Polyglutamated antifolates and uses thereof

Abstract

Compositions are provided for treating diseases including hyperproliferative diseases such as cancer, disorders of the immune system such as rheumatoid arthritis, and infectious diseases such as HIV. [Solution] Provided are polyglutamated antifolates, formulations containing liposomes loaded with polyglutamated antifolates, methods for preparing polyglutamated antifolates and liposome-containing formulations, and methods for treating hyperproliferative disorders (e.g., cancer) and immune system disorders (e.g., autoimmune diseases such as rheumatoid arthritis) using formulations containing polyglutamated antifolates and liposomes.

Description

[Technical Field]

[0001] Related Projects This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 62 / 374,458, filed August 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 August 11, 2017, and U.S. Application No. 15 / 675,701, filed August 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 a delivery vehicle, such as a targeted or non-targeted 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.

[0003] Folate is essential for cell proliferation and tissue regeneration. Mammalian cells do not synthesize folate de novo but rely on extracellular folates taken up by three major folate uptake pathways: the reduced folate carrier (RFC) system, the folate receptor (FR) α and β systems, and the folate cotransporter (PCFT) system. Antifolates that target folate-dependent biosynthetic pathways function as antiproliferative agents. Naturally occurring folates are present intracellularly 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 major purposes: (1) promotes the accumulation of intracellular folates in a vast excess of monoglutamate pools that are freely mobile in and out of cells; (2) allows selective intracellular retention of these relatively large anionic molecules; and (3) 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 first developed over 70 years ago as "folate-mimetic" cytotoxic agents. The rationale was to design a molecular family that counteracts the effects of folate in rapidly replicating cells, such as cancer cells, by exploiting physiological folate transport mechanisms and their stimulatory intracellular mechanism of action on DNA replication during cell division. 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 on DNA and RNA synthesis, exerting 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 these enzymes suppresses new nucleotide biosynthesis, resulting in an imbalance between purine and pyrimidine precursors, preventing cells from accurately replicating DNA and ultimately leading to cell death. It is therefore not surprising that inhibitors of the folate metabolic pathway 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 fused 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 polyglutamated by folylpoly-γ-glutamate synthetase. The polyglutamated form of pemetrexed has higher intracellular retention and higher affinity for TS and GARFT compared with 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 effects of such agents on normal healthy cells. To address the toxicity of antifolates in normal cells, WO2016 / 25882, for example, reports a liposomal formulation of antifolates that is targeted to cancer cells using an antibody with specific affinity for the folate receptor expressed by many cancer cells. This formulation can reduce and / or minimize the effects 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 remains 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 relates generally 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, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV. The 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, immune system disorders, and infectious diseases.

[0011] By way of example and not limitation, the present disclosure describes liposomal compositions containing 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 improved efficacy and safety for delivering antifolates to cancer cells by preferentially delivering a higher cytotoxic payload (e.g., polyglutamylated antifolates) compared to the cytotoxicity of the antifolates in their monoglutamylated forms. The present disclosure also provides targeted liposomal compositions containing a targeting moiety with specific affinity for an epitope (antigen) expressed on the surface of a target cell of interest. 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 present 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 entrapped with a polyglutamated antifolate. In some embodiments, the PLPA liposomes have a diameter ranging from 20 to 200 nm, 30 to 175 nm, or 50 to 150 nm. In some embodiments, the PLPA liposomes have diameters in the range of 30 to 175 nm or 50 to 150 nm, hi further embodiments, the PLPA liposomes have diameters 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 hexaglutamated PMX, MTX, RTX, or LTX. In one embodiment, the PLPA composition comprises polyglutamated PMX. In a further embodiment, the PLPA composition comprises pentaglutamated PMX. In a further embodiment, the PLPA composition comprises hexaglutamated 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 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 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 an antibody fragment. 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 specific affinity for an epitope on a tumor cell surface antigen that is present on tumor cells but absent or inaccessible on non-tumor cells. In some embodiments, the targeting moiety-PLPA further comprises one or more of an immunostimulatory agent, 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 diameters ranging from 20 to 200 nm. In further embodiments, the liposomes have diameters ranging from 80 to 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 is greater than or equal to 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 methods for 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 methods are performed in vivo. In some embodiments, the methods are 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, kidney cancer, bile duct cancer, gallbladder cancer, and hematological malignancies.

[0026] In additional embodiments, the present 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 administered 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 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 polyglutamated PMX, MTX, RTX, and / or LTX. In further embodiments, the administered composition comprises pentaglutamated forms of PMX, MTX, RTX, and / or LTX. In further embodiments, the administered composition comprises hexaglutamated forms of PMX, MTX, RTX, and / or LTX. In one embodiment, the administered composition comprises pentaglutamated forms of PMX. In one embodiment, the administered composition comprises hexaglutamated forms of PMX. In another embodiment, the administered composition comprises pentaglutamated forms of MTX. In another embodiment, the administered composition comprises hexaglutamated forms of MTX. In another embodiment, the administered composition comprises pentaglutamated forms of RTX.In another embodiment, the administered composition comprises a hexaglutamated form of RTX. In a further embodiment, the administered composition comprises a pentaglutamated form of LTX. In a further embodiment, the administered composition comprises a hexaglutamated form of LTX.

[0027] In additional embodiments, the present disclosure provides methods 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 administered 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 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 polyglutamated PMX, MTX, RTX, and / or LTX. In some embodiments, the administered composition comprises pentaglutamated PMX, MTX, RTX, and / or LTX. In some embodiments, the administered composition comprises hexaglutamated PMX, MTX, RTX, and / or LTX. In a further embodiment, the administered composition comprises pentaglutamated PMX. In a further embodiment, the administered composition comprises hexaglutamated PMX. In another further embodiment, the administered composition comprises pentaglutamated MTX. In another further embodiment, the administered composition comprises pentaglutamated MTX. In another further embodiment, the administered composition comprises pentaglutamated RTX. In another further embodiment, the administered composition comprises hexaglutamated RTX.In yet a further embodiment, the administered composition comprises a pentaglutamylated form of LTX. In yet a further embodiment, the administered composition comprises a hexaglutamylated form of LTX.

[0028] In some embodiments, the present disclosure provides methods 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 present disclosure provides methods for treating cancer, comprising administering a liposomal polyglutamated antifolate composition to a subject having or at risk of having a cancer that expresses a folate receptor on its surface that is bound by the targeting moiety, comprising a polyglutamated antifolate and a targeting moiety having specific binding affinity for the folate receptor. In further embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ).

[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 administered 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 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 polyglutamated forms of PMX, MTX, RTX, and / or LTX. In some embodiments, the administered composition comprises pentaglutamated forms of PMX, MTX, RTX, and / or LTX. In some embodiments, the administered composition comprises hexaglutamated forms of PMX, MTX, RTX, and / or LTX. In further embodiments, the administered composition comprises pentaglutamated forms of PMX. In further embodiments, the administered composition comprises hexaglutamated forms of PMX. In another further embodiment, the administered composition comprises pentaglutamated forms of MTX. In another further embodiment, the administered composition comprises hexaglutamated forms of MTX. In another further embodiment, the administered composition comprises pentaglutamated forms of RTX. In another further embodiment, the administered composition comprises hexaglutamated forms of RTX. In yet a further embodiment, the administered composition comprises a pentaglutamylated form of LTX. In yet a further embodiment, the administered composition comprises a hexaglutamylated form of LTX.

[0030] In additional embodiments, the present disclosure provides methods 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 present 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 polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate composition comprising a liposomal polyglutamated (e.g., pentaglutamated or hexaglutamated) 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 administered polyglutamate 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 further embodiments, the administered composition comprises a pentaglutamated antifolate. In further embodiments, the administered composition comprises a hexaglutamate antifolate. In some embodiments, the administered composition comprises polyglutamated PMX, MTX, RTX, and / or LTX. In some embodiments, the administered composition comprises pentaglutamated forms of PMX, MTX, RTX, and / or LTX. In some embodiments, the administered composition comprises pentaglutamated forms of PMX, MTX, RTX, and / or LTX. In another further embodiment, the administered composition comprises hexaglutamated forms of MTX. In a further embodiment, the administered composition comprises pentaglutamated forms of PMX. In a further embodiment, the administered composition comprises hexaglutamated forms of PMX. In another further embodiment, the administered composition comprises pentaglutamated forms of RTX.In another further embodiment, the administered composition comprises a hexaglutamated form of RTX. In an additional further embodiment, the administered composition comprises a pentaglutamated form of LTX. In an additional further embodiment, the administered composition comprises a hexaglutamated form of LTX.

[0032] The present disclosure also provides a method for delivering a polyglutamate antifolate to a tumor, comprising administering to a subject having a tumor a liposomal polyglutamated (e.g., pentaglutamated and / or hexaglutamated) 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 at a therapeutically effective dose.

[0033] In additional embodiments, the present disclosure provides methods for preparing compositions comprising liposomal polyglutamated antifolate compositions, the methods 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 polyglutamated (e.g., pentaglutamated and / or hexaglutamated) antifolate (LPA) compositions and / or pegylated LPA (PLPA) compositions, optionally further including a targeting moiety that has specific affinity for a surface antigen on the surface of a target cell of interest. [Brief explanation 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 polyglutamated in the cytosol and mitochondria, while hydrolysis occurs in liposomes, a competing process. Figure 1 is adapted from Gonen et al., Drug Resistance Updates 15:183-210 (2012). [Figure 2]

[0013] Figure 2 is a diagram showing 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] Figure 3 shows the disruption of cell polarity and disorganization, which are characteristic of advanced epithelial tumors. As shown in Figure 3A, normal simple epithelium contains a single layer of individual cells that exhibit clear apical-basal polarity. The cells are tightly packed and connected to each other by apical junctional complexes that separate the apical and basal membrane regions. In normal cells where polarity is preserved, FR-α is bound to the apical surface of the cells, where it is distant from and does not directly contact circulating folate. As shown in Figure 3B, high-grade epithelial tumors show a loss of apical-basal polarity and overall disorganization, bringing FR-α into direct contact with circulating folate. [Figure 4] Molecular mechanisms underlying antifolate resistance in cancer. Figure 4 is adapted from Gonen et al., Drug Resistance Updates 15:183-210 (2012). [Figure 5] FIG. 1 shows the chemical formula of L-α hexaglutamated pemetrexed. [Figure 6] FIG. 1 shows the chemical formula of an example L-gamma polyglutamated antifolate composition encompassed by the present disclosure. [Figure 7]Figure 7 shows the dose-response relationship of free pemetrexed L-γ hexaglutamate (gG6), liposomal pemetrexed L-γ hexaglutamate (liposomal gG6), pemetrexed, and the folate receptor α-targeting antibody (FR1Ab) liposomal pemetrexed L-γ hexaglutamate (liposomal gG6-FR1Ab) in NCI H2342 non-small cell lung cancer (NSCLC) adenocarcinoma subtype. Results are cell viability as measured by luciferase luminescence. As summarized in Figure 7, free pemetrexed gG6 appears to have the lowest potency in inhibiting cell viability 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. [Figure 8] FIG. 1 shows the dose-response relationship of free pemetrexed L-γ hexaglutamate (gG6), liposomal pemetrexed L-γ hexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha-targeting antibody (FR1Ab) liposomal pemetrexed L-γ hexaglutamate (liposomal gG6-FR1Ab) in adenocarcinoma subtype NCI H2342 non-small cell lung cancer (NSCLC) as percent of viable cells 48 hours after treatment. [Figure 9] FIG. 1 shows an example of the dose-response relationship of free pemetrexed L-γ hexaglutamate (gG6), liposomal pemetrexed L-γ hexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha-targeting antibody (FR1Ab) liposomal pemetrexed L-γ hexaglutamate (liposomal gG6-FR1Ab) in adenocarcinoma subtype NCI H2342 non-small cell lung cancer (NSCLC) as percent of viable cells 48 hours after treatment. [Figure 10]This figure shows the dose-response relationship at 48 hours in HT-29 (colon cancer) patients for free pemetrexed L-γ hexaglutamate (gG6), liposomal pemetrexed L-γ hexaglutamate (liposomal gG6), pemetrexed, and the folate receptor α-targeting antibody (FR1Ab) liposomal pemetrexed L-γ hexaglutamate (liposomal gG6-FR1Ab). Free pemetrexed gG6 appears to have the lowest potency. Furthermore, liposomal pemetrexed gG6 is twice as potent as pemetrexed, and liposomal pemetrexed gG6-FR1Ab is five times more potent than free pemetrexed. [Figure 11] FIG. 1 shows an example of the dose-response relationship at 48 hours in HT-29 (colon cancer) for free pemetrexed L-γ hexaglutamate (gG6), liposomal pemetrexed L-γ hexaglutamate (liposomal gG6), pemetrexed, and the folate receptor alpha-targeting antibody (FR1Ab) liposomal pemetrexed L-γ hexaglutamate (liposomal gG6-FR1Ab). [Figure 12] Figure 1 shows the effects 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 drug. Liposomal pemetrexed hexa gG6 can enter cells and inhibit the proliferation of ovarian cancer OAW28 cells more efficiently than pemetrexed hexa gG6. [Figure 13] Figure 1 shows the effect of free pemetrexed L-γ hexaglutamate (hexa gG6) and liposomal pemetrexed L-γ 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 hours of 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-γ hexaglutamate (Lps hexa gG6) on H292 non-small cell lung cancer cells after 48 hours of exposure, compared to pemetrexed. [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 of liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6) and pemetrexed at different doses ranging from 16 to 128 nM on H292 non-small cell lung cancer cells after 48 hours of exposure. At each dose range tested, the liposomal pemetrexed gG6 formulation is superior to pemetrexed in inhibiting H292 non-small cell lung cancer cells. [Figure 19]

[0023] 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]

[0023] Figure 1 shows the therapeutic effects of liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6) and pemetrexed at a range of concentrations on OAW28 ovarian cancer cells after 48 hours of exposure. At a dose of 128 nM, pemetrexed appears to 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 superior therapeutic efficacy to pemetrexed, and at 16 nM, the therapeutic efficacy of liposomal pemetrexed gG6 is comparable to that of 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]

[0023] Figure 1 shows the effects of liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6) and the equivalent pemetrexed drug at 16 nM, 32 nM, 64 nM, and 128 nM on AML12 hepatocytes after 48 hours of exposure. Remarkably, there appears to be no toxicity to AML12 hepatocytes after treatment with liposomal pemetrexed gG6 or the liposomal drug at any of the doses tested. In contrast, pemetrexed treatment results in a reduction in AML12 hepatocyte count of approximately 40% at all doses tested. [Figure 23] 1 shows the effect of liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6) and pemetrexed equivalents 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 resulted in a reduction of CCD841 colonic epithelial cell number by approximately 50% or more, compared to a reduction of cell number of approximately 20% or less after treatment with liposomal gG6. DETAILED DESCRIPTION OF THE INVENTION

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

[0037] More specifically, the present application describes encapsulated polyglutamated antifolates, such as, but not limited to, pemetrexed polyglutamate and lometrexol polyglutamate. In a non-limiting example, the encapsulated polyglutamated antifolate (e.g., pemetrexed polyglutamate) is targeted using 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 untargeted.

[0038] Folate is a water-soluble B vitamin. Its primary role in the body / cell is as a cofactor for various methyltransferases involved in serine, methionine, thymidine, and purine biosynthesis, critical processes involved in cell division. Folate's primary 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 nucleotide biosynthesis, folate plays an important role in DNA synthesis, stability, and integrity, as well as repair. Folate also provides the primary 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). For this reason, antifolates have been developed as cancer treatments to block the effects of folate, thereby inhibiting cell division, DNA / RNA synthesis and repair, and protein synthesis, as described in more detail below.

[0039] In nature, folic acid occurs in a polyglutamated form in animal products and leafy vegetables. Polyglutamic acid is not suitable for physiological transport. Natural polyglutamic acid is degraded primarily to monoglutamic acid in the jejunum before physiological transport and cellular uptake. Dietary folic acid in the polyglutamated 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 recycling. 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 decrease in serum folate within 6 hours, whereas dietary restriction does not produce a comparable decrease over 3 weeks, probably because total body stores of folate are estimated to be between 500 and 20,000 mcg. This observation indicates a substantial 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. Upon absorption 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 critical process involved in cell division.

[0042] Polyglutamation involves the addition of a glutamic acid group in a gamma linkage to the terminal carboxyl group of a neighboring 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 contains more than one glutamic acid residue. Several terms are used in the literature to refer to glutamic acid residues, including glutamic acid, glutamyl group, and glutamyl radical. Each glutamic acid residue (glutamyl group) 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 (monoglutamated). The addition of glutamic acid residues to pemetrexed as described herein results in polyglutamated 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), and is referred to herein as pemetrexed hexaglutamate or hexaglutamated pemetrexed. In the literature, this material is also 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 on 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 on the alpha carbon of the glutamic acid side chain.

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

[0047] Antifolates were developed over 70 years ago as "folate-mimetic" cytotoxic agents. The rationale was to design a molecular family that counteracts the effects of folate in rapidly replicating cells, such as cancer cells, by exploiting physiological folate transport mechanisms and their stimulatory intracellular mechanism of action on DNA replication during cell division. 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 on DNA and RNA synthesis, exerting 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.

[0048] Generally, there are four types of transporters for folates and antifolates in the human body: the reduced folate carrier (RFC), the folate receptor (FR), the proton-coupled folate transporter (PCFT), and the 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 that utilize high transmembrane anion gradients, particularly the organic phosphate gradient, regulated by the energy state of the cell, to achieve the difficult transport of folate into cells.

[0050] The transport of cytotoxic antifolates by RFC is not tumor-specific: because RFC is widely expressed in normal tissues and is highly active at neutral pH, which is characteristic of most normal tissues, antifolates transported 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 anchored to the cell membrane by glycosylphosphatidylinositol (GPI) anchor regions. FRs have a particularly high affinity for FAs. They transport folates into cells via an endocytic mechanism. In the cytoplasm, when vesicles are acidified 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. FR-α has a higher affinity for its preferred substrate than RFC, but 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] The proton-coupled folate transporter (PCFT) is a folate-H+ symporter that functions most efficiently in acidic extracellular environments, particularly those characteristic of hypoxic environments. A key difference between RFC and PCFT is their optimal pH, which affects their affinity for transport substrates. At pH 7.4, RFC activity is optimal and PCFT activity is minimized, so antifolate transport is primarily mediated by RFC. Alternatively, PCFT activity is more pronounced as the pH decreases. 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 at neutral pH, PCFT may play a role in folate receptor-mediated endocytosis.

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

[0054] Intracellular folates are converted to polyglutamates by FPGS, while GGH removes the terminal glutamate, thereby facilitating the export of folate out of the cell and back into the extracellular circulation by ATP-binding cassette transporters, often referred to as folate efflux pumps. While not wishing to be bound by theory, it is believed that polyglutamated folates are better retained in cells because they are poor substrates for ATP-binding cassette transporters. Furthermore, while not wishing to be bound by theory, it is believed that polyglutamated folates are better substrates for intracellular folate-dependent enzymes than monoglutamates.

[0055] As in the case of folate, antifolates such as MTX, PMX, and RTX are thought to be retained in tumor and normal cells through FPGS-induced polyglutamation, which is then exported from the cell after hydrolysis to monoglutamate by GGH. Like physiological folate, polyglutamated antifolates are retained in cells longer, thereby increasing their cytotoxicity by extending the duration 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 their monoglutamate counterparts. Therefore, FPGS and GGH are thought 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 cells, folates and antifolates (such as, but not limited to, pemetrexed and pralatrexate) are polyglutamated to polyglutamates by FPGS. This process is necessary for biological activity. For example, polyglutamination promotes retention (increasing intracellular concentrations) 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] Applicant undertook 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 (discussed in more detail below), 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 can result from the excretion of excess antifolate in cancer cells.

[0058] In particular, excess amounts of antifolates present in cancer cells can be returned to the circulation via the highly efficient ATP-dependent efflux pumps of folic acid and antifolates. This is true even when antifolates are 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 antifolates to cancer cells while largely avoiding normal cells, but the excess antifolate delivered can flow back into the circulation and be taken up by normal cells, resulting in undesirable toxicity. Although these toxicities are significantly reduced compared to the toxicities associated with conventional administration of free antifolates in the patient's circulation, they are still undesirable. The antifolates and delivery systems described herein can further reduce the significantly reduced toxic effects on normal cells achieved by WO2016 / 25882, resulting in even greater reductions in toxicity to normal cells.

[0059] The present inventors have recognized the potential benefits of using polyglutamated antifolates for cancer treatment. For example, polyglutamates, particularly pentaglutamate, can enable cells to more efficiently retain their folate pools. However, due to their high negative charge and other properties, pentaglutamate currently has no known substrates for transport across cell membranes.

[0060] The present disclosure describes advanced and improved techniques for delivering various classes of antifolates (e.g., polyglutamates) to cancer cells that previously could not be taken up by cancer cells. Due to the lack of transport across the cell membrane, polyglutamates delivered to cancer cells are effectively retained within the cancer cells. This reduces and / or eliminates the toxicity associated with non-polyglutamated antifolates, such as monoglutamated antifolates, because polyglutamates are 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 forms of many cytotoxic agents, particularly antifolates, are polyglutamated. Therefore, delivery of previously undeliverable polyglutamated antifolates means that the drugs are immediately active. Furthermore, some solid tumors are known to have low or no FPGS activity and polyglutamation. This means that tumors with low FPGS activity are more resistant to chemotherapy mediated by non-polyglutamated drugs, because the drugs still rely on low endogenous FPGS activity for activation after cellular uptake.

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

[0064] Third, because polyglutamated antifolates are retained intracellularly and not transported extracellularly, administration of polyglutamated antifolates (e.g., intravenous administration) can be performed for only a limited period of time, thus further reducing the potential for undesired toxicity to normal tissues. A shortened administration period also improves patient comfort and reduces costs by allowing for a relatively shorter administration period in clinical settings. For example, retention of polyglutamated drugs within tumor cells means that tumor cells are unlikely to recover from treatment, even after the drug is removed from the extracellular environment (e.g., by stopping intravenous administration).

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

[0066] For all of the foregoing reasons, liposomal polyglutamated antifolates have a higher therapeutic index than non-polyglutamated antifolates delivered in the same manner. That is, we predict that the drugs will have minimized / reduced toxicity, allowing patients to tolerate higher doses of the drug with fewer side effects. These effects are not present when non-polyglutamated drugs are administered.

[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 antifolate 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 components of a polyglutamated antifolate composition (e.g., polyglutamic acid and a delivery vehicle, such as a liposome, comprising polyglutamic acid) 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, polyglutamated antifolate composition components that are isolated are substantially pure. Isolated compositions are free of, 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). Polyglutamated antifolate compositions can be formulated with a diluent or adjuvant and, for practical purposes, can be isolated; for example, polyglutamated antifolate compositions are typically mixed with a pharmaceutically acceptable carrier or diluent when used in diagnosis or therapy. In some embodiments, the isolated polyglutamated antifolate composition (eg, polyglutamic acid and a delivery vehicle, such as a liposome, comprising 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 liposome compositions are pentaglutamated. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated antifolates in the provided compositions are pentaglutamated. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated antifolates in the provided liposome compositions are pentaglutamated. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated antifolates in the provided compositions are hexaglutamated. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated antifolates in the liposome compositions are hexaglutamated. In some embodiments, greater than 70%, 80%, or 90% of the polyglutamated antifolates in the liposome compositions have 5-10 or greater than 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 a delivery vehicle, such as a liposome, comprising 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 100 ng / mL, 250 ng / mL, 500 ng / mL, 750 ng / mL, 1 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 750 μg / mL, 1 mg / mL, 100 mg / mL, 250 mg / mL, or greater than 500 mg / mL. In some embodiments, the concentration of the liposome comprising 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 antifolate 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 multitargeted 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 is 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. Pemetrexed has demonstrated efficacy comparable to docetaxel in second-line treatment of non-small cell lung cancer, but with significantly lower toxicity. Pemetrexed's most common and serious toxicities—myelosuppression and mucositis—have been significantly ameliorated with folic acid and vitamin B12 supplementation, but still present undesirable toxicities. In some specific embodiments, the polyglutamated antifolate is hexaglutamated pemetrexed (PMX). In a further embodiment, the polyglutamated antifolate is pentaglutamated PMX. In a further embodiment, the polyglutamated antifolate is hexaglutamated PMX.

[0075] These toxicities can 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 through direct inhibition of its glycinamide ribonucleotide transferase (GARFT) activity. In certain embodiments, the polyglutamated antifolate is polyglutamated lometrexol (LTX). In further embodiments, the polyglutamated antifolate is pentaglutamated LTX. In further embodiments, the polyglutamated antifolate is hexaglutamated LTX.

[0078] As discussed herein, the use of antifolates such as pemetrexed in clinical settings has presented a significant clinical dilemma. Rapidly regenerating normal tissue cells, such as bone marrow, GI tract, and oral mucosal 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 settings, the use of antifolates often results in severe and life-threatening hematologic and non-hematologic toxic side effects. Furthermore, toxicity is considered to be dose-limiting on the one hand and to hinder the ability to achieve superior efficacy on the other. Due to their associated severe and life-threatening toxicities, promising antifolates either failed during development, such as lometrexol, or have limited use in clinical settings, such as 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 offer a strategy for overcoming the pharmacological challenges associated with the dose-limiting toxicity of antifolates. The provided methods deliver conventionally undeliverable (due to lack of transport mechanisms) polyglutamate forms of antifolate payloads to tumor cells while (1) minimizing / reducing exposure to normal tissue cells, (2) optimizing / improving the cytotoxic effects of antifolates on cancer cells, and (3) minimizing / reducing the effects of efflux pumps.

[0081] The polyglutamated chemicals provided herein achieve many benefits over comparable antifolates that are not delivered to cells in polyglutamated form. For example, polyglutamated antifolates are administered as a bolus, avoiding the FPGS-induced polyglutamation activity required for antifolates such as PMX, MTX, RTX, and LTX to have increased activity (via increased binding affinity for 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 the extracellular agent, continuing 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 mitigating clinical and pharmacological challenges associated with the use of antifolates for cancer therapy. Furthermore, novel chemical entities minimize tumor cell resistance to therapy mediated by cellular efflux pumps.

[0083] By way of example, and not limitation, a targeted liposomal polyglutamated antifolate composition can include 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), wherein the amino acid chain comprises a plurality of amino acids, and the targeting moiety has specific affinity for at least one type of folate receptor, and the targeting moiety is attached to one or both of the PEG and the exterior of the liposome. In exemplary embodiments, the liposome-encapsulated polyglutamated antifolate (LPA) can be pentaglutamated pemetrexed or any suitable pentaglutamated antifolate. In exemplary embodiments, the liposome-encapsulated polyglutamated antifolate (LPA) can be hexaglutamated pemetrexed or any suitable hexaglutamated antifolate.

[0084] By way of example, and without limitation, a non-targeted liposomal polyglutamated antifolate composition can include PEGylated liposomes containing an entrapped and / or encapsulated polyglutamated antifolate. In exemplary embodiments, the liposome-encapsulated polyglutamated antifolate (LPA) can be pentaglutamated pemetrexed or any suitable pentaglutamated antifolate. In exemplary embodiments, the liposome-encapsulated polyglutamated antifolate (LPA) can be hexaglutamated pemetrexed or any suitable hexaglutamated antifolate.

[0085] In some embodiments, the optionally PEGylated targeted liposomal 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), wherein the amino acid chain comprises a plurality of amino acids, and the targeting moiety has 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 targeting moiety can be bound to one or both of the PEG and the exterior of the liposome, and can be determined to have an equilibrium dissociation constant (Kd) in the molar [0.05 nanomolar to 10 micromolar] range.

[0086] In some embodiments, the optionally PEGylated targeted liposome composition 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), wherein 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. In some embodiments, the specific affinity of the targeting moiety is 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 major limitations to the use of free cytotoxic agents, such as antifolates, that require polyglutamation for enhanced activity. The novel 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 results from cellular uptake of the monoglutamate form, e.g., via RFC or PCFT, followed by its intracellular conversion by FPGS to a gamma-polyglutamated form with enhanced cytotoxicity, accompanied by prolonged cellular retention in normal tissue cells. The second limitation is the insufficient delivery of most active polyglutamated forms of the drug, due to the inability of such compounds to cross the cell membrane. Instead, their intracellular availability depends on the cell's ability to polyglutamate the monoglutamate form of the drug 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 polyglutamation, and in some cases, increase the degradation of polyglutamylated forms by GGH and the upregulation of efflux pumps (ATP cassettes) that have the ability to excrete mono- and other less glutamylated forms (1-, 2-, 3-) glutamate from cells, but not the more highly (e.g., pentaglutamylated and hexaglutamylated) forms of folates and their analogs.

[0088] The polyglutamated antifolate novel 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 novel chemical entities reduce / minimize the pharmacological challenges associated with systemic transport, cellular uptake, and intratumoral accumulation of active agents. The novel chemical entities of the present disclosure preferentially target tumor cells for exposure to pentaglutamated and hexaglutamated antifolates and / or minimize exposure of such antifolate cocktails to normal cells, particularly those with high turnover (e.g., rapid replication) in the epithelial lining of the bone marrow and gastrointestinal tract.

[0089] In some embodiments, the present 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 polyglutaminated member is hexaglutamated.

[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 present 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] In 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, a targeted PEGylated liposomal polyglutamated antifolate is provided. In some embodiments, the targeted PEGylated liposomal polyglutamated antifolate may 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., antibody or 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 cells, wherein 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 can be any liposome known in the art. However, it will be understood by those skilled in the art that liposomal encapsulation of any particular drug, such as, but not limited to, the polyglutamated antifolates described herein, can involve fairly routine experimentation to achieve a useful and functional liposome formulation. Generally, the provided liposomes can have any liposome structure, such as a structure having an interior space separated from the external medium by one or more lipid bilayers, or any microcapsule having a semipermeable membrane with a lipophilic core separating the interior. The lipid bilayer can be any arrangement of amphiphilic molecules characterized by a hydrophilic portion (hydrophilic moiety) and a hydrophobic portion (hydrophobic moiety). Typically, the amphiphilic molecules in the bilayer are arranged in a two-dimensional sheet with the hydrophobic portion facing inward and the hydrophilic portion facing outward. The amphiphilic molecules forming the provided liposomes can be any known or later discovered amphiphilic molecules, such as synthetic or naturally occurring lipids or biocompatible lipids. Liposomes can also be formed from amphiphilic polymers and surfactants, such as polymersomes and niosomes. For purposes of this disclosure, 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 a dry powder or dry solid. The dry powder or dry solid may be first dried, e.g., under lyophilization conditions, or optionally, both the dry solid and dry powder may be first dried only, or both first dried and second dried. In dry formulations, the powder or solid may have, for example, 1-6% moisture, e.g., 2-5% moisture, or 2-4% moisture. One exemplary method of drying is lyophilization (also known as freeze-drying or cryodesication). Any of the disclosed compositions and methods 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 glycol, trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol, or aminoglycosides. In further embodiments, the lyoprotectant or cryoprotectant comprises up to 10% or up to 20% of the solution outside the liposome, inside the liposome, or both outside and inside the liposome.

[0106] In some embodiments, liposomes contain steric stabilizers that extend their lifespan in the circulation. One or more steric stabilizers, such as hydrophilic polymers (polyethylene glycol (PEG)), glycolipids (monosialoganglioside (GM1)), or others, occupy the space immediately adjacent to the liposome surface and exclude other macromolecules from this space. As a result, access and binding of plasma opsonins to the liposome surface is hindered, thereby inhibiting macrophage interaction with such liposomes or any other clearance mechanism, and enhancing the lifespan of 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 architecture, such as linear, branched, star-shaped, or comb-shaped, and are commercially available.

[0107] The diameter of the disclosed liposomes is not particularly limited. In some embodiments, the liposomes have diameters in the range of, for example, 30 to 150 nm (nanometers). In other embodiments, the liposomes have diameters in the range of 40 to 70 nm.

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

[0109] In some embodiments, cationic lipids are used to prepare cationic liposomes, which are commonly used as gene transfection agents.The positive charge on cationic liposomes can interact with the negative charge on the cell surface.After the cationic liposomes bind to cells, the liposomes are transported into the cell 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 a neutral lipid, such as HSPC, and an anionic lipid, such as PEG-DSPE, results in the formation of anionic liposomes that are less likely to nonspecifically bind to normal cells. Specific binding to tumor cells can be achieved by using tumor-targeting antibodies, such as folate receptor antibodies, including, for example, folate receptor α antibodies, folate receptor β antibodies, and / or folate receptor δ antibodies.

[0111] For example, at least one type (or some) of the lipids is an amphipathic lipid, defined as having a hydrophilic and a hydrophobic portion (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, phosphate, carboxyl, sulfate, amino, sulfhydryl, nitro, hydroxy, and other groups. The hydrophobic portion may include nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted with one or more aromatic, cycloaliphatic, 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, including, but not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, etc. It should be understood that other lipid membrane components, such as cholesterol, sphingomyelin, and cardiolipin, can 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 a 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 limited to such.Examples of non-cationic lipids include lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoyloleoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoyloleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearo ...oleoylphosphatidylcholine (DSPC), dioleoyloleo 16-O-trans PE, 16-O-dimethyl PE, 18-I-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 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 liposomes 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-maleimidePEG-2000·Na.

[0116] Lipid derivatives, for example, include at least one or more steric stabilizers and / or functional groups attached (preferably covalently) to a liposome component, after which the steric stabilizer and / or functional group is considered to be part of the liposome component. The functional group includes a group that can be used to attach the liposome component to another moiety, such as a protein. Such functional groups include at least maleimide. These steric stabilizers include at least one member selected from the group consisting of polyethylene glycol (PEG), poly-L-lysine (PLL), monosialoganglioside (GM1), poly(vinylpyrrolidone) (PVP), poly(acrylamide) (PAA), poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), phosphatidylpolyglycerol, poly[N-(2-hydroxypropyl)methacrylamide], amphiphilic poly-N-vinylpyrrolidone, L-amino acid-based polymers, and polyvinyl alcohol.

[0117] Liposome components can include any molecule (i.e., chemical / reagent / protein) attached thereto, and thus in some embodiments, provided liposome components 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, provided liposome components include DSPE, DSPE-PEG, DSPE-maleimide, HSPC, HSPC-PEG, HSPC-maleimide, cholesterol, cholesterol-PEG, and cholesterol-maleimide. In exemplary embodiments, the liposome components making up the liposome 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. Maleimide groups can cross-link with each other in the presence of dithiol cross-linking agents, 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 described 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 immunostimulator, refers to a substance that stimulates the immune system by inducing activation or increasing the activity of one of its components. These immunostimulants may include one or more of the following: heptanes, adjuvants, protein immunostimulants, nucleic acid immunostimulants, and chemical immunostimulants. Many adjuvants contain substances designed to stimulate the immune response, such as lipid A, proteins derived from Bordetella pertussis, or Mycobacterium tuberculosis. Certain adjuvants are commercially available, such as Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Michigan), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, New Jersey), AS-2 (SmithKline Beecham, Philadelphia, Pennsylvania), 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 exemplary embodiments, the immunostimulant may be at least one selected from the group consisting of fluorescein, DNP, β-glucan, β-1,3-glucan, and 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 the liposome. For example, the immunostimulatory agent and / or detectable marker can be bound to the liposome membrane by hydrophobic interactions or ionic bonds such as avidin / biotin bonds or metal chelation bonds (e.g., Ni-NTA). Alternatively, the immunostimulatory agent or detectable marker can be covalently bound to the exterior of the liposome, for example, by being covalently bound 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, provided liposomes 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 provided herein. In some embodiments, the interior space further includes a pharmaceutically 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 buffer. In further embodiments, the buffer is a HEPES buffer or a citrate buffer. In yet 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-500 mM.

[0127] In some embodiments, the targeted PEGylated liposomal polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate comprises a liposome comprising an interior space; an aqueous polyglutamated 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, wherein the targeting moiety is disposed on the exterior of 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 contain steric stabilizers that can extend their lifespan in the 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), phosphatidylpolyglycerol, poly[N-(2-hydroxypropyl)methacrylamide], amphiphilic poly-N-vinylpyrrolidone, L-amino acid-based polymers, and polyvinyl alcohol. In some embodiments, the steric stabilizer or group 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 5,000 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 containing fewer 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 interior space of the LPA or PLPA contains less than 200,000 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 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 interior 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 comprises 10,000 to 100,000 molecules of polyglutamylated RTX. In some embodiments, the liposome comprises 10,000 to 100,000 molecules of pentaglutamylated RTX. In some embodiments, the liposome comprises 10,000 to 100,000 molecules of hexaglutamylated RTX.

[0133] In some embodiments, the interior space of the LPA or PLPA contains less than 200,000 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 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 interior space of the LPA or PLPA contains less than 200,000 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 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 liposomes contain less than 200,000 molecules of hexaglutamylated RTX. In some embodiments, the liposomes contain 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 containing less than 200,000 molecules of the polyglutamated antifolate. In some embodiments, the PLPA or PLPNA comprises 10,000 to 100,000 molecules of the polyglutamated antifolate. In further embodiments, the PLPA or PLPNA comprises 10,000 to 100,000 molecules of the pentaglutamated antifolate. In further embodiments, the PLPA or PLPNA comprises 10,000 to 100,000 molecules of the hexaglutamated antifolate.

[0136] In some embodiments, the interior space of the PLPA or PLPNA contains less than 200,000 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 less than 200,000 molecules of polyglutamated PMX. In some embodiments, the PLPA or PLPNA contains less than 200,000 molecules of polyglutamated MTX. In some embodiments, the 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 interior 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 to 100,000 molecules of polyglutamylated LTX. In some embodiments, the PLPA or PLPNA comprises 10,000 to 100,000 molecules of pentaglutamylated LTX. In some embodiments, the PLPA or PLPNA comprises 10,000 to 100,000 molecules of hexaglutamylated LTX.

[0138] In further embodiments, the interior space of the PLPA or PLPNA contains less than 200,000 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 less than 200,000 molecules of pentaglutamated PMX. In some embodiments, the PLPA or PLPNA contains 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 contains less than 200,000 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 less than 200,000 molecules of hexaglutamated PMX. In some embodiments, the PLPA or PLPNA contains 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 interior 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, wherein the liposome is PEGylated and comprises a 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. Such liposomes may be generally referred to herein as "targeted liposomes," e.g., the liposomes comprise one or more targeting moieties or biodistribution modifiers attached to the surface of the liposome or otherwise attached to the liposome. The targeting moiety of the targeted liposome may be any moiety or agent capable of specifically binding a desired target (e.g., an antigen target expressed on the surface of the target cell of interest). In one embodiment, the targeted liposome specifically and preferentially binds to a target on the surface of a desired target cell into which the targeted liposome is internalized, and the encapsulated polyglutamated cytotoxic agent (e.g., a polyglutamated antifolate, such as pentaglutamated or hexaglutamated PMX, LTX, and MTX) exerts its cytotoxic effect. In a further embodiment, the target cell is a cancer cell, tumor cell, or metastatic cell. In some embodiments, the targeted liposome is an immunoliposome.

[0144] The terms conjugate or conjugated refer to any type of bond, including, for example, a covalent bond, an ionic bond (e.g., avidin-biotin), bond through hydrophobic interactions, and bond via a functional group such as maleimide or a linker such as PEG. For example, detectable markers, steric stabilizers, liposomes, liposome components, and immunostimulatory agents can be directly conjugated to each other through maleimide functional groups or through 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 an antibody fragment. 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 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 PEG and one or more of an immunostimulatory agent, a detectable marker, and a maleimide disposed on at least one of 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 diameters ranging from 20 to 200 nm. In further embodiments, the liposomes have diameters ranging from 80 to 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 is greater than or equal to 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, wherein 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 serve as targeting moieties for the disclosed targeted liposomes include one or more of the following: 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 can have a combination of the above properties. For example, a humanized antibody can be an antigen-binding fragment and can be pegylated and multiplexed.

[0150] The term "humanized antibody" refers to a specific immunoglobulin chain, chimeric immunoglobulin, or a non-human (e.g., murine) form thereof that contains minimal non-human (e.g., murine) sequence. Generally, humanized antibodies are human immunoglobulins in which residues from the complementarity-determining regions (CDRs) are replaced with residues from CDRs of non-human species (e.g., mouse, rat, rabbit, and hamster) that have 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 human immunoglobulins are replaced with corresponding residues in antibodies from non-human species that have the desired specificity, affinity, and capacity. Humanized antibodies can be further modified by substituting 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 will contain substantially all of at least one, and typically two or three, variable regions, including all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, where all or substantially 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 make 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 deliver 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 α, folate receptor β, and folate receptor δ. Antibodies to folate receptor α can generally be generated using techniques known in the art. Furthermore, the sequences of many anti-folate receptor antibodies are publicly and / or commercially available and are readily available.

[0152] Murine antibodies against folate receptors are examples of antibodies that can be used as targeting moieties for the disclosed targeted liposomes.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 sequences already disclosed in the public domain, antibody genes were synthesized and placed into transient expression vectors, and the antibodies were produced in a HEK-293 transient expression system.The antibodies can be whole antibodies, Fabs, or any of the various antibody variants described herein or otherwise known in the art.

[0153] In some embodiments, the targeted liposome comprises 30-500 targeting moieties (e.g., 30-250 targeting moieties or 30-200 targeting moieties). In some embodiments, provided targeted liposomes 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 of the targeting moieties are attached to one component of the liposome, such as PEG. In other embodiments, the targeting moieties are attached to another component of the liposome. For example, some targeting moieties may be attached to a lipid component or cholesterol, some targeting moieties may be attached to a steric stabilizer (e.g., PEG), and still other targeting moieties may 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 further embodiments, the targeting moiety of the targeted liposome has affinity and specificity for an antigen selected from the group consisting of folate receptor α, folate receptor β, and folate receptor δ. 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 α, folate receptor β, and folate receptor δ. In a further embodiment, the targeting moiety has specific affinity for at least two antigens selected from the group consisting of folate receptor α, folate receptor β, and folate receptor δ. In another embodiment, the targeting moiety has specific affinity for three antigens, e.g., folate receptor α, folate receptor β, and folate receptor δ. Because targeting moieties sometimes do not bind the entire antigen but only one epitope among many epitopes on an antigen, a targeting moiety can have affinity and specificity for one epitope on an antigen.

[0156] The terms "epitope" and "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 an 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 comprise at least three, more usually at least five or eight to ten 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 structural changes in cancer, causing cancer cell-specific epitopes to be presented. Targeting moieties with specific affinity for the epitopes described herein are useful and are encompassed by the disclosed compositions and methods. In some embodiments, tumor cells bearing 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 pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers that may be 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 to 7.5. In some embodiments, the pH is 6.3 to 7.0. In further embodiments, the pH is 6.5. Ideally, the pH of the pharmaceutical composition allows for both stability of the liposome membrane lipids and retention of the entrapped substance. Examples of buffer substances include histidine, hydroxyethylpiperazine-ethylsulfonic acid (HEPES), morpholinoethylsulfonic acid (MES), succinic acid, tartaric acid, and citric acid, typically at concentrations of 2 to 20 mM. Other suitable carriers include, for example, water, buffered aqueous solution, 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 level of 0.25-0.35 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 (e.g., filtration) known in the art. The resulting aqueous solution can be packaged for use or filtered under aseptic conditions and lyophilized, with the lyophilized preparation being combined with a sterile aqueous medium prior to administration.

[0159] The provided pharmaceutical liposome compositions may also contain other pharmaceutically 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. Furthermore, the liposome suspension may contain a lipid-protecting agent that protects lipids against free radical and lipid-peroxidation damage during 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 compositions can vary widely as needed, typically from about 0.05% or less by weight, or at least about 2-10% to 30-50% by weight, and can be selected primarily based on the liquid volume and viscosity, depending on the particular administration regimen selected. For example, the concentration can be increased to reduce the fluid burden associated with treatment, which may be particularly desirable in patients with congestive heart failure or severe hypertension associated with atherosclerosis. Alternatively, liposome pharmaceutical compositions composed of irritating lipids can be diluted to low concentrations to reduce inflammation at the administration site.

[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 art of drug testing. See, for example, DB Budman, AHCalvert, and EK Rowinsky (eds.), Handbook of Anticancer Drug Development, LWW, 2003. Therapeutically effective dosages for 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 achieved by administering the same amount of therapeutic agent in its conventional non-liposomal formulation. Typically, the dosage for the liposomal pharmaceutical composition ranges, 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 administration of a drug sufficient to produce a medically desirable result. The effective amount will vary depending on factors such as the desired outcome, the particular condition being treated or prevented, the age and physical condition of the subject being treated, the severity of the 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 an agent (e.g., a polyglutamated antifolate) that reduces tumor volume or burden (e.g., as 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 bodily fluids or tissues (e.g., biopsies). 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] Terms such as "treat" or "treatment" or "to treat" refer to both (a) therapeutic measures that cure, delay, reduce, and / or halt the progression of 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 already with the cancer or condition, those at risk of having the cancer or condition, and those with an infection or condition to be prevented. In certain embodiments, a subject is successfully "treated" by the methods provided herein if the subject, for example, exhibits all, partial, or temporary improvement or disappearance of symptoms associated with the disease or condition (e.g., cancer, rheumatoid arthritis).

[0167] Pharmaceutical compositions containing the provided polyglutamated antifolate compositions (e.g., liposomes containing pentaglutamated or hexaglutamated antifolates) are also provided. The pharmaceutical compositions are sterile compositions containing sample liposomes and, preferably, an antifolate, preferably in a pharmaceutically acceptable carrier.

[0168] The term "delivery vehicle" generally refers to any composition, e.g., 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 "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration to, for example, a human or other subject.

[0170] The term "carrier" refers to an organic or inorganic material, natural or synthetic, with which the liposome 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 specified 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 performed 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, PLPA and / or targeted PLPA are prepared as an infusion composition, an injection composition, a parenteral composition, or a topical composition. In further embodiments, the injection includes one or more of intraperitoneal injection, direct intratumoral injection, intraarterial injection, intravenous injection, subcutaneous injection, intramuscular injection, transdermal and intranasal delivery. In further embodiments, PLPA and / or targeted PLPA are in 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, targeted PEGylated liposomal polyglutamated antifolates are administered to tumors of the central nervous system using slow, sustained intracranial infusion 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 chemically or environmentally 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 desirable to deliver the compositions systemically, they can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable formulations can be provided in unit dosage form, for example, in ampoules or multi-dose containers. Parenteral formulations of pharmaceuticals include aqueous solutions of the raw 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 antifolate may be in powder or lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0177] Provided compositions (e.g., polyglutamated antifolates and polyglutamated antifolate-containing liposomes) can also be formulated in 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 applications in vivo, ex vivo, and in vitro. In some embodiments, the compositions have in vivo applications. In vivo uses can include uses such as cell culture and tissue engineering, where selective treatment of a subpopulation of cells is desired. For example, when culturing stem cells from a normal patient or a patient with cancer, the cells can be treated with the sample composition or sample liposomes discussed to address a cancerous subpopulation of cells. A cancerous subpopulation can arise because the donor originally has cancer or because the cells spontaneously transform during in vitro treatment.

[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 liposomes, and instructions, e.g., procedures or information related to using the liposome composition in one or more applications. Such instructions can be provided in any medium, e.g., hard paper copy, electronic media, or through a database or website that contains instructions.

[0180] In some embodiments, the present 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 antifolate comprises 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 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 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 highly proliferative cells selected from the group consisting of: CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98.

[0181] In some embodiments, the present disclosure provides a method of inhibiting tumor cell proliferation, comprising contacting tumor 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 antifolate comprises 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 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 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 present 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 administered 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 antifolate comprises 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 administered 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 administered polyglutamated antifolate comprises pentaglutamated PMX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated PMX.In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated RTX. In some embodiments, the administered pentaglutamated antifolate comprises hexaglutamated RTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated LTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated LTX. In some embodiments, the administered 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 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 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.

[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 administered polyglutamate 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 antifolate comprises 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 administered 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 administered polyglutamated antifolate comprises pentaglutamated PMX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated PMX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated LTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated LTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated pralatrexate (PTX). In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated pralatrexate (PTX). In some embodiments, the administered 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 to 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-α, folate receptor-β, mucin 1, folate receptor-δ, 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 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.

[0184] In some embodiments, the present 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 administered 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 antifolate comprises 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 administered 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 administered polyglutamated antifolate comprises pentaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated PMX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated PMX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated LTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated LTX. In some embodiments, the administered 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 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 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.

[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 administered polyglutamate 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 antifolate comprises 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 administered 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 certain embodiments, the administered polyglutamated antifolate comprises pentaglutamated PMX.In certain embodiments, the administered polyglutamated antifolate comprises hexaglutamated PMX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated RTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated RTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated LTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated LTX. In some embodiments, the administered delivery vehicle 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 lung cancer (e.g., non-small cell lung cancer) cells. 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 administered 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 antifolate comprises 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 administered 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 certain embodiments, the administered polyglutamated antifolate comprises pentaglutamated PMX. In certain embodiments, the administered polyglutamated antifolate comprises hexaglutamated PMX.In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated RTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated RTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated LTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated LTX. In some embodiments, the administered delivery vehicle 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 a further embodiment, 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 δ, mesothelin, guanylate cyclase C (GCC), SLC44A4, 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 TACSTD2 (TROP2), mucin 1, folate receptor δ, 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)) 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 administered 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 antifolate comprises 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 administered 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 certain embodiments, the administered polyglutamated antifolate comprises pentaglutamated PMX. In certain embodiments, the administered polyglutamated antifolate comprises hexaglutamated PMX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated MTX. In some embodiments, the polyglutamated antifolate administered comprises hexaglutamated MTX.In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated RTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated RTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated LTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated LTX. In some embodiments, the administered delivery vehicle 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 breast cancer cells. 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 δ, 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 δ, GONMB, and nectin 4.

[0188] In some embodiments, provided compositions (e.g., liposomes comprising a polyglutamated antifolate) are administered to a subject having or at risk of having a hematological cancer identifiable by the expression of a tumor-specific or tumor-associated antigen on the cell surface. Thus, in some embodiments, the present disclosure provides methods 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 tumor, and / or metastasis identifiable by the expression of a tumor-specific or tumor-associated antigen on the cell surface of the cancer, wherein 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 administered polyglutamate 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 antifolate comprises 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 administered 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 certain embodiments, the administered polyglutamated antifolate comprises pentaglutamated PMX. In certain embodiments, the administered polyglutamated antifolate comprises hexaglutamated PMX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated RTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated RTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated LTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated LTX. In some embodiments, the administered delivery vehicle 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 hematological cancer cells. 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 present disclosure provides use of a composition comprising a polyglutamated antifolate for the manufacture of a medicament for treating 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 tumor, 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 to a subject having or at risk of having a cancer, solid tumor, and / or metastasis that is identifiable by the expression of a tumor-specific or tumor-associated antigen on the cell surface of the cancer an effective amount of a delivery vehicle (e.g., liposome) comprising a targeting moiety and a polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate, wherein 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 administered polyglutamate 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 antifolate comprises 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 administered 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 certain embodiments, the administered polyglutamated antifolate comprises pentaglutamated PMX. In certain embodiments, the administered polyglutamated antifolate comprises hexaglutamated PMX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated RTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated RTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated LTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated LTX. In some embodiments, the administered delivery vehicle 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 cancer, solid tumor, and / or metastatic cells.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 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 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.

[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 their 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 α, folate receptor β, or folate receptor δ. In some embodiments, the administered polyglutamate 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 antifolate comprises 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 administered 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 certain embodiments, the administered polyglutamated antifolate comprises pentaglutamated PMX. In certain embodiments, the administered polyglutamated antifolate comprises pentaglutamated PMX. In certain embodiments, the administered polyglutamated antifolate comprises pentaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated MTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated RTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated RTX. In some embodiments, the administered polyglutamated antifolate comprises pentaglutamated LTX. In some embodiments, the administered polyglutamated antifolate comprises hexaglutamated LTX. In some embodiments, the administered delivery vehicle is a liposome. In further embodiments, the liposome is pegylated. As disclosed herein, folate receptor-targeted PEGylated liposomes containing polyglutamated antifolates can deliver higher amounts of polyglutamated antifolates to cancer cells, particularly cancer cells that express folate receptors, compared to normal cells (i.e., cells that, unlike cancer cells, do not actively take up liposomes and / or do not express folate receptors). Any cancer that expresses folate receptors can be treated according to the disclosed methods. It should be noted that while some cancers may express folate receptors in early stages, many cancers may express folate receptors in later stages.

[0192] Cancers that can be treated by the method of the present invention include carcinoma, sarcoma, and melanoma.Carcinomas include, but are 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 that arise 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 schwannoma, neurofibrosarcoma, or neurogenic sarcoma), Ewing's tumor (including Ewing's sarcoma of bone, extraskeletal (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 lymphomas 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 practiced in 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, such as domestic pets (e.g., dogs, cats, rabbits, and ferrets), livestock or farm animals (e.g., cows, pigs, sheep, chickens, and other poultry), horses, including 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 provided compositions. In some embodiments, such conditions include cancer (e.g., solid tumor cancer or a 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. Determining whether a cell type expresses a folate receptor can be done 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 assays, urine screening tests, DNA probe hybridization, and serological tests. Physicians will also generally consider a complete medical 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 antifolates 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 methods for making the liposome compositions disclosed herein. In one embodiment, the method includes preparing a mixture containing (1) liposome components and (2) a polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate in aqueous solution. In a further embodiment, the mixture includes PEGylated liposome components. The mixture is then homogenized to form liposomes in aqueous solution. Furthermore, the mixture can be extruded through a membrane to form liposomes surrounding the polyglutamated antifolate in aqueous solution. It is understood that the liposome components of the present disclosure can include any lipid (including cholesterol), including functionalized lipids and lipids conjugated to targeting moieties, a detectable label, and a steric stabilizer, or any subset of all of these. Furthermore, it is noted that the bioactive polyglutamated antifolate in aqueous solution can include any reagents and chemicals described herein or otherwise known in the art for the interior or exterior of liposomes, 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 containing (1) liposome components, (2) a 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 then 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 bound to a targeting moiety, a detectable label, and a steric stabilizer, or any subset of all of these. It is further noted that targeted PEGylated liposomes can contain any of the reagents and chemicals described herein or otherwise known in the art on the interior or exterior of the liposome, including, for example, buffers, salts, and cryoprotectants.

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

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

[0206] Although specific exemplary 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. Liposome size can be routinely controlled by controlling the pore size of the membrane used for low-pressure extrusion or pressurization, the pressure and permeation rate used in microfluidization, or other suitable methods known in the art.

[0207] Generally, the polyglutamated antifolate is contained within the liposome, i.e., in the interior (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 art (e.g., dilution, ion exchange chromatography, size exclusion chromatography, dialysis, ultrafiltration, and precipitation). Thus, the above-described or otherwise known methods for making liposomes can 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 receptors, comprising a liposome having an internal space, a polyglutamated antifolate disposed within the internal space, a steric stabilizer molecule attached to the exterior of the liposome, and a targeting moiety comprising a protein having specific affinity for at least one type of folate receptor, wherein the targeting moiety is attached to at least one of the steric stabilizer and the exterior of the liposome. The components of this embodiment may 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, which may be PEG, are as described elsewhere in the present disclosure.

[0209] In some embodiments, the present disclosure provides a method for preparing a non-targeted composition comprising liposomes containing entrapped and / or encapsulated polyglutamated antifolates, the method comprising: forming a mixture comprising liposome components, the polyglutamated antifolate in solution; homogenizing the mixture to form liposomes in the solution; and treating the mixture to form liposomes that entrap and / or encapsulate the polyglutamated antifolate. In some embodiments, the treatment comprises one or more of: thin film hydration, extrusion, in-line mixing, and agitation; once particles are formed, the particles can be further modified in 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 methods for 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 comprising 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 liposomes are defined to have an equilibrium dissociation constant (Kd) in the molar range (0.05 nanomolar to 10 μmolar), and the targeting moiety is attached to one or both of PEG and the exterior of the liposome, and the method includes preparing a mixture including liposome components and a polyglutamated antifolate in solution, homogenizing the mixture to form liposomes in the solution, treating the mixture to form liposomes that entrap and / or encapsulate the polyglutamated antifolate, and providing a targeting moiety on the surface of the liposomes that entrap and / or encapsulate the polyglutamated antifolate, wherein the targeting moiety has 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 processing 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 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, but not limit, the present disclosure in any way, shape, or form, either explicitly or implicitly. While they are typical of those that may be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used. The exemplary composition includes exemplary liposomes. Both the exemplary composition and exemplary liposomes were used in the experiments described in the Examples section and are specific embodiments of the present disclosure throughout this disclosure, but are not intended to define the full scope of the present disclosure.

[0212] Figure 5 shows the L- α The chemical formula for pentaglutamated pemetrexed is shown. Once inside the cell, pemetrexed is converted to hydroxybenzoates by the enzyme folylpolyglutamate synthetase. gamma Converted to polyglutamic acid Here, Each glutamic acid teeth L-shaped do. Furthermore, each bond is through the gamma carboxyl of glutamic acid.

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

[0214] method Preparation of hexaglutamated pemetrexed (HGP) liposomes Briefly, γHGP (gG6) was encapsulated in liposomes by the following procedure. First, the lipid components of the liposome membrane were weighed and mixed as a concentrated solution in ethanol at approximately 65°C. In this example, the lipids used were homogenized soybean 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 approximately 3:2:0.15. Next, gG6 was dissolved in aqueous buffer at a concentration of 20 mg / mL at a pH of 6.5–6.9. The drug solution was heated to 65°C. The ethanolic lipid solution was injected into the gG6 via a small-bore needle. During this step, the drug solution was thoroughly stirred using a magnetic stirrer. Mixing was carried out 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, of 51–54 °C). As a result, the liquid hydrated and formed multiple bilayer (multilamellar) vesicles (MLVs) containing gG6 in an aqueous core.

[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 200 nm pore sizes and six layers with 100 nm pore sizes. During extrusion, the temperature was maintained above Tm to ensure the plasticity of the liquid membrane. As a result of extrusion, 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 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 gG6-containing ULVs were prepared, the extraliposomal gG6 was removed using a small-volume column or tangential flow diafiltration against a buffer suitable for large volumes. While any buffer solution can be used, in this example, the buffer used was 5 mM HEPES, 145 mM sodium chloride, pH 6.7. At the end of the purification, sterile filtration was performed using a 0.22 micron filter.

[0217] antibody conjugation Activated liposomes were prepared by adding DSPE-PEG-maleimide to a lipid composition containing four different lipids: hydrogenated soybean 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 using 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 of incubation at room temperature. The thiolated antibody was added to activated liposomes at a ratio of 60 g / mol phosphate lipid, 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 days 3 (48 hours) and 4 (72 hours) using the CellTiter-Glo® (CTG) luminescent cell viability assay. This assay measures the number of viable cells in culture by 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 by pemetrexed, HGP and liposomes were examined in different cancer cell growths using the 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. A lower IC50 indicated a more potent test substance in inhibiting cancer cell growth.

[0220] Cells were seeded into 96-well plates at a cell density of 5 × 10 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. Additionally, 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 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 number PCS-800-012). Cells were thawed at 37°C for 1 minute and then placed on ice. Cells were then resuspended in StemSpan SFEM (Stem Cell Tech catalog number 9650) plus 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 + 100 ng / mL human stem cell factor (Sigma, Cat. No. H8416), 20 ng / mL human granulocyte colony-stimulating factor (Sigma, Cat. No. H5541), and 10 ng / mL human recombinant IL3 (Sigma, SRP3090) at a density of 2.5 x 10 cells / well. Cells were incubated at 37°C for 10 days. Fresh medium was added every two days. Mature neutrophils were then collected and seeded into 96-well plates at a density of 1 x 10 cells / well and incubated overnight at 37°C. The following 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 colon epithelial cells) is similar to that used for cancer cells.

[0224] result Figure 7 shows 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. Results are cell viability as measured by luciferase luminescence. As shown in Figure 7, free pemetrexed gG6 appears to have the lowest potency as measured by IC50. Liposomal pemetrexed gG6 and liposomal pemetrexed gG6-FR1Ab are both 7- 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 are 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 twice as potent as pemetrexed, and liposomal pemetrexed gG6-FR1Ab is five times 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 above data, 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] The relative potencies of all of the above derivatives compared to pemetrexed after 48 hours of exposure are shown in Figure 14. The relative potencies of treatment with the various derivatives were calculated for each cell line by dividing the IC50 of pemetrexed by the IC50 of liposomal pemetrexed hexaglutamate, as shown in the figure. As shown in the figure, the potency of liposomal pemetrexed hexaglutamate far 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 dose of 4% or less of liposomal pemetrexed hexaglutamate has the same therapeutic effect as a 100% dose of pemetrexed.

[0230] Cancer cell viability studies comparing liposomal pemetrexed hexaglutamate (liposomal 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 demonstrate that liposomal pemetrexed hexaglutamate is more potent than pemetrexed. Furthermore, as an indication of efficacy, results from experiments on 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, pemetrexed at a dose of 128 nM appears to be as effective as liposomal pemetrexed hexaglutamate (see Figure 20), while at doses of 32 nM and 64 nM, liposomal pemetrexed hexaglutamate has better therapeutic effects than pemetrexed, and at 16 nM, the therapeutic effect is less, and similar, for liposomal pemetrexed hexaglutamate and pemetrexed.

[0231] The primary toxicity observed in patients treated with pemetrexed is bone marrow suppression, manifested as a drop in blood cell counts, including neutrophil counts (a type of white blood cell). There are also several adverse effects on the lining of the oral cavity and gastrointestinal tract, manifested as diarrhea and mucositis, as well as adverse effects on the liver in some cases. To assess this toxicity, liposomal pemetrexed hexaglutamate and pemetrexed treatment 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 than pemetrexed. This is also supported by the better maintenance of neutrophil counts after treatment with this derivative compared to pemetrexed at doses ranging from 16 to 128 nM (Figure 21). Strikingly, there appears to be no toxicity to hepatocytes after treatment with liposomal pemetrexed hexaglutamate at the dose levels tested (Figure 22). In contrast, pemetrexed at all doses tested results in an approximately 40% reduction in hepatocyte count. And finally, the same trend is observed after treatment of epithelial colonocytes (Figure 23). As shown in this figure, pemetrexed at all doses tested results in a greater than approximately 50% reduction in cell count, compared to a less than approximately 20% reduction after 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.

[0234] In the composition of either of the above two paragraphs, the polyglutamated antifolate 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 include a polyglutamated antifolate, which may be polyglutamated 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 six paragraphs above, 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 folate.

[0240] In the LPA or PLPA composition of either of the above two paragraphs, the polyglutamated antifolate 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 folate.

[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 can be attached to one or both of the PEG and the exterior of the liposome, and the targeting moiety can have 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 immunostimulatory agent, 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 x 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 0.1 to 0.5.

[0250] In the LPA or PLPA of any 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 can 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 the

[0254] The method may include administering an effective amount of any of the polyglutamated 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] 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 described in paragraphs

[0254] to

[0272] .

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

[0254] -

[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 polyglutamated antifolate compositions of paragraphs

[0254] through

[0272] .

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

[0254] through

[0272] .

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

[0254] through

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

[0260] A non-limiting exemplary method for 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] -

[0272] .

[0262] While 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 literature 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 set forth 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 (RT), X), a liposome composition selected from the group consisting of polyglutamated 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 does not contain a targeting moiety having specific affinity for a surface antigen of a target cell.

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

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

4. The liposome composition of claim 1 , wherein the polyglutamated antifolate comprises a pentaglutamated antifolate or a hexaglutamated antifolate.

5. 2. The liposome composition of claim 1, wherein the liposomes have a diameter ranging from 20 nm to 200 nm or from 80 nm to 120 nm.

6. The liposomes are 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:

7. 7. The liposome composition of claim 6, wherein one or more liposome components further comprises 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), phosphatylpolyglycerol, poly[N-(2-hydroxypropyl)methacrylamide], amphiphilic poly-N-vinylpyrrolidone, L-amino acid based polymers, and polyvinyl alcohol.

8. The liposome composition of claim 1, wherein the polyethylene glycol has a number average molecular weight (Mn) of 200 to 5000 Daltons.

9. 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.

10. 2. The liposome composition of claim 1, wherein the liposome has an interior space containing the polyglutamated antifolate and a water-soluble pharmaceutically acceptable carrier.

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

12. 2. The liposome composition of claim 1, wherein the polyglutamated antifolate has a pH of 5-8.

13. The liposome composition of claim 1, wherein the polyglutamated antifolate contains 4 to 10 glutamic acid residues containing gamma carboxyl linkages.

14. 14. The liposome composition of claim 13, further comprising one or more of an immunostimulant, a detectable marker, and a maleimide.

15. The liposome composition according to claim 14, wherein the immunostimulant is at least one selected from the group consisting of fluorescein; fluorescein isothiocyanate (FITC); DNP; β-glucan; β-1,3-glucan; and β-1,6-glucan.

16. 16. The liposome composition of claim 15, 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 of claim 1.

19. The pharmaceutical composition of claim 18, wherein the liposome has a zeta potential of less than zero.

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

21. 20. The pharmaceutical composition of claim 19, wherein the polyglutamated antifolate comprises a pentaglutamated antifolate or a hexaglutamated antifolate.

22. 20. 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. 20. The pharmaceutical composition of claim 19, wherein the polyglutamated antifolate contains 4 to 10 glutamic acid residues containing gamma carboxyl linkages.

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