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-alpha, folate receptor-beta, mucin 1, folate receptor-delta, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV The antibody comprises a targeting moiety that specifically binds a cell surface antigen on the surface of a highly proliferative cell selected from the group consisting of: CD49, CD51, CD61, CD70, CD81, CD161, CD170, CD19, CD22, CD33, and CD98.

[0181] In some embodiments, the present disclosure provides a method of inhibiting tumor cell proliferation comprising contacting the tumor cell with a delivery vehicle (e.g., liposome) comprising a polyglutamylated (e.g., pentaglutamylated or hexaglutamylated) antifolate. In some embodiments, the polyglutamylated antifolate is a member selected from the group consisting of polyglutamylated methotrexate (MTX), polyglutamylated pemetrexed (PMX), polyglutamylated lometrexol (LTX), polyglutamylated AG2034, polyglutamylated raltitrexed (RTX), polyglutamylated piritrexim, polyglutamylated pralatrexate, polyglutamylated AG2034, polyglutamylated GW1843, polyglutamylated aminopterin, and polyglutamylated LY309887. In some embodiments, the polyglutamated antioxidant comprises a pentaglutamated antioxidant selected from the group consisting of pentaglutamated MTX, pentaglutamated PMX, pentaglutamated LTX, pentaglutamated AG2034, pentaglutamated RTX, pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887. In some embodiments, the polyglutamated antifolate comprises a hexaglutamated antifolate selected from the group consisting of hexaglutamated MTX, hexaglutamated PMX, hexaglutamated LTX, hexaglutamated AG2034, hexaglutamated RTX, hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887. In some embodiments, the polyglutamated antifolate comprises pentaglutamated PMX. In some embodiments, the polyglutamated antifolate comprises hexaglutamated PMX. In some embodiments, the polyglutamated antifolate comprises pentaglutamated MTX.In some embodiments, the polyglutamated antifolate comprises hexaglutamated MTX. In some embodiments, the polyglutamated antifolate comprises pentaglutamated RTX. In some embodiments, the polyglutamated antifolate comprises hexaglutamated RTX. In some embodiments, the polyglutamated antifolate comprises pentaglutamated LTX. In some embodiments, the polyglutamated antifolate comprises hexaglutamated LTX. In some embodiments, the delivery vehicle is a liposome. In further embodiments, the liposome is PEGylated. In additional embodiments, the delivery vehicle comprises a targeting moiety on its surface that specifically binds an antigen on the surface of the hyperproliferative cell. In further embodiments, the delivery vehicle is selected from the group consisting of GONMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-alpha, folate receptor-beta, mucin 1, folate receptor-delta, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP 6), CGEN-15027, comprises a targeting moiety that specifically binds a cell surface antigen on the surface of a tumor cell selected from the group consisting of P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, EGFR, CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98.In a further embodiment, the delivery vehicle is a liposome, and the liposome is capable of expressing any of the following proteins: GONMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-alpha, folate receptor-beta, mucin 1, folate receptor-delta, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LI The antibody comprises a targeting moiety that specifically binds a cell surface antigen on the surface of a tumor cell selected from the group consisting of V-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, EGFR, CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98.

[0182] In some embodiments, the disclosure provides a method for treating a hyperproliferative disease comprising administering to a subject having or at risk of having a hyperproliferative disease an effective amount of a delivery vehicle (e.g., a liposome) comprising a polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate. In some embodiments, the polyglutamated antifolate administered is a member selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the administered polyglutamated antioxidant comprises a pentaglutamated antioxidant selected from the group consisting of pentaglutamated MTX, pentaglutamated PMX, pentaglutamated LTX, pentaglutamated AG2034, pentaglutamated RTX, pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887. In some embodiments, the polyglutamated antifolate administered comprises a hexaglutamated antifolate selected from the group consisting of hexaglutamated MTX, hexaglutamated PMX, hexaglutamated LTX, hexaglutamated AG2034, hexaglutamated RTX, hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated PMX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated PMX.In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated RTX. In some embodiments, the pentaglutamated antifolate administered comprises hexaglutamated RTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated LTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated LTX. In some embodiments, the delivery vehicle administered is a liposome. In further embodiments, the liposome is pegylated. In additional embodiments, the delivery vehicle comprises a targeting moiety on its surface that specifically binds an antigen on the surface of a target cell of interest. In further embodiments, the delivery vehicle is selected from the group consisting of GONMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-alpha, folate receptor-beta, mucin 1, folate receptor-delta, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV- 1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, EGFR, CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98.In a further embodiment, the delivery vehicle is a liposome, and the liposome is capable of expressing any of the following receptors: GONMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-alpha, folate receptor-beta, mucin 1, folate receptor-delta, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue The targeting moiety specifically binds a cell surface antigen selected from the group consisting of: LIV-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, EGFR, CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98.

[0183] In some embodiments, the present disclosure provides a method for treating a disease of the immune system (e.g., an autoimmune disease such as rheumatoid arthritis) comprising administering to a subject having or at risk of having an autoimmune disease an effective amount of a delivery vehicle (e.g., a liposome) comprising a polyglutamylated (e.g., pentaglutamylated or hexaglutamylated) antifolate. In some embodiments, the disease of the immune system is an autoimmune disease. In further embodiments, the disease of the immune system is rheumatoid arthritis. In some embodiments, the polyglutamated antifolate administered is a member selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the administered polyglutamated antioxidant comprises a pentaglutamated antioxidant selected from the group consisting of pentaglutamated MTX, pentaglutamated PMX, pentaglutamated LTX, pentaglutamated AG2034, pentaglutamated RTX, pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887. In some embodiments, the administered polyglutamated antioxidant comprises a hexaglutamated antioxidant selected from the group consisting of hexaglutamated MTX, hexaglutamated PMX, hexaglutamated LTX, hexaglutamated AG2034, hexaglutamated RTX, hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887.In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated PMX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated PMX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated LTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated LTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated pralatrexate (PTX). In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated pralatrexate (PTX). In some embodiments, the delivery vehicle administered is a liposome. In further embodiments, the liposome is pegylated. In additional embodiments, the delivery vehicle comprises a targeting moiety on its surface that specifically binds an antigen on the surface of a target cell of interest. In further embodiments, the delivery vehicle comprises a targeting moiety selected from the group consisting of GONMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-alpha, folate receptor-beta, mucin 1, folate receptor-delta, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV- 1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, EGFR, CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98.In a further embodiment, the delivery vehicle is a liposome, and the liposome is capable of expressing any of the following receptors: GONMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-alpha, folate receptor-beta, mucin 1, folate receptor-delta, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue The targeting moiety specifically binds a cell surface antigen selected from the group consisting of: LIV-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, EGFR, CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98.

[0184] In some embodiments, the disclosure provides a method for treating cancer comprising administering to a subject having or at risk of having cancer an effective amount of a delivery vehicle (e.g., a liposome) comprising a polyglutamylated (e.g., pentaglutamylated or hexaglutamylated) antifolate. In some embodiments, the cancer is selected from the group consisting of lung (e.g., non-small cell lung cancer), pancreatic, breast, ovarian, lung, prostate, head and neck, stomach, gastrointestinal tract, colon, esophagus, cervix, kidney, bile duct, gallbladder, and hematological malignancies (e.g., leukemia or lymphoma). In some embodiments, the polyglutamated antioxidant administered is a member selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the administered polyglutamated antioxidant comprises a pentaglutamated antioxidant selected from the group consisting of pentaglutamated MTX, pentaglutamated PMX, pentaglutamated LTX, pentaglutamated AG2034, pentaglutamated RTX, pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887.In some embodiments, the polyglutamated antifolate administered comprises a hexaglutamated antifolate selected from the group consisting of hexaglutamated MTX, hexaglutamated PMX, hexaglutamated LTX, hexaglutamated AG2034, hexaglutamated RTX, hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated PMX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated PMX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated LTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated LTX. In some embodiments, the delivery vehicle administered is a liposome. In further embodiments, the liposome is pegylated. In additional embodiments, the delivery vehicle comprises a targeting moiety on its surface that specifically binds an antigen on the surface of a target cell of interest.In further embodiments, the delivery vehicle is selected from the group consisting of GONMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-alpha, folate receptor-beta, mucin 1, folate receptor-delta, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV- 1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, EGFR, CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98. In a further embodiment, the delivery vehicle is a liposome, and the liposome is capable of expressing any of the following receptors: GONMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-alpha, folate receptor-beta, mucin 1, folate receptor-delta, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue The targeting moiety specifically binds a cell surface antigen selected from the group consisting of: LIV-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, EGFR, CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98.

[0185] In some embodiments, the present disclosure provides a method for treating lung cancer (e.g., non-small cell lung cancer), comprising administering to a subject having or at risk of having lung cancer an effective amount of a delivery vehicle (e.g., a liposome) comprising a polyglutamylated (e.g., pentaglutamylated or hexaglutamylated) antifolate. In certain embodiments, the cancer is non-small cell lung cancer. In some embodiments, the polyglutamated antifolate administered is a member selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the administered polyglutamated antioxidant comprises a pentaglutamated antioxidant selected from the group consisting of pentaglutamated MTX, pentaglutamated PMX, pentaglutamated LTX, pentaglutamated AG2034, pentaglutamated RTX, pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887. In some embodiments, the polyglutamated antifolate administered comprises a hexaglutamated antifolate selected from the group consisting of hexaglutamated MTX, hexaglutamated PMX, hexaglutamated LTX, hexaglutamated AG2034, hexaglutamated RTX, hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887. In certain embodiments, the polyglutamated antifolate administered comprises pentaglutamated PMX.In certain embodiments, the polyglutamated antifolate administered comprises hexaglutamated PMX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated RTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated RTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated LTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated LTX. In some embodiments, the delivery vehicle administered is a liposome. In further embodiments, the liposome is pegylated. In some embodiments, the liposome is non-targeted. In additional embodiments, the delivery vehicle comprises a targeting moiety on its surface that specifically binds an antigen on the surface of a lung cancer (e.g., non-small cell lung cancer) cell. In further embodiments, the delivery vehicle comprises a targeting moiety that specifically binds a cell surface antigen selected from the group consisting of mucin 1, folate receptor δ, nectin 4, NaPi2b, CD56, EGFR, and SC-16. In further embodiments, the delivery vehicle is a liposome, and the liposome comprises a targeting moiety that specifically binds a cell surface antigen selected from the group consisting of mucin 1, folate receptor δ, nectin 4, NaPi2b, CD56, EGFR, and SC-16.

[0186] In some embodiments, the present disclosure provides a method for treating pancreatic cancer comprising administering to a subject having or at risk of having pancreatic cancer an effective amount of a delivery vehicle (e.g., a liposome) comprising a polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate. In some embodiments, the polyglutamated antifolate administered is a member selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the administered polyglutamated antioxidant comprises a pentaglutamated antioxidant selected from the group consisting of pentaglutamated MTX, pentaglutamated PMX, pentaglutamated LTX, pentaglutamated AG2034, pentaglutamated RTX, pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887. In some embodiments, the polyglutamated antifolate administered comprises a hexaglutamated antifolate selected from the group consisting of hexaglutamated MTX, hexaglutamated PMX, hexaglutamated LTX, hexaglutamated AG2034, hexaglutamated RTX, hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887. In certain embodiments, the polyglutamated antifolate administered comprises pentaglutamated PMX. In certain embodiments, the polyglutamated antifolate administered comprises hexaglutamated PMX.In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated RTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated RTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated LTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated LTX. In some embodiments, the delivery vehicle administered is a liposome. In further embodiments, the liposome is pegylated. In some embodiments, the liposome is non-targeted. In additional embodiments, the delivery vehicle comprises a targeting moiety on its surface that specifically binds an antigen on the surface of a pancreatic cancer cell. In further embodiments, the delivery vehicle comprises a targeting moiety that specifically binds a cell surface antigen selected from the group consisting of TACSTD2 (TROP2), mucin 1, folate receptor delta, mesothelin, guanylate cyclase C (GCC), SLC44A4, and nectin 4. In further embodiments, the delivery vehicle is a liposome, and the liposome comprises a targeting moiety that specifically binds a cell surface antigen selected from the group consisting of TACSTD2 (TROP2), mucin 1, folate receptor delta, mesothelin, guanylate cyclase C (GCC), SLC44A4, and nectin 4.

[0187] In additional embodiments, the present disclosure provides a method for treating breast cancer (e.g., triple-negative breast cancer (estrogen receptor agonist or vasopressin-dependent) cancer) comprising administering to a subject having or at risk of having breast cancer an effective amount of a delivery vehicle (e.g., liposome) comprising a polyglutamylated (e.g., pentaglutamylated or hexaglutamylated) antifolate. - , progesterone receptor - , and HER2 -In some embodiments, the polyglutamated antifolate is a member selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the administered polyglutamated antioxidant comprises a pentaglutamated antioxidant selected from the group consisting of pentaglutamated MTX, pentaglutamated PMX, pentaglutamated LTX, pentaglutamated AG2034, pentaglutamated RTX, pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887. In some embodiments, the polyglutamated antifolate administered comprises a hexaglutamated antifolate selected from the group consisting of hexaglutamated MTX, hexaglutamated PMX, hexaglutamated LTX, hexaglutamated AG2034, hexaglutamated RTX, hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887. In certain embodiments, the polyglutamated antifolate administered comprises pentaglutamated PMX. In certain embodiments, the polyglutamated antifolate administered comprises hexaglutamated PMX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated MTX.In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated RTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated RTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated LTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated LTX. In some embodiments, the delivery vehicle administered is a liposome. In further embodiments, the liposome is pegylated. In some embodiments, the liposome is non-targeted. In additional embodiments, the delivery vehicle comprises a targeting moiety that specifically binds an antigen on the surface of a breast cancer cell. In further embodiments, the delivery vehicle comprises a targeting moiety on its surface that specifically binds a cell surface antigen selected from the group consisting of LIV-1 (ZIP6), EGFR, HER2, HER3, mucin 1, folate receptor delta, GONMB, and nectin 4. In a further embodiment, the delivery vehicle is a liposome, and the liposome comprises a targeting moiety that specifically binds a cell surface antigen selected from the group consisting of LIV-1 (ZIP6), EGFR, HER2, HER3, mucin 1, folate receptor delta, GONMB, and nectin 4.

[0188] In some embodiments, provided compositions (e.g., liposomes comprising polyglutamated antifolates) are administered to a subject having or at risk of having a hematological cancer that is identifiable by expression of a tumor-specific or tumor-associated antigen on the cell surface. Thus, in some embodiments, the present disclosure provides a method for treating cancer comprising administering an effective amount of a delivery vehicle (e.g., liposomes) comprising a targeting moiety and a polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate to a subject having or at risk of having a cancer, solid cancer, and / or metastasis that is identifiable by expression of a tumor-specific or tumor-associated antigen on the cell surface of the cancer, where the targeting moiety is displayed on the surface of the delivery vehicle and specifically binds the tumor-specific or tumor-associated antigen. In some embodiments, the polyglutamated antifolate administered is a member selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the administered polyglutamated antioxidant comprises a pentaglutamated antioxidant selected from the group consisting of pentaglutamated MTX, pentaglutamated PMX, pentaglutamated LTX, pentaglutamated AG2034, pentaglutamated RTX, pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887.In some embodiments, the polyglutamated antifolate administered comprises a hexaglutamated antifolate selected from the group consisting of hexaglutamated MTX, hexaglutamated PMX, hexaglutamated LTX, hexaglutamated AG2034, hexaglutamated RTX, hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887. In certain embodiments, the polyglutamated antifolate administered comprises pentaglutamated PMX. In certain embodiments, the polyglutamated antifolate administered comprises hexaglutamated PMX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated RTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated RTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated LTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated LTX. In some embodiments, the delivery vehicle administered is a liposome. In further embodiments, the liposome is pegylated. In additional embodiments, the delivery vehicle comprises a targeting moiety that specifically binds a cell surface antigen expressed on the surface of a hematological cancer cell. In additional embodiments, the targeting moiety specifically binds a cell surface antigen selected from the group consisting of CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98. In a further embodiment, the delivery vehicle is a liposome, and the liposome comprises a targeting moiety that specifically binds a cell surface antigen selected from the group consisting of CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98.

[0189] In some embodiments, the disclosure provides for the use of a composition comprising a polyglutamated antifolate for the manufacture of a medicament for the treatment of a hyperproliferative disease. In some embodiments, the polyglutamated antifolate comprises five or more glutamyl groups. In some embodiments, the polyglutamated antifolate is pentaglutamated or hexaglutamated. In some embodiments, the polyglutamated antifolate is a member selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the polyglutamated antifolate is methotrexate (MTX). In some embodiments, the polyglutamated antifolate is polyglutamated pemetrexed (PMX). In some embodiments, the polyglutamated antifolate is polyglutamated lometrexol (LTX). In some embodiments, the polyglutamated antifolate is polyglutamated AG2034. In some embodiments, the polyglutamated antifolate is polyglutamated raltitrexed (RTX). In some embodiments, the polyglutamated antifolate is polyglutamated pralatrexate. In some embodiments, the polyglutamated antifolate is polyglutamated AG2034. In some embodiments, the polyglutamated antifolate is polyglutamated GW1843. In some embodiments, the polyglutamated antifolate is polyglutamated aminopterin. In some embodiments, the polyglutamated antifolate is polyglutamated LY309887. In some embodiments, the polyglutamated antifolate is in a liposome. In some embodiments, the hyperproliferative disease is cancer.In some embodiments, the cancer is selected from the group consisting of lung (e.g., non-small cell lung cancer), pancreatic, breast cancer, ovarian, lung, prostate, head and neck, stomach, gastrointestinal tract, colon, esophagus, cervix, kidney, bile duct, gallbladder, and hematological malignancies. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the hyperproliferative disease is an autoimmune disease. In some embodiments, the hyperproliferative disease is rheumatoid arthritis.

[0190] In some embodiments, the disclosed compositions (e.g., liposomes comprising polyglutamated antifolates) are administered to a subject having or at risk of having a cancer, solid cancer, and / or metastasis that is identifiable by the expression of a tumor-specific or tumor-associated antigen on the cell surface. Thus, in some embodiments, the present disclosure provides a method for treating cancer comprising administering an effective amount of a delivery vehicle (e.g., liposome) comprising a targeting moiety and a polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate to a subject having or at risk of having a cancer, solid cancer, and / or metastasis that is identifiable by the expression of a tumor-specific or tumor-associated antigen on the cell surface of the cancer, where the targeting moiety is displayed on the surface of the delivery vehicle and specifically binds the tumor-specific or tumor-associated antigen. In some embodiments, the polyglutamated antifolate administered is a member selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the administered polyglutamated antioxidant comprises a pentaglutamated antioxidant selected from the group consisting of pentaglutamated MTX, pentaglutamated PMX, pentaglutamated LTX, pentaglutamated AG2034, pentaglutamated RTX, pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887.In some embodiments, the polyglutamated antifolate administered comprises a hexaglutamated antifolate selected from the group consisting of hexaglutamated MTX, hexaglutamated PMX, hexaglutamated LTX, hexaglutamated AG2034, hexaglutamated RTX, hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887. In certain embodiments, the polyglutamated antifolate administered comprises pentaglutamated PMX. In certain embodiments, the polyglutamated antifolate administered comprises hexaglutamated PMX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated RTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated RTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated LTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated LTX. In some embodiments, the delivery vehicle administered is a liposome. In further embodiments, the liposome is pegylated. In additional embodiments, the delivery vehicle comprises a targeting moiety that specifically binds a cell surface antigen expressed on the surface of a cancer, solid tumor, and / or metastatic cell.In additional embodiments, the targeting moiety is selected from the group consisting of GONMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-alpha, folate receptor-beta, mucin 1, folate receptor-delta, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, L Specifically binds a cell surface antigen selected from the group consisting of IV-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, EGFR, CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98. In a further embodiment, the delivery vehicle is a liposome, and the liposome is capable of expressing any of the following receptors: GONMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-alpha, folate receptor-beta, mucin 1, folate receptor-delta, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue The targeting moiety specifically binds a cell surface antigen selected from the group consisting of: LIV-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, EGFR, CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98.

[0191] In a further embodiment, the present disclosure provides a method for treating cancer comprising administering to a subject having or at risk of having a cancer comprising cells expressing a folate receptor on the cell surface an effective amount of a delivery vehicle (e.g., a liposome) comprising a targeting moiety on its surface that specifically binds the folate receptor and a polyglutamylated (e.g., pentaglutamylated or hexaglutamylated) antifolate agent. In a further embodiment, the folate receptor is folate receptor alpha, folate receptor beta, or folate receptor delta. In some embodiments, the polyglutamated antifolate administered is a member selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887. In some embodiments, the administered polyglutamated antioxidant comprises a pentaglutamated antioxidant selected from the group consisting of pentaglutamated MTX, pentaglutamated PMX, pentaglutamated LTX, pentaglutamated AG2034, pentaglutamated RTX, pentaglutamated piritrexim, pentaglutamated pralatrexate, pentaglutamated AG2034, pentaglutamated GW1843, pentaglutamated aminopterin, and pentaglutamated LY309887. In some embodiments, the administered polyglutamated antioxidant comprises a hexaglutamated antioxidant selected from the group consisting of hexaglutamated MTX, hexaglutamated PMX, hexaglutamated LTX, hexaglutamated AG2034, hexaglutamated RTX, hexaglutamated piritrexim, hexaglutamated pralatrexate, hexaglutamated AG2034, hexaglutamated GW1843, hexaglutamated aminopterin, and hexaglutamated LY309887.In certain embodiments, the polyglutamated antifolate administered comprises pentaglutamated PMX. In certain embodiments, the polyglutamated antifolate administered comprises pentaglutamated PMX. In certain embodiments, the polyglutamated antifolate administered comprises pentaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated RTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated RTX. In some embodiments, the polyglutamated antifolate administered comprises pentaglutamated LTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated LTX. In some embodiments, the delivery vehicle administered is a liposome. In further embodiments, the liposome is pegylated. As disclosed herein, PEGylated liposomes targeting the folate receptor containing polyglutamated antifolates can deliver high amounts of polyglutamated antifolates to cancer cells, particularly cancer cells expressing folate receptors, compared to normal cells (i.e., cells that do not actively take up liposomes and / or do not express folate receptors, unlike cancer cells). Any cancer that expresses folate receptors can be treated according to the disclosed method. It should be noted that while some cancers may express folate receptors at early stages, many cancers may express folate receptors at later stages.

[0192] The cancer that can be treated by the method of the present invention includes carcinoma, sarcoma and melanoma.Carcinoma includes but is not limited to basal cell carcinoma, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS cancer, colorectal cancer, kidney or renal cell carcinoma, laryngeal cancer, liver cancer, small cell lung cancer, non-small cell lung cancer (NSCLC, including adenocarcinoma, giant (or oat) cell carcinoma and squamous cell carcinoma), oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer (including basal cell carcinoma and squamous cell carcinoma), stomach cancer, testicular cancer, thyroid cancer, uterine cancer, rectal cancer, respiratory system cancer and urinary system cancer.

[0193] Sarcomas are mesenchymal neoplasms arising in bone (osteosarcoma) and soft tissue (fibrosarcoma). Sarcomas include, but are not limited to, liposarcoma (including myxoid liposarcoma and pleomorphic liposarcoma), leiomyosarcoma, rhabdomyosarcoma, malignant peripheral nerve sheath tumor (also called malignant nerve sheath tumor, neurofibrosarcoma, or neurogenic sarcoma), Ewing's tumor (including Ewing's sarcoma of bone, extraosseous (i.e., not in bone), Ewing's sarcoma, and primitive neuroectodermal tumor), synovial sarcoma, angiosarcoma, angiosarcoma, lymphangiosarcoma, Kaposi's sarcoma, hemangioendothelioma, desmoid tumor (also called progressive fibromatosis), dermatofibrosarcomatous ureteritis (DFSP), malignant fibrous histiocytoma (MFH), hemangiopericytoma, malignant mesenchymoma, soft tissue sarcoma, epithelioid sarcoma, clear cell sarcoma, desmoplastic small cell tumor, gastrointestinal stromal tumor (GIST), and chondrosarcoma.

[0194] Melanoma is a tumor arising from the melanocytic system of the skin and other organs. Examples of melanoma include, but are not limited to, lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, and acral lentiginous melanoma.

[0195] In some embodiments, the cancer treated by one or more of the methods disclosed herein is a solid tumor lymphoma. Examples of solid tumor lymphoma include Hodgkin's lymphoma, non-Hodgkin's lymphoma, and B-cell lymphoma.

[0196] In some embodiments, the cancer treated by one or more of the methods disclosed herein is bone cancer, brain cancer, breast cancer, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasia, melanoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, prostate cancer, retinoblastoma, or rhabdomyosarcoma.

[0197] The disclosed methods can be performed on any subject that may benefit from delivery of the compositions contemplated herein (e.g., polyglutamated antifolates, such as liposomes containing pentaglutamated or hexaglutamated antifolates). Mammalian subjects, particularly human subjects, are preferred. In some embodiments, subjects also include animals, including domestic pets (e.g., dogs, cats, rabbits, and ferrets), livestock or farm animals (e.g., cows, pigs, sheep, chickens, and other poultry), horses, such as purebred horses, laboratory animals (e.g., mice, rats, and rabbits), and other animals. In other embodiments, subjects include fish and other aquatic species.

[0198] The subject to whom the agent is delivered may be a normal subject. Alternatively, the subject may have or be at risk of developing a condition that can be diagnosed or benefit from delivery of one or more of the compositions provided. In some embodiments, such conditions include cancer (e.g., solid tumor cancer or non-solid cancer such as lymphoma). In some embodiments, these conditions (e.g., cancer) include cells expressing antigens that can be specifically bound by the targeted PEGylated liposomal polyglutamated antifolates disclosed herein. In further embodiments, these antigens specifically bind and internalize the targeted PEGylated liposomal polyglutamated antifolates into the cells. In some embodiments, the targeted PEGylated liposomal polyglutamated antifolates specifically bind folate receptors (e.g., folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ)) expressed on the surface of cancer cells.

[0199] Tests for diagnosing conditions treatable by the provided compositions are known in the art and will be familiar to physicians. Determination of whether a cell type expresses a folate receptor can be made using commercially available antibodies. These laboratory tests include, but are not limited to, microscopic analysis, growth-dependent tests (such as culture), and nucleic acid detection tests. These include wet mounts, stain-enhanced microscopy, immunomicroscopy (e.g., FISH), hybridization microscopy, particle agglutination, enzyme-linked immunosorbent assay, urine screening test, DNA probe hybridization, and serological tests. Physicians will also generally consider a full history and perform a complete physical examination in addition to performing the above laboratory tests.

[0200] A subject with cancer may be, for example, a subject with detectable cancer cells.A subject at risk of developing cancer may be, for example, a subject with a higher than normal probability of developing cancer.These subjects include, for example, subjects with genetic abnormalities that have been shown to be associated with a high probability of developing cancer, subjects with a familial predisposition to cancer, subjects exposed to cancer-causing agents (i.e., carcinogens), such as tobacco, asbestos, or other chemical toxins, and subjects who have previously been treated for cancer and are in apparent remission.

[0201] In some embodiments, the present disclosure provides methods for selectively delivering folate receptor-targeted PEGylated liposomal polyglutamated antioxidants to a high percentage of tumor cells that express folate receptors on their surface (at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, or at least 5-fold higher than cells that do not express folate receptors on their cell surface).

[0202] In some embodiments, the present disclosure provides a method of making the liposome composition disclosed herein. In one embodiment, the method includes preparing a mixture including (1) liposome components, and (2) a polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate in an aqueous solution. In a further embodiment, the mixture includes PEGylated liposome components. The mixture is then homogenized to form liposomes in the aqueous solution. Furthermore, the mixture can be extruded through a membrane to form liposomes surrounding the polyglutamated antifolate in the aqueous solution. It is understood that the liposome components of the present disclosure can include any lipid (including cholesterol), including functionalized lipids and lipids bound to targeting moieties, detectable labels, and steric stabilizers, or any subset of all of these. Furthermore, it is noted that the bioactive polyglutamated antifolate in the aqueous solution can include any reagents and chemicals described herein or otherwise known in the art for the interior or exterior of the liposome, including, for example, buffers, salts, and cryoprotectants.

[0203] In some embodiments, the present disclosure provides a method for making the targeted PEGylated liposomal polyglutamated antifolate (targeted PLPA) or non-targeted PLPA disclosed herein. In one embodiment, the method includes preparing a mixture including (1) liposome components, (2) polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate in aqueous solution, and (3) a targeting moiety. The mixture is then homogenized to form liposomes in aqueous solution. The mixture can further be extruded through a membrane to form liposomes surrounding the polyglutamated antifolate in aqueous solution. It is understood that the targeted PEGylated liposome components can include any lipid (including cholesterol), including functionalized lipids and lipids that are bound to targeting moieties, detectable labels, and steric stabilizers, or any subset of all of these. It is further noted that targeted PEGylated liposomes may contain any of the reagents and chemicals described herein or otherwise known in the art on the interior or exterior of the liposomes, including, for example, buffers, salts, cryoprotectants, and the like.

[0204] The above method optionally further comprises the step of freeze-drying the composition after the step of removing to prepare a freeze-dried composition.As mentioned above, the targeted PTPLA or non-targeted PTPLA in aqueous solution can include a cryoprotectant as described herein or otherwise known in the art.When the composition is freeze-dried, a cryoprotectant may be preferred.

[0205] Additionally, after the lyophilization step, the method optionally further comprises a step of reconstituting the lyophilized composition by dissolving the composition in a solvent after the lyophilization step. Methods of reconstitution are known in the art. One exemplary solvent is water. Other solvents include saline and buffered solutions.

[0206] Although specific example embodiments are discussed herein, it is understood that liposomes can be prepared by any method known in the art. See, for example, G. Gregoriadis (editor), Liposome Technology, vol. 1-3, 1st edition, 1983; 2nd edition, 1993, CRC Press, 45 Boca Raton, Fla. Examples of suitable methods for making liposome compositions include extrusion, reverse phase evaporation, sonication, solvent (e.g., ethanol) injection, microfluidization, detergent dialysis, ether injection, and dehydration / rehydration. The size of liposomes can be routinely controlled by controlling the pore size of the membrane used for low pressure extrusion or pressurization and the pressure and permeation number used in microfluidization, or by other suitable methods known in the art.

[0207] Generally, the polyglutamated antifolate is contained within the liposome, i.e., in the inner (internal) space. In one embodiment, the substituted ammonium is partially or almost completely removed from the external medium surrounding the liposome. Such removal can be achieved by any suitable means known in the prior art (e.g., dilution, ion exchange chromatography, size exclusion chromatography, dialysis, ultrafiltration, and precipitation). Thus, the above-mentioned otherwise known methods of making liposomes may optionally further comprise a step of removing the polyglutamated antifolate in the aqueous solution outside the liposome after the extrusion step.

[0208] In another embodiment, the present disclosure provides a targeted PEGylated liposomal polyglutamated antifolate (PLPA) that selectively targets folate receptor, which comprises a liposome that comprises an internal space, a polyglutamated antifolate disposed in the internal space, a steric stabilizer molecule that is bound to the outside of the liposome, and a targeting moiety that comprises a protein having specific affinity for at least one type of folate receptor, and the targeting moiety is bound to at least one of the steric stabilizer and the outside of the liposome.The components of this embodiment can be the same as those described in other embodiments of the present disclosure.For example, the targeted PEGylated liposomal polyglutamated antifolate and the steric stabilizer that can be PEG are as described in other parts of the present disclosure.

[0209] In some embodiments, the disclosure provides a method of preparing a non-targeted composition comprising liposomes containing entrapped and / or encapsulated polyglutamated antifolate, the method comprising forming a mixture comprising liposome components, polyglutamated antifolate in solution; homogenizing the mixture to form liposomes in solution; and treating the mixture to form liposomes entrapping and / or encapsulating the polyglutamated antifolate. In some embodiments, the treatment comprises one or more of: thin film hydration, extrusion, in-line mixing, and agitation, and once the particles are formed, the particles can be further modified in their size by one or more of extrusion and sonication. In some embodiments, the non-targeted composition comprises at least 10% liposome-entrapped polyglutamated antifolate. In some embodiments, the liposomes are anionic or neutral. In some embodiments, the liposomes are cationic.

[0210] In some embodiments, the present disclosure provides a method of preparing a targeted composition comprising a PEGylated liposome containing an entrapped and / or encapsulated polyglutamated antifolate, wherein the targeting moiety is an amino acid chain, the amino acid chain comprises a plurality of amino acids, and the targeting moiety has a specific affinity for at least one type of folate receptor, the specific affinity being greater than or equal to 0.5×10 for at least one type of folate receptor.-10 ~10×10 -6 The liposome composition is determined to have an equilibrium dissociation constant (Kd) in the range of 0.05 nanomolar to 10 micromolar, the targeting moiety being attached to one or both of the PEG and the exterior of the liposome, and the method includes preparing a mixture including liposome components, a polyglutamated antifolate in solution, homogenizing the mixture to form liposomes in the solution, treating the mixture to form liposomes that capture and / or encapsulate the polyglutamated antifolate, and providing a targeting moiety on the surface of the liposomes that capture and / or encapsulate the polyglutamated antifolate, the targeting moiety having a specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ). In some embodiments, the treatment includes one or more of thin film hydration, extrusion, in-line mixing, and agitation, and once the particles are formed, the particles can be further modified in their size by one or more of extrusion and sonication. In some embodiments, the targeting composition comprises at least 10% liposome-entrapped polyglutamated antifolate. In some embodiments, the liposome is anionic or neutral. In some embodiments, the targeting moiety has specific affinity for one or more of folate receptor alpha (FR-α), folate receptor beta (FR-β) and folate receptor delta (FR-δ). In further embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α) and folate receptor beta (FR-β). In additional embodiments, the targeting moiety has specific affinity for an epitope on a tumor cell surface antigen that is present on tumor cells but not present or accessible on non-tumor cells.

[0211] example The following examples are intended to illustrate the present disclosure in any manner, shape, or form, without being limited thereto, explicitly or implicitly. These are typical of those that may be used, but other procedures, methodologies, or techniques known to those skilled in the art may be used instead. The exemplary composition includes exemplary liposomes. Both the exemplary composition and the exemplary liposomes are used in the experiments described in the Examples section and are specific embodiments of the present disclosure throughout the present disclosure, and are not intended to define the full scope of the present disclosure.

[0212] Figure 5 shows the chemical formula of L-alpha pentaglutamated pemetrexed. Once inside the cell, pemetrexed is converted by the enzyme folylpolyglutamate synthetase to gamma polyglutamate form, where each glutamic acid is in the L form. Furthermore, each bond is through the gamma carboxyl of a glutamic acid.

[0213] 6 shows the chemical formula of an example L-gamma polyglutamated antioxidant composition encompassed by this disclosure. The glutamic acids are in the L form and are linked through the gamma carboxyl of the glutamic acid.

[0214] method Preparation of hexaglutamylated pemetrexed (HGP) liposomes Briefly, γHGP (gG6) was encapsulated in liposomes by the following procedure. First, the lipid components of the liposomal membrane were weighed out and mixed as concentrated solutions in ethanol at a temperature of about 65 °C. In this example, the lipids used were homogenized soy phosphatidylcholine, cholesterol, and DSPE-PEG-2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). The molar ratio of HSPC:cholesterol:PEG-DSPE was about 3:2:0.15. Next, gG6 was dissolved in an aqueous buffer at a pH of 6.5-6.9 at a concentration of 20 mg / mL. The drug solution was heated to 65 °C. The ethanol lipid solution was injected into gG6 through a small-bore needle. During this step, the drug solution was thoroughly stirred by a magnetic stirring device. Mixing was performed at elevated temperatures (63-72 °C) to ensure that the lipids were in a liquid crystalline state (as opposed to a gel state, which is reached below the liquid transition temperature, Tm = 51-54 °C). As a result, the liquid became hydrated and formed multiple bilayer (multilamellar) vesicles (MLVs) containing gG6 in their aqueous cores.

[0215] Size reduction of MLVs using filter extrusion MLVs were fractionated into unilamellar (single bilamellar) vesicles of the desired size by high-pressure extrusion using two passes through a laminated (track-etched polycarbonate) membrane. The laminated membrane had two layers with a pore size of 200 nm and six layers with a pore size of 100 nm. During extrusion, the temperature was maintained above the Tm to ensure the plasticity of the liquid membrane. As a result of extrusion, the MLVs, which were highly heterogeneous in size and stratification, became small, uniform (100-120 nm) unilamellar vesicles (ULVs) that sequestered the drug within them. A Malvern Zetasizer Nano ZS instrument (Southborough, MA) with a backscatter detector (90°) was used to measure the hydrodynamic size (diameter) in a quartz microcuvette at 25°C. Samples were diluted 50-fold in the formulation matrix before analysis.

[0216] Liposome purification After the ULVs containing gG6 were prepared, the extraliposomal gG6 was removed using a small volume column or tangential flow diafiltration against a buffer suitable for large volumes. Any buffer solution can be used, but in this example, the buffer used was 5 mM HEPES, 145 mM sodium chloride, pH 6.7. At the end of the purification, filtration sterilization was performed using a 0.22 micron filter.

[0217] Antibody conjugation Activated liposomes were prepared by adding DSPE-PEG-maleimide to the lipid composition. The liposomes contained four different lipids: hydrogenated soy phosphatidylcholine (HSPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG-2000), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG-maleimide), in a ratio of 3:2:0.1125:0.0375.

[0218] Antibody thiolation was performed by the use of Traut's reagent (2-iminothiolane) to couple sulfhydryl groups to primary amines. Antibodies were suspended in PBS at concentrations of 0.9-1.6 mg / mL. Traut's reagent (14 mM) was added to the antibody solution at a final concentration of 1-5 mM and then removed by dialysis after 1 h incubation at room temperature. The thiolated antibody was added to the activated liposomes at a ratio of 60 g / mol phospholipid and the reaction mixture was incubated for 1 h at room temperature and overnight at 4°C. The reaction was stopped using L-cysteine ​​and unconjugated antibody was removed by dialysis. [Table 1]

[0219] Dose-response study of HGP (pentaglutamated pemetrexed) and liposomes Cell viability was measured on day 3 (48 hours) and day 4 (72 hours) by CellTiter-Glo® (CTG) luminescent cell viability assay. This assay measures the number of live cells in culture based on quantifying the ATP present therein, which in turn indicates the presence of metabolically active cells. The CTG assay uses luciferase as a readout. To evaluate the dose-response inhibition of cell viability of pemetrexed, HGP and liposomes in different cancer cell proliferation were examined using CellTiter-Glo® luminescent cell viability assay. Human cancer cells were harvested, counted and plated at the same cell density on day 0. Eight serial dilutions of each test substance were added to the cells on day 1. Dose-response curves were generated and fitted using GraphPad Prism, and the IC50 of each test substance was calculated. The lower the IC50, the more potent the test substance was in inhibiting cancer cell proliferation.

[0220] Cells were seeded into 96-well plates at a cell density of 5 x 104 cells per well in 100 μL of fresh medium on day 0. Eight two-fold serial dilutions of each test substance in culture medium were prepared and added to the cells in triplicate on day 1. In addition, three wells of cells were treated with vehicle alone (drug-free HBS or empty liposomes for liposomal HGP) as controls.

[0221] On days 3 and 4, 100 μL of CellTiterGlo® was added to each well and incubated at room temperature for 15 minutes. Luciferase luminescence was recorded for each well. Additionally, eight two-fold serial dilutions of vehicle (HBS or empty liposomes) in culture medium were added to blank wells and included in the assay to generate background luminescence signals. Luciferase signals were normalized by subtracting the background luminescence signal from each reading.

[0222] Human normal primary bone marrow CD34+ cells were obtained from ATCC (ATCC, Catalog No. PCS-800-012). Cells were thawed for 1 min at 37°C and then placed on ice. Cells were then resuspended in StemSpan SFEM (Stem Cell Tech Catalog No. 9650) + 10% heat-inactivated fetal bovine serum (Corning, 35-015-CV). Cells were seeded into 96-well culture plates at a density of 2.5 x 104 cells / well. The next day, viable cells were collected by centrifugation and resuspended in neutrophil growth medium (StemSpan SFEM) + 10% heat-inactivated fetal bovine serum + 100ng / mL human stem cell factor (Sigma, Cat. No. H8416), 20ng / mL human granulocyte colony-stimulating factor (Sigma, Cat. No. H5541), and 10ng / mL human recombinant IL3 (Sigma, SRP3090) at a density of 2.5x104 cells / well. Cells were incubated at 37°C for 10 days. Fresh medium was added every 2 days. Mature neutrophils were then collected and seeded in 96-well plates at a density of 1x104 cells / well and incubated overnight at 37°C. The next day, test substances or vehicle were resuspended in neutrophil medium and added to the plates. Cells were then incubated at 37°C for either 48 or 72 hours before being measured at each time point using the Cell Titer Glo Assay (Promega, Cat. No. G7572).

[0223] The methodology used with the cell lines AML12 (non-cancerous hepatic cells) and CCD841 (non-cancerous colonic epithelial cells) is similar to that used for cancer cells.

[0224] result The dose-response relationships of free pemetrexed gamma hexaglutamate (gG6), (non-targeted) liposomal pemetrexed gamma hexaglutamate (liposomal gG6), pemetrexed, and the folate receptor alpha targeted antibody (FR1Ab) liposomal pemetrexed gamma hexaglutamate (liposomal gG6-FR1Ab) in NCI H2342 non-small cell lung cancer (NSCLC), adenocarcinoma subtype, are shown in Figure 7. The outcome is cell viability as measured by luciferase luminescence. As shown in this Figure 7, free pemetrexed gG6 appears to be the least potent as measured by IC50. Both liposomal pemetrexed gG6 and liposomal pemetrexed gG6-FR1Ab are 7-fold and 40-fold more potent than free pemetrexed, respectively. Figures 8 and 9 show similar data as percent viable cells 48 hours after treatment.

[0225] Similar data is shown in Figures 10 and 11, which show cell viability expressed as a percentage, for the HT-29 colon cancer cell line. As shown in these figures, free pemetrexed gG6 appears to be the least potent. In this example, liposomal pemetrexed gG6 is 2-fold more potent than pemetrexed, and liposomal pemetrexed gG6-FR1Ab is 5-fold more potent than free pemetrexed.

[0226] Additional cell lines, namely OAW28 ovarian cancer cells and SW260 colon cancer cells, were also treated with pemetrexed gamma G5 (also called gG6) formulations. As shown in Figure 12, consistent with the data above, free pemetrexed gG6 was the least effective, and liposomal pemetrexed gG6 appears to be more effective than pemetrexed.

[0227] FIG. 13 shows that liposomal pemetrexed gG6-FR1Ab results in numerically greater inhibition of cancer cells than non-targeted liposomal pemetrexed gG6 and free pemetrexed gG6.

[0228] In another series of dose-response experiments, six cell lines representing different types of cancer were tested: HT-29 (colon cancer), H2342 (NSCLC, adenocarcinoma subtype), H292 (NSCLC, adenocarcinoma subtype), SW620 (CRC), H1806 (triple-negative breast cancer), and OAW28 (ovarian cancer) (Figure 14). Treatment consisted of a 48-hour exposure with liposomal pemetrexed hexaglutamate (liposomal gG6).

[0229] After 48 hours of exposure, the relative potency of all of the above derivatives compared to pemetrexed is shown in Figure 14. The relative potency of treatment with the various derivatives was calculated by dividing the IC50 of pemetrexed by the IC50 of liposomal pemetrexed hexaglutamate for each cell line, as shown in the figure. As shown in the figure, the potency of liposomal pemetrexed hexaglutamate well exceeded that of pemetrexed in all cell lines. For example, consider the NSCLC cell line H292. As shown in the figure, the potency of liposomal pemetrexed hexaglutamate ranged from 25 to 50 times greater than that of pemetrexed. This suggests that a 4% or lower dose of liposomal pemetrexed hexaglutamate has the same therapeutic effect as a 100% dose of pemetrexed.

[0230] Cancer cell viability studies comparing liposomal pemetrexed hexaglutamate (Liposome gG6 / Lps Hexa gG6) with pemetrexed for cytotoxic activity against representative cell lines in breast, lung, and ovarian cancer are shown in Figures 15-17. These data indicate that liposomal pemetrexed hexaglutamate is more potent than pemetrexed. Additionally, as an indication of efficacy, results of experiments in the same cell lines are shown at various doses ranging from 16 to 128 nM in Figures 18-20. As shown in these figures, at each of these dose ranges, liposomal pemetrexed hexaglutamate is superior to pemetrexed in inhibiting cancer cells in lung and breast cancer cell lines (Figures 18 and 19, respectively). In ovarian cancer cell lines, at a dose of 128 nM pemetrexed appears to be as effective as liposomal pemetrexed hexaglutamate (see FIG. 20), while at doses of 32 nM and 64 nM liposomal pemetrexed hexaglutamate has better therapeutic efficacy than pemetrexed, and at 16 nM the therapeutic efficacy is less, being comparable for liposomal pemetrexed hexaglutamate and pemetrexed.

[0231] The main toxicity observed in patients treated with pemetrexed is myelosuppression, which manifests as a drop in blood cell counts, including neutrophil counts (a type of white blood cell). There are also some adverse effects on the lining of the oral cavity and digestive tract, manifested as diarrhea and mucositis, as well as in some cases adverse effects on the liver. To assess the above toxicity, liposomal pemetrexed hexaglutamate and pemetrexed treatments were measured at 48 hours in neutrophils, CCD841 colonic epithelial cells, and CD34+ cells differentiated into AML12 hepatocytes. As shown in Figure 21, liposomal pemetrexed hexaglutamate is significantly less toxic to differentiating human neutrophils compared to pemetrexed. This is also supported by the neutrophil counts, which are better maintained after treatment with the derivative compared to pemetrexed in the dose range of 16-128 nM (Figure 21). Strikingly, there does not appear to be any toxicity to hepatocytes following treatment with liposomal pemetrexed hexaglutamate at the dose levels tested (Figure 22). In contrast, pemetrexed at all doses tested results in approximately a 40% decrease in hepatocyte count. And finally, the same trend is observed following treatment of epithelial colonocytes (Figure 23). As shown in this figure, pemetrexed at all doses tested results in approximately a 50% or greater decrease in cell count compared to approximately a 20% or less decrease following treatment with liposomal pemetrexed hexaglutamate.

[0232] In a non-limiting exemplary embodiment of the present disclosure, a composition comprising a polyglutamated antifolate is provided.

[0233] In the composition of the immediately preceding paragraph, the composition may include a pentaglutamated or hexaglutamated antifolate agent.

[0234] In the composition of either of the above two paragraphs, the polyglutamated antioxidant can be one or more members selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887.

[0235] In the composition of any of the above three paragraphs, the composition may comprise a polyglutamylated antifolate, which may be polyglutamylated PMX, MTX, RTX, or LTX.

[0236] In the composition of any of the above four paragraphs, the composition may include a polyglutamated antifolate, which may include a pentaglutamated or hexaglutamated antifolate.

[0237] In the composition of any of the above five paragraphs, the composition may include a polyglutamated antifolate, which may include pentaglutamated or hexaglutamated PMX, MTX, RTX, and / or LTX.

[0238] A non-limiting example liposomal polyglutamated antifolate (LPA) composition can include any of the compositions in the above six paragraphs, where the liposomes can optionally be pegylated (PLPA).

[0239] In the LPA or PLPA composition of the immediately preceding paragraph, the polyglutamated antifolate may comprise a pentaglutamated or hexaglutamated antifolate.

[0240] In the LPA or PLPA composition of either of the above two paragraphs, the polyglutamated antioxidant can be one or more members selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887.

[0241] In the LPA or PLPA composition of any of the above three paragraphs, the polyglutamated antifolate is polyglutamated PMX, MTX, RTX, and / or LTX.

[0242] In the LPA or PLPA composition of any of the above four paragraphs, the polyglutamated antifolate may comprise a pentaglutamated or hexaglutamated antifolate.

[0243] In the LPA or PLPA composition of any of the above five paragraphs, the polyglutamated antifolate may comprise pentaglutamated or hexaglutamated PMX, MTX, RTX, and / or LTX.

[0244] In the LPA or PLPA compositions of any of the above six paragraphs, the liposomes can be anionic or neutral.

[0245] In the LPA or PLPA compositions of any of the seven paragraphs above, a targeting moiety may be attached to one or both of the PEG and the exterior of the liposome, and the targeting moiety may have a specific affinity for a surface antigen on a target cell of interest.

[0246] In the LPA or PLPA compositions of any of the above eight paragraphs, the targeting moiety may be attached to one or both of the PEG and the exterior of the liposome, and may be a polypeptide.

[0247] In the LPA or PLPA compositions of any of the above nine paragraphs, the targeting moiety may be attached to one or both of the PEG and the exterior of the liposome, and may be an antibody or a fragment of an antibody.

[0248] In the LPA or PLPA of any of the above 10 paragraphs, one or more of the immunostimulant, detectable marker, and maleimide may be disposed on at least one of the PEG and the exterior of the liposome.

[0249] In any of the LPAs or PLPAs of paragraph 11 above, the polypeptide has a molecular weight of 0.5×10 as measured using Biacore analysis. -10 ~10×10 -6 The antigen can be bound with an equilibrium dissociation constant (Kd) in the range of

[0250] In any of the LPAs or PLPAs of the above 12 paragraphs, the polypeptide can specifically bind 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).

[0251] A non-limiting exemplary method of killing hyperproliferative cells may include contacting the hyperproliferative cells with any of the liposomal polyglutamated antifolate compositions of paragraphs

[0254] -

[0272] .

[0252] In the method of the immediately preceding paragraph, the hyperproliferative cells can be cancer cells.

[0253] A non-limiting exemplary method for treating cancer is administering to a subject having or at risk of having cancer a therapeutic agent as described in paragraph 1.

[0254] The method may include administering an effective amount of any of the polyglutamylated antifolates listed in any one of the following:

[0254] In the method of the immediately preceding paragraph, the cancer can be one or more selected from the group consisting of lung cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colon cancer, esophageal cancer, cervical cancer, kidney cancer, bile duct cancer, gallbladder cancer, and hematological malignancies.

[0255] A non-limiting exemplary maintenance therapy for a subject undergoing or having undergone cancer therapy may include administering to a subject undergoing or having undergone cancer therapy an effective amount of any of the polyglutaminated antifolates of paragraphs

[0254] to

[0272] .

[0256] Non-limiting exemplary pharmaceutical compositions may include any of the polyglutamated antifolate compositions of paragraphs

[0254] to

[0272] .

[0257] A non-limiting exemplary method for treating an immune system disorder can include administering to a subject having or at risk of having an immune system disorder an effective amount of any of the polyglutaminated antifolate compositions of paragraphs

[0254] to

[0272] .

[0258] A non-limiting exemplary method for treating an infectious disease may include administering to a subject having or at risk of having an infectious disease an effective amount of any of the polyglutaminated antifolate compositions of paragraphs

[0254] to

[0272] .

[0259] A non-limiting exemplary method of delivering a polyglutamated antifolate to a tumor expressing a folate receptor on its surface may include administering to a subject having a tumor a polyglutamated antifolate composition of any of paragraphs

[0254] to

[0272] in an amount that delivers a therapeutically effective dose of the polyglutamated antifolate to the tumor.

[0260] A non-limiting exemplary method of preparing a liposomal polyglutamated antifolate composition, which may include the polyglutamated antifolate composition of any of the paragraphs, includes forming a mixture containing liposome components, a polyglutamated antifolate in solution, homogenizing the mixture to form liposomes in the solution, and treating the mixture to form liposomes containing the polyglutamated antifolate.

[0261] Non-limiting exemplary pharmaceutical compositions may include any of the polyglutamated antifolate compositions of paragraphs

[0254] to

[0272] .

[0262] Although the present disclosure has been described with reference to several different embodiments, it should be understood that various modifications can be made without departing from the spirit of the present disclosure. Accordingly, the scope of the present disclosure should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all cited documents and references, including patent applications and publications, are incorporated herein by reference for all purposes.

[0263] Various novel chemical entities, methods and apparatus for making these chemical entities are described below in the accompanying claims.

Claims

1. A liposome composition comprising a liposome encapsulating a polyglutamated antifolate, the polyglutamated antifolate comprising 2 to 10 glutamic acid residues containing gamma carboxyl linkages, the polyglutamated antifolate being selected from the group consisting of polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed (RTX), polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamated lometrexol (LTX), ... methotrexate (MTX), polyglutam TX), polyglutaminated piritrexim, polyglutamated pralatrexate, polyglutamated AG2034, polyglutamated GW1843, polyglutamated aminopterin, and polyglutamated LY309887, wherein the liposome comprises polyethylene glycol, has a zeta potential of zero or less, and contains a targeting moiety having specific affinity for a surface antigen of a target cell.

2. The liposome composition described in claim 1, wherein the polyglutamated antifolate is polyglutamated pemetrexed (PMX).

3. The liposome composition of claim 1, wherein the polyglutamylated antifolate is polyglutamylated MTX, polyglutamylated RTX, or polyglutamylated LTX.

4. The liposome composition described in claim 1, wherein the polyglutamated antifolate contains a pentaglutamated antifolate or a hexaglutamated antifolate.

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

6. The liposome composition of claim 1, wherein the targeting moiety is a polypeptide or one or more selected from the group consisting of an antibody, a humanized antibody, an antigen-binding fragment of an antibody, a single-chain antibody, a single-domain antibody, a bispecific antibody, a synthetic antibody, a pegylated antibody, and a multivalent antibody.

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

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

9. The liposome formed from liposome components, the liposome components comprising: at least one of anionic lipids and neutral lipids; At least one selected from the group consisting of DSPE, DSPE-PEG-maleimide, HSPC, HSPC-PEG, cholesterol, cholesterol-PEG, and cholesterol-maleimide; or At least one selected from the group consisting of DSPE, DSPE-PEG-FITC, DSPE-PEG-maleimide, cholesterol, and HSPC The liposome composition of claim 1 , comprising:

10. The liposome composition of claim 9, wherein one or more liposome components further comprise at least one steric stabilizer selected from the group consisting of 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.

11. The liposome composition of claim 1, wherein the polyethylene glycol has a number average molecular weight (Mn) of 200 to 5,000 daltons.

12. The liposome composition of claim 1, wherein the liposome has a zeta potential of 0 to -150 mV, or a zeta potential of -30 to -50 mV.

13. The liposome composition of claim 1, wherein the liposome contains less than 200,000, or between 10,000 and 100,000 molecules of polyglutamated antifolate.

14. The liposome composition of claim 1, wherein the polyglutamated antifolate has a pH of 5 to 8.

15. The liposome composition of claim 1, wherein the polyglutamated antifolate agent contains 4 to 10 glutamic acid residues including gamma carboxyl group bonds.

16. The liposome composition of claim 1, further comprising at least one cryoprotectant selected from the group consisting of mannitol, trehalose, sorbitol, and sucrose.

17. The liposome composition of claim 1, which is a unit dosage form.

18. A pharmaceutical composition comprising the liposome composition described in claim 1.

19. The pharmaceutical composition of claim 1, wherein the liposome has a zeta potential of less than zero.

20. The pharmaceutical composition of claim 19, wherein the polyglutamylated antifolate is polyglutamylated PMX, polyglutamylated MTX, polyglutamylated RTX, or polyglutamylated LTX.

21. The pharmaceutical composition described in claim 19, wherein the polyglutamated antifolate contains a pentaglutamated antifolate or a hexaglutamated antifolate.

22. The pharmaceutical composition of claim 19, wherein the liposomes have a diameter in the range of 20 nm to 200 nm or 80 nm to 120 nm, and the liposomes have a zeta potential of 0 to -150 mV or -30 to -50 mV.

23. The pharmaceutical composition of claim 19, wherein the polyglutamated antifolate agent contains 4 to 10 glutamic acid residues including gamma carboxyl group bonds.

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