EA045904B1 - METHODS FOR TREATING EPITHELIOID CELL TUMORS - Google Patents

Description

Cross reference to related applications

This application claims the benefit of priority to U.S. Provisional Patent Application No. 62/186,252, filed June 29, 2015, the contents of which are incorporated herein by reference in its entirety.

Field of technology to which the invention relates

The present invention relates to methods and compositions for treating proliferative diseases belonging to the family of epithelioid cell tumors, such as perivascular epithelioid cell tumors (PEComas), by administering compositions comprising nanoparticles containing an mTOR inhibitor, such as a limus drug, and albumin.

State of the art

Perivascular epithelioid cell tumors (PEComas) are a family of rare mesenchymal neoplasms composed of histologically and immunohistochemically characteristic epithelioid cells. The PEComa family of tumors includes, for example, lymphangioleiomyomatosis (LAM), angiomyolipoma (AML), clear cell sugar tumors of the lung, and PEComas of other sites (PEComas-NOS), a term referring to less well-characterized PEComas of other anatomic origins). PEComas have a characteristic cell type, namely perivascular epithelioid cells, and are often structured as nests and sheets, or sometimes spindle cells, with a focal association with the walls of blood vessels. See Hornick J.L. et al., Histopathology, 48:75-82 (2006); Wildgruber M. et al., World J. Surg. Oncol., 12:1-4 (2014).

Although the majority of PEComas are benign tumors, a subgroup of aggressive PEComas exhibit a malignant nature and form, for example, locally invasive or distant metastases. See Gennatas S. et al., World J. Surg. Oncol., 10:1-4 (2012); Wagner A.J. et al., J. Clin. Oncol., 28:835-840 (2010); Koenig A.M. et al., J. Med. Case Reports, 3:1-5 (2009).

The first-line treatment with PECom is surgical resection. See Martignoni G. et al., Virkhows Arch., 452:119-132 (2008). The treatment regimen with chemotherapy and/or radiotherapy remains controversial. The rarity of registered PECom has hampered the use of designed therapeutic trials to investigate a new PECom treatment regimen. See Selvaggi F. et al., Navy Surg., 11 (2011); Waters P.S. et al., Int. J. Surg. Case Reports, 3:89-91 (2012). There is currently no second-line treatment for unresectable, aggressive, advanced, locally advanced, metastatic, or malignant PEComas. Consequently, these subgroups of PEComa remain very difficult to treat. The prognosis for patients in these patient subgroups is poor, with median survival estimated at 12–17 months after diagnosis of the advanced type of disease. In addition, for example, the 5-year survival rate of patients with metastatic uterine PEComa is approximately 16%. See Khaja F. et al., Case Reports in Medicine, 2013:1-4 (2013).

The disclosure of all publications, patents, patent applications and published patent applications mentioned herein is incorporated by reference in their entirety.

The essence of the invention

The present invention, in some embodiments, provides a method of treating an epithelioid tumor in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor and albumin. In some embodiments, the epithelioid tumor is a perivascular epithelioid cell tumor (PEComa). In some embodiments, PEComa is selected from the group consisting of clear cell sugar lung tumor, and PEComa of other sites (PEComa-NOS), angiomyolipoma, and lymphangioleiomyomatosis. In some embodiments, the epithelioid tumor is malignant. In some embodiments, the epithelioid tumor is locally advanced. In some embodiments, the epithelioid tumor is metastatic.

In some embodiments, according to any of the methods described above, the mTOR inhibitor is a limus drug, such as sirolimus.

In some embodiments, according to any of the methods described above, the effective amount of mTOR inhibitor in the nanoparticle composition is from about 10 to about 100 mg/m ² (including, for example, about 45-100 mg/m ² , about 75-100 mg/m m ² or about 45, 50, 75 or 100 mg/m ² ). In some embodiments, the nanoparticle composition is administered weekly. In some embodiments, the nanoparticle composition is administered 2 times every 3 weeks. In some embodiments, the nanoparticle composition is administered 3 times every 4 weeks. In some embodiments, the nanoparticle composition is administered on days 1 and 8 of a 21-day cycle. In some embodiments, the nanoparticle composition is administered on days 1, 8, and 15 of a 28-day cycle.

In some embodiments, according to any of the methods described above, the nanoparticle composition is administered intravenously, intraarterially, intraperitoneally, intravesicularly, subcutaneously, intrathecally, intrapulmonary, intramuscularly, intratracheally, intraocularly, transdermally, orally, intraportally, intrahepatically, by hepatic artery infusion, or inhalation . In some embodiments, the nanoparticle composition is administered intravenously. In neko

- 1 045904 In other embodiments, the nanoparticle composition is administered subcutaneously.

In some embodiments, according to any of the methods described above, the nanoparticles in the composition have an average diameter of no more than about 150 nm, such as no more than about 120 nm.

In some embodiments, according to any of the methods described above, the mTOR inhibitor in the nanoparticles is bound (eg, coated) with albumin.

In some embodiments, according to any of the methods described above, the subject is a human.

In some embodiments, according to any of the methods described above, a subject is selected for treatment based on a level (e.g., a high level) of a melanocyte marker (including, for example, HMB45, MelanA, and microphthalmia-associated transcription factor) and a smooth muscle marker (including, for example, , smooth muscle actin, pan-muscle actin, h-caldesmon and calponin). In some embodiments, the level of melanocyte marker and smooth muscle marker is determined by immunohistochemistry.

In some embodiments, according to any of the methods described above, a subject is selected for treatment based on the mutational status of a gene selected from the group consisting of TSC1, TSC2, TFE3, RHEB, MTOR, AKT, PIK3CA, and PTEN. In some embodiments, a subject is selected for treatment if the subject has a mutation in a gene, for example, as determined by gene sequencing. In some embodiments, gene sequencing is based on sequencing circulating or cell-free DNA in a blood sample. In some embodiments, gene sequencing is based on DNA sequencing in a tumor sample.

In some embodiments, according to any of the methods described above, a subject is selected for treatment based on the phosphorylation status of a protein selected from the group consisting of AKT, S6, S6K, and 4EBP1. In some embodiments, a subject is selected for treatment if the subject's protein is phosphorylated. In some embodiments, a subject is selected for treatment if the subject's protein is not phosphorylated. In some embodiments, the protein phosphorylation status is determined by immunohistochemistry.

In some embodiments, according to any of the methods described above, a subject is selected for treatment based on the level of a proliferation marker or an apoptosis marker, such as the Ki-67 proliferation marker or an apoptosis marker, PARP, or a fragment thereof. In some embodiments, a subject is selected for treatment based on the level of a proliferation marker and an apoptosis marker, for example, as determined by immunohistochemistry.

In some embodiments, according to any of the methods described above, the subject has not previously been treated with an mTOR inhibitor.

In some embodiments, according to any of the methods described above, the subject has previously been treated with chemotherapy, radiation therapy, or surgery.

These and other aspects and advantages of the present invention will become apparent from the following detailed description and the appended claims.

It should be understood that one, some, or all of the properties of the various embodiments described herein can be combined to form other embodiments of the present invention.

Detailed Description of the Invention

The present invention relates to methods and compositions for treating epithelioid cell tumors, such as perivascular epithelioid cell tumors (PEComas), in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (hereinafter also referred to as an mTOR nanoparticle composition) and albumin. In some embodiments, the composition contains a limus drug and albumin (hereinafter also referred to as a nanoparticle composition of a limus drug).

In some embodiments, a method of treating PEComa in a subject in need thereof is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin. In some embodiments, the limus drug is sirolimus. In some embodiments, the albumin is human albumin (such as human serum albumin). In some embodiments, the nanoparticles comprise sirolimus bound to (eg, coated with) albumin. In some embodiments, the nanoparticles have an average particle size in the nanoparticle composition of no more than about 150 nm (eg, no more than about 120 nm). In some embodiments, the composition includes an albumin-stabilized sirolimus nanoparticle composition. In some embodiments, the composition is Nab-sirolimus.

In some embodiments, a method of treating PEComa in a subject is provided, comprising administering to the subject an effective amount of a nanoparticle composition comprising

- 2 045904 between a medicinal product from the limus group and albumin, where the medicinal product from the limus group is bound (for example, coated) with albumin. In some embodiments, a method of treating PEComa in a subject is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a limus drug and albumin, wherein the nanoparticles have an average particle size in the nanoparticle composition of no more than about 150 nm (e.g., less than about 120 nm). In some embodiments, a method of treating PEComa in a subject is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a limus drug and albumin, wherein the limus drug is coated with albumin and wherein the nanoparticles have an average particle size in the nanoparticle composition of no more than less than about 150 nm (eg, no more than about 120 nm). In some embodiments, a method of treating PEComa in a subject is provided, comprising administering to the subject an effective amount of a Nab-sirolimus-containing composition. In some embodiments, a method of treating PEComa in a subject is provided, comprising administering to the subject an effective amount of Nab-sirolimus.

In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered intraportally. In some embodiments, the composition is administered intra-arterially. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intrahepatically. In some embodiments, the composition is administered by infusion into the hepatic artery. In some embodiments, the composition is administered intravesicularly. In some embodiments, the composition is administered subcutaneously. In some embodiments, the composition is administered intrathecally. In some embodiments, the composition is administered intrapulmonary. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered intratracheally. In some embodiments, the composition is administered intraocularly. In some embodiments, the composition is administered transdermally. In some embodiments, the composition is administered orally. In some embodiments, the composition is administered by inhalation.

PEComas that can be treated by the methods described herein include, but are not limited to, lymphangioleiomyomatosis (LAM), angiomyolipoma (AML), clear cell sugar tumors of the lung, PEComas of other sites (PEComas-NOS) and their malignant forms. In some embodiments, PEComa is any early-stage PEComa, non-metastatic PEComa, primary PEComa, advanced PEComa, locally advanced PEComa, metastatic PEComa, PEComa in remission, recurrent PEComa, PEComa in the adjuvant setting, or PEComa in the neoadjuvant setting. In some embodiments, PEComa is a treatment-resistant non-nanoparticle chemotherapeutic agent composition (such as a non-nanoparticle limus drug composition).

The methods described herein can be used for any one or more of the following purposes: alleviating one or more symptoms of PEComa, delaying progression of PEComa, reducing tumor size in a patient with PEComa, inhibiting PEComa tumor growth, prolonging overall survival, prolonging disease-free survival, prolonging time before disease progression for PEComa, preventing or delaying PEComa tumor metastasis, reducing previously occurring PEComa tumor metastasis, reducing the incidence or burden of prior PEComa tumor metastasis, and preventing recurrence of PEComa.

Also provided are compositions (such as pharmaceutical compositions), drug, kits and unit dosage forms suitable for the methods described herein.

Also provided are methods of treating PECom according to any of the methods described above, where the treatment is based on the level of expression or activity or mutational status of one or more biomarkers. Biomarkers include, but are not limited to, mTOR pathway genes including, but not limited to, PIK3CA, TsC1, TSC2, AKT, PTEN, MTOR, RHEB, and TFE3, phosphoproteins including, but not limited to, p-AKT, p-S6, p -S6K, p-4EBP1 and p-SPARC, proliferation markers including but not limited to Ki-67, and apoptosis markers including but not limited to PARP.

Definitions

As used herein, the terms treat or treatment represent an approach to obtain useful or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: relief of one or more symptoms resulting from a disease, reduction in the progression of a disease, stabilization of a disease (eg, preventing or delaying the exacerbation of a disease), prevention or delaying the spread (eg, metastasis) of a disease, preventing or delaying the recurrence of a disease, delaying or slowing the progression of a disease, reducing the severity of a disease, causing remission (partial or complete) of a disease, reducing the dose of one or more other drugs needed to treat a disease, delay disease progression, improved quality of life and/or

- 3 045904 survival time. The term treatment also covers the reduction of the pathological consequences of PEComa. The methods of the invention provide for any one or more of these treatment aspects.

The term subject refers to a mammal and includes, but is not limited to, human, bovine, equine, feline, canine, rodent or primate. In some embodiments, the subject is a human.

As used herein, a subject at risk is a subject who is at risk of developing PEComa. The subject at risk may or may not have a detectable disease and may or may not have a detectable disease prior to application of the treatment methods described herein. At risk means that a subject has one or more so-called risk factors, which are measurable parameters that correlate with the development of PEComa, described here. A subject having one or more of these risk factors has a higher likelihood of developing cancer than a subject without these risk factors.

An adjuvant regimen refers to a clinical situation in which the subject has a history of PEComa and typically (but not necessarily) responds to therapy, which includes, but is not limited to, surgery (eg, surgical resection), radiation therapy, and chemotherapy. However, due to their history of PEComa, these subjects are considered at risk for further disease progression. Treatment or administration in the adjuvant mode refers to the subsequent mode of treatment. The degree of risk (eg, whether a subject in the adjuvant setting is considered high risk or low risk) depends on several factors, most often the extent of disease progression upon first treatment.

Neoadjuvant regimen refers to the clinical situation in which the method is performed before primary/definitive therapy.

As used herein, delaying the development of PEComa means postponing, preventing, slowing, delaying, stabilizing and/or delaying the development of the disease. This delay may vary in length of time, depending on the medical history and/or the subject being treated. As will be apparent to one of ordinary skill in the art, a sufficient or significant delay may, in effect, include a warning because the subject does not develop the disease. A method that delays the development of PEComa is a method that reduces the likelihood of developing the disease in a certain period of time and/or reduces the degree of development of the disease in a certain period of time compared to not using the method. Such comparisons are usually based on the results of clinical studies using a statistically significant number of subjects. The development of PEComa can be detected using standard methods including, but not limited to, computerized axial tomography (CAT Scan), magnetic resonance imaging (MRI), abdominal ultrasound, blood clotting test, arteriography, or biopsy. Development may also refer to the progression of PEComa, which may initially be undetectable and includes appearance, recurrence, and onset.

As used herein, the term effective amount refers to an amount of a compound or composition sufficient to treat a particular disorder, condition or disease, eg, suppress, attenuate, reduce and/or delay one or more symptoms thereof. With respect to PEComa, an effective amount includes an amount sufficient to cause a reduction in tumor size and/or a decrease in the rate of tumor growth (eg, suppression of tumor growth) or prevention or delay of other unwanted cellular proliferation in PEComa. In some embodiments, the effective amount is an amount sufficient to delay the progression of PEComa. In some embodiments, the effective amount is an amount sufficient to prevent or delay recurrence. An effective amount may be administered in one or more administrations. In the case of PECom, an effective amount of the drug or composition can (i) reduce the number of epithelioid cells; (ii) reduce tumor size; (iii) inhibit, delay, slow down to some extent and preferably stop the infiltration of malignant PEComa cells into peripheral organs; (iv) inhibit (eg, slow and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the onset and/or recurrence of a tumor and/or (vii) alleviate to some extent one or more symptoms associated with PEComa.

As used herein, the term pharmaceutically acceptable or pharmacologically compatible means a substance that is not biologically or otherwise undesirable, for example, the substance can be included in a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a harmful manner with any another component of the composition in which it is included. Pharmaceutically acceptable carriers or excipients preferably meet required toxicological and manufacturing testing standards and/or are included in the Inactive Ingredients Guide issued by the US Food and Drug Administration.

As used here, the term Nab means albumin-associated nanoparticle. For example,

- 4 045904

Nab-sirolimus is an albumin-bound nanoparticle formulation of sirolimus. Nab-sirolimus is also known as Nab-rapamycin, which has previously been described, for example, in international applications WO 2008/109163 A1, WO 2014/151853, WO 2008/137148 A2 and WO 2012/149451 A1.

As used herein, the term mutational status refers to the status of a gene sequence (eg, containing a mutation) relative to the wild-type or reference gene sequence.

It will be understood that aspects and embodiments of the invention described herein include and/or predominantly include aspects and embodiments.

A reference to about before a value or parameter here includes (and describes) options that target that value or parameter as such. For example, a description referring to about X includes a description of X.

As used herein and in the accompanying claims, singular forms include plural forms unless the context clearly dictates otherwise.

Method of treatment with PEK

The present invention provides methods for treating PEComa in a subject (such as a human) comprising administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a limus drug) and albumin. In some embodiments, the invention provides methods for treating PEComa in a subject (eg, a human), comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin. In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (eg, coated) with the albumin. In some embodiments, the method comprises administering to a subject an effective amount of a composition comprising nanoparticles containing a lymus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm). In some embodiments, the average or median diameter of the nanoparticles is from about 10 to about 150 nm. In some embodiments, the average or median diameter of the nanoparticles is from about 40 nm to about 120 nm. In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the nanoparticles comprise a limus drug bound (e.g., coated) with albumin, wherein the nanoparticles have an average particle size of no more than less than about 150 nm (eg, no more than about 120 nm). In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g. , not more than about 120 nm, for example, about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (for example, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

As used herein, the term mTOR inhibitor refers to mTOR inhibitors. mTOR is a serine/threonine-specific protein kinase downstream of the phosphatidylinositol 3kinase (PI3K)/Akt (protein kinase B) pathway and is a key regulator of cell survival, proliferation, stress and metabolism. Dysregulation of the mTOR pathway has been found in many human carcinomas, and inhibition of mTOR had a significant suppressive effect on tumor progression. The mTOR inhibitors described herein include, but are not limited to, BEZ235 (NVP-BEZ235), everolimus (also known as RAD001, Zortress, Certican and Afinitor), rapamycin (also known as sirolimus or Rapamune), AZD8055, temsirolimus (also known as CCI-779 and Torizel), PI-103, Ku-0063794, INK 128, AZD2014, NVP-BGT226, PF-04691502, CH5132799, GDC-0980 (RG7422), Torin 1, WAY-600, WYE-125132, WYE - 687, GSK2126458, PF-05212384 (PKI-587), PP-121, OSI-027, Palomid 529, PP242, XL765, GSK1059615, WYE-354 and eforolimus (also known as ridaforolimus or deforolimus).

In some embodiments, the mTOR inhibitor is a limus drug, which includes sirolimus and analogs thereof. Examples of limus drugs include, but are not limited to, temsirolimus (CCI-779), everolimus (RAD001), ridaforolimus (AP-23573), deforolimus (MK-8669), zotarolimus (ABT-578), pimecrolimus, and tacrolimus (FK-578). 506). In some embodiments, the limus drug is selected from the group consisting of temsirolimus (CCI-779), everolimus (RAD001), ridaforolimus (AP-23573), deforolimus (MK-8669), zotarolimus (ABT-578), pimecrolimus, and tacrolimus (FK-506).

In some embodiments, PEComa represents lymphangioleiomyomatosis. In some embodiments, PEComa is an angiomyolipoma. In some embodiments it will be carried out

- 5 045904 phenomena PEComa is a clear cell sugar tumor of the lung. In some embodiments, PEComa is an extrapulmonary clear cell sugar tumor. In some embodiments, PEComa represents PEComa-NOS. In some embodiments, PEComa is malignant.

In some embodiments, PEComa represents early-stage PEComa, non-metastatic PEComa, non-invasive PEComa, invasive PEComa, primary PEComa, advanced PEComa, locally advanced PEComa, metastatic PEComa, recurrent PEComa, or PEComa in remission. In some embodiments, PEComa was resistant to prior therapy. In some embodiments, PEComa is resistant to treatment with a non-nanoparticle chemotherapeutic agent drug (such as a non-nanoparticle mTOR inhibitor drug, such as a limus drug).

In some embodiments, PEComa is localized resectable (e.g., refers to tumors that can be completely removed by surgery), localized inoperable (e.g., refers to localized tumors that are inoperable due to the fact that key vascular structures are involved in the pathological process vessels) or inoperable (for example, the characteristics of the tumor or the patient’s condition do not allow surgical removal of the tumor).

In some embodiments, PEComa is located in or closely associated with the kidneys, bladder, prostate, uterus, ovary, vulva, vagina, lung, pancreas, liver, lymph node, and/or skin.

In some embodiments, PEComa is a TNM tumor of Stage I (single tumor without vascular invasion), Stage II tumor (single tumor with vascular invasion or multiple tumors less than or equal to 5 cm in diameter), or Stage III tumor (multiple tumors greater than 5 cm in diameter). ), stage IV tumor (tumors with direct invasion of adjacent organs other than the gallbladder, or invasion of the visceral peritoneum), N1 tumor (metastases in regional lymph nodes) or M1 tumor (distant metastases). In some embodiments, PEComa is, according to AJCC (American Joint Commission on Cancer) criteria, PEComa stage T1, T2, T3, or T4. In some embodiments, the subject is in clinical stage Ta, Tis, T1, T2, T3a, T3b, or T4. In some embodiments, the subject is in clinical stage Tis, CIS, Ta, or T1.

In some embodiments, PEComa is characterized by immunohistochemically positive expression of a melanocyte marker and a smooth muscle marker. In some embodiments, the melanocyte marker is selected from the group consisting of HMB45, MelanA, and microphthalmia-associated transcription factor. In some embodiments, the smooth muscle marker is selected from the group consisting of smooth muscle actin, pan-muscle actin, h-caldesmon, and calponin. In some embodiments, PEComa characterization is performed based on a biopsy of the primary tumor. In some embodiments, characterization of PEComa is based on a biopsy of the metastatic tumor. In some embodiments, the subject is diagnosed as having PEComa based on the characteristics of the tumor being immunohistochemically positive for the expression of a melanocyte marker and a smooth muscle marker. In some embodiments, the melanocyte marker is selected from the group consisting of HMB45, MelanA, and microphthalmia-associated transcription factor. In some embodiments, the smooth muscle marker is selected from the group consisting of smooth muscle actin, pan-muscle actin, caldesmon, and calponin. In some embodiments, the diagnosis is made based on a biopsy of the primary tumor. In some embodiments, the diagnosis is made based on a biopsy of the metastatic tumor.

In some embodiments, a method of treating localized, resectable PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, the invention provides methods for treating localized, operable PEComa in a subject (eg, a human), comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin. In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (eg, coated) with the albumin. In some embodiments, the method comprises administering to a subject an effective amount of a composition comprising nanoparticles containing a lymus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm). In some embodiments, the method comprises administering to a subject an effective amount of a composition comprising nanoparticles comprising a limus drug and albumin, wherein the nanoparticles comprise a limus drug bound (e.g.,

- 6 045904 coated) with albumin, where the nanoparticles have an average particle size of no more than about 150 nm (for example, no more than about 120 nm). In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g. , not more than about 120 nm, for example, about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (for example, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus. In some embodiments, PEComa is resectable, requiring multiple resections.

In some embodiments, a method of treating localized, inoperable PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, the invention provides methods for treating localized, inoperable PEComa in a subject (eg, a human), comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin. In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (eg, coated) with the albumin. In some embodiments, the method comprises administering to a subject an effective amount of a composition comprising nanoparticles containing a lymus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm). In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the nanoparticles comprise a limus drug bound (e.g., coated) with albumin, wherein the nanoparticles have an average particle size of no more than less than about 150 nm (eg, no more than about 120 nm). In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g. , not more than about 120 nm, for example, about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (for example, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

Thus, for example, in some embodiments, a method of treating inoperable PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a limus drug) and albumin. In some embodiments, the invention provides methods for treating inoperable PEComa in a subject (eg, a human), comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin. In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (eg, coated) with the albumin. In some embodiments, the method comprises administering to a subject an effective amount of a composition comprising nanoparticles containing a lymus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm). In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the nanoparticles comprise a limus drug bound (e.g., coated) with albumin, wherein the nanoparticles have an average particle size of no more than less than about 150 nm (eg, no more than about 120 nm). In some embodiments, the method comprises administering to a subject an effective amount of a composition comprising nanoparticles containing sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g. , not more than about 120 nm, for example, about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (for example, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

Thus, for example, in some embodiments, a method of treating

- 7 045904 malignant PEComa in a subject (eg, a human), comprising administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, the invention provides methods for treating malignant PEComa in a subject (eg, a human), comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin. In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (eg, coated) with the albumin. In some embodiments, the method comprises administering to a subject an effective amount of a composition comprising nanoparticles containing a lymus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm). In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles comprising a limus drug and albumin, wherein the nanoparticles comprise a limus drug bound (e.g., coated) with albumin, wherein the nanoparticles have an average particle size of no more than less than about 150 nm (eg, no more than about 120 nm). In some embodiments, the method comprises administering to a subject an effective amount of a composition comprising nanoparticles containing sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g. , not more than about 120 nm, for example, about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (for example, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

Thus, for example, in some embodiments, a method of treating metastatic PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, the invention provides methods for treating metastatic PEComa in a subject (eg, a human), comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin. In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (eg, coated) with the albumin. In some embodiments, the method comprises administering to a subject an effective amount of a composition comprising nanoparticles containing a lymus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm). In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the nanoparticles comprise a limus drug bound (e.g., coated) with albumin, wherein the nanoparticles have an average particle size of no more than less than about 150 nm (eg, no more than about 120 nm). In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g. , not more than about 120 nm, for example, about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (for example, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

Thus, for example, in some embodiments, a method of treating recurrent PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, the invention provides methods for treating recurrent PEComa in a subject (eg, a human), comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin. In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (eg, coated) with the albumin. In some embodiments, the method comprises administering to a subject an effective amount of a composition comprising nanoparticles containing a lymus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm).

- 8 045904

In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the nanoparticles comprise a limus drug bound (e.g., coated) with albumin, wherein the nanoparticles have an average particle size of no more than less than about 150 nm (eg, no more than about 120 nm). In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g. , not more than about 120 nm, for example, about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (for example, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

Thus, for example, in some embodiments, a method of treating locally advanced PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a limus drug) and albumin. In some embodiments, the invention provides methods for treating locally advanced PEComa in a subject (eg, a human), comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin. In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (eg, coated) with the albumin. In some embodiments, the method comprises administering to a subject an effective amount of a composition comprising nanoparticles containing a lymus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm). In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the nanoparticles comprise a limus drug bound (e.g., coated) with albumin, wherein the nanoparticles have an average particle size of no more than less than about 150 nm (eg, no more than about 120 nm). In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g. , not more than about 120 nm, for example, about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (for example, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

Thus, for example, in some embodiments, a method is provided for stabilizing PEComa (e.g., preventing or delaying the exacerbation of the disease) in a subject (e.g., a human), comprising administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a drug from the group limus) and albumin. In some embodiments, the invention provides methods for stabilizing PEComa (eg, preventing or delaying the exacerbation of the disease) in a subject (eg, a human), comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin. In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (eg, coated) with the albumin. In some embodiments, the method comprises administering to a subject an effective amount of a composition comprising nanoparticles containing a lymus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm). In some embodiments, the method includes administering to a subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the nanoparticles comprise a limus drug bound (e.g., coated) with albumin, wherein the nanoparticles have an average particle size of no more than less than about 150 nm (eg, no more than about 120 nm). In some embodiments, the method comprises administering to a subject an effective amount of a composition comprising nanoparticles containing sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g. , not more than about 120 nm, for example about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or

- 9 045904 lower (for example, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nabsirolimus.

The methods provided herein can be used to treat a subject (eg, a human) who has been diagnosed or suspected of having PEComa. In some embodiments, the subject is a human. In some embodiments, the age of the subject is at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 years. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject has undergone tumor resection. In some embodiments, the subject refuses surgery. In some embodiments, the subject is medically inoperable.

In some embodiments, the subject is a person who exhibits one or more symptoms associated with PEComa. In some embodiments, the subject is in the early stages of PEComa. In some embodiments, the subject is in the stage of advanced PEComa. In some embodiments, the subject is genetically or otherwise predisposed (eg, has a risk factor) to developing PEComa. Subjects at risk for developing PEComa include, for example, subjects whose risk is determined by analysis of genetic or biochemical markers. Such risk factors include, but are not limited to, age, sex, race, diet, pre-existing medical history, presence of pre-existing disease, genetic factors (eg, tuberous sclerosis complex (TSC) mutation status, and environmental exposures. In some embodiments, subjects susceptible to at risk for developing PEComa include, for example, subjects who have a relative with PEComa and subjects whose risk is determined by analysis of genetic or biochemical markers (eg, expression and activity levels, or mutational status).Biomarkers include, but are not limited to, pathway genes mTOR including but not limited to PIK3CA, TSC1, TSC2, AKT, PTEN, MTOR, RHEB and TFE3, phosphoproteins including but not limited to p-AKT, p-S6, p-S6K, p-4EBP1, p- SPARC, proliferation markers, including, but not limited to, the Ki67 marker, and apoptosis markers, including, but not limited to, PARP or fragments thereof.In some embodiments, a blood sample or a tumor biopsy sample from the subject is used to evaluate the biomarkers.

The methods provided herein can be practiced in an adjuvant setting. In some embodiments, the method is practiced in a neoadjuvant setting, i.e. the method can be carried out before primary/definitive therapy. In some embodiments, the method is used to treat a subject who has previously been treated. In some embodiments, the subject has not previously been treated. In some embodiments, the method is used as first-line therapy. In some embodiments, the method is used as second-line therapy. In some embodiments, the subject has not previously been treated with an mTOR inhibitor. In some embodiments, the subject has not previously been treated with a limus drug.

In some embodiments, a method is provided for prolonging the time until progression of PEComa in a subject, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor and albumin. In some embodiments, the method prolongs the time to disease progression by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

In some embodiments, a method of prolonging the survival of a subject having PEComa is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor and albumin. In some embodiments, the method prolongs the survival of a subject by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

In some embodiments, a method of reducing one or more symptoms in a subject having PEComa is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

In some embodiments, a method is provided to improve the quality of life of a subject

- 10 045904 project having PEComa, comprising administering to a subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor and albumin. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

In some embodiments, the subject has previously been treated for PEComa (also refers to prior therapy). In some embodiments, the subject is resistant to treatment of PEComa with other agents (such as mTOR inhibitor drugs other than nanoparticles). In some embodiments, the subject initially responded to treatment for PEComa with other agents, but the tumor progressed after treatment. Previous treatments include, but are not limited to, chemotherapy, radiation therapy, and surgery. In some embodiments, prior therapy is discontinued more than or 28 days prior to initiation of the methods described herein. In some embodiments, prior therapy is discontinued for a period greater than five times the half-life of the prior therapy agents prior to initiation of the methods described herein.

In some embodiments, the subject has recurrent PEComa after prior therapy. For example, a subject may initially respond to prior therapy but develop PEComa approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, or 60 months after discontinuation of prior therapy .

In some embodiments, the subject is resistant to prior therapy.

In some embodiments, the subject has had disease progression while on a prior therapy during treatment. For example, the subject has had disease progression approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after treatment with the prior therapy.

In some embodiments, the subject is resistant to prior therapy.

In some embodiments, the subject is unsuitable to continue prior therapy, for example, due to lack of response to treatment and/or due to toxicity.

In some embodiments, the subject does not respond to prior therapy.

In some embodiments, the subject partially responds to prior therapy or exhibits a less than desirable degree of responsiveness to treatment.

In some embodiments, a method of treating lymphangioleiomyomatosis in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, a method of treating lymphangioleiomyomatosis in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a lymus drug and albumin. In some embodiments, a method of treating lymphangioleiomyomatosis in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (e.g., coated) with albumin. In some embodiments, a method of treating lymphangioleiomyomatosis in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a lymus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm). In some embodiments, a method of treating lymphangioleiomyomatosis in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a limus drug and albumin, wherein the nanoparticles comprise a limus drug bound (e.g., coated) with albumin and wherein the nanoparticles have an average particle size of no more than about 150 nm (eg, no more than about 120 nm). In some embodiments, a method of treating lymphangioleiomyomatosis in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or less (e.g., about 9:1 or approximately 8:1). In some embodiments, a method of treating lymphangioleiomyomatosis in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a Nab-sirolimus-containing composition. In some embodiments, a method of treating lymphangioleiomyomatosis in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of Nab-sirolimus. In some embodiments, the inhibitor

- 11 045904 mTOR (such as a limus drug) is administered at a dose of about 10 to about 150 mg/m ² , including, for example, about 45 to about 100 mg/m ² and about 75 to 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 75 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 56 mg/m ² . In some embodiments, the limus drug nanoparticle composition is administered weekly. In some embodiments, the nanoparticle drug composition from the limus group is administered twice every three weeks (for example, on days 1 and 8 of a 21-day cycle). In some embodiments, the limus drug nanoparticle composition is administered three times every four weeks (eg, on days 1, 8, and 15 of a 28-day cycle). In some embodiments, the limus drug nanoparticle composition is administered by intravenous infusion over 30 minutes.

In some embodiments, a method of treating angiomyolipoma in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, a method of treating angiomyolipoma in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a lymus drug and albumin. In some embodiments, a method of treating angiomyolipoma in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (e.g., coated) with albumin. In some embodiments, a method of treating angiomyolipoma in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a lymus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm). In some embodiments, a method of treating angiomyolipoma in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a lymus drug and albumin, wherein the nanoparticles comprise a lymus drug bound (e.g., coated) with albumin and wherein the nanoparticles have an average particle size of no more than about 150 nm (eg, no more than about 120 nm). In some embodiments, a method of treating angiomyolipoma in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) to human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or less (e.g., about 9:1 or approximately 8:1). In some embodiments, a method of treating angiomyolipoma in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a Nab-sirolimus-containing composition. In some embodiments, a method of treating angiomyolipoma in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of Nab-sirolimus. In some embodiments, the limus drug is administered at a dose of about 10 to about 150 mg/m ² , including, for example, about 45 to about 100 mg/m ² and about 75 to 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 75 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 56 mg/m ² . In some embodiments, the limus drug nanoparticle composition is administered weekly. In some embodiments, the nanoparticle drug composition from the limus group is administered twice every three weeks (for example, on days 1 and 8 of a 21-day cycle). In some embodiments, the limus drug nanoparticle composition is administered three times every four weeks (eg, on days 1, 8, and 15 of a 28-day cycle). In some embodiments, the limus drug nanoparticle composition is administered by intravenous infusion over 30 minutes.

In some embodiments, a method of treating clear cell sugary lung tumors in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, a method of treating clear cell sugar lung tumors in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a nanoparticle composition comprising

- 12 045904 medicinal product from the limus group and albumin. In some embodiments, a method of treating clear cell sugary lung tumors in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug is bound in the nanoparticles (e.g., coated) with albumin. In some embodiments, a method of treating clear cell sugary lung tumors in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a lymus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (eg, no more than about 120 nm). In some embodiments, a method of treating clear cell sugary lung tumors in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a lymus drug and albumin, wherein the nanoparticles comprise a lymus drug bound (e.g. , coated) with albumin and where the nanoparticles have an average particle size of no more than about 150 nm (for example, no more than about 120 nm). In some embodiments, a method of treating clear cell sugary lung tumors in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (e.g., about 9 :1 or approximately 8:1). In some embodiments, a method of treating clear cell sugary lung tumors in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a Nab-sirolimus-containing composition. In some embodiments, a method of treating clear cell sugary lung tumors in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of Nab-sirolimus. In some embodiments, the limus drug is administered at a dose of about 10 to about 150 mg/m ² , including, for example, about 45 to about 100 mg/m ² and about 75 to 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 75 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 56 mg/m ² . In some embodiments, the limus drug nanoparticle composition is administered weekly. In some embodiments, the nanoparticle drug composition from the limus group is administered twice every three weeks (for example, on days 1 and 8 of a 21-day cycle). In some embodiments, the limus drug nanoparticle composition is administered three times every four weeks (eg, on days 1, 8, and 15 of a 28-day cycle). In some embodiments, the limus drug nanoparticle composition is administered by intravenous infusion over 30 minutes.

In some embodiments, a method of treating PEComa-NOS in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug) and albumin. In some embodiments, a method of treating PEComaNOS in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a lymus drug and albumin. In some embodiments, a method of treating PEComa-NOS in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (e.g., coated ) with albumin. In some embodiments, a method of treating PEComa-NOS in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a limus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm ( for example, no more than about 120 nm). In some embodiments, a method of treating PEComa-NOS in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a limus drug and albumin, wherein the nanoparticles comprise a limus drug bound (e.g., coated) with albumin and wherein the nanoparticles have an average particle size of no more than about 150 nm (for example, no more than about 120 nm). In some embodiments, a method of treating PEComa-NOS in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein nanoparticles have an average

- 13 045904 particle size of not more than about 150 nm (for example, not more than about 120 nm, for example, about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (for example, about 9: 1 or approximately 8:1). In some embodiments, a method of treating PEComa-NOS in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a Nab-sirolimus-containing composition. In some embodiments, a method of treating PEComa-NOS in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of Nab-sirolimus. In some embodiments, the limus drug is administered at a dose of about 10 to about 150 mg/m ² , including, for example, about 45 to about 100 mg/m ² and about 75 to 100 mg/m ² In some embodiments implementation, a drug from the limus group is administered at a dose of approximately 100 mg/ ^m2 . In some embodiments, the limus drug is administered at a dose of about 75 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 56 mg/m ² . In some embodiments, the limus drug nanoparticle composition is administered weekly. In some embodiments, the nanoparticle drug composition from the limus group is administered twice every three weeks (for example, on days 1 and 8 of a 21-day cycle). In some embodiments, the limus drug nanoparticle composition is administered three times every four weeks (eg, on days 1, 8, and 15 of a 28-day cycle). In some embodiments, the limus drug nanoparticle composition is administered by intravenous infusion over 30 minutes.

In some embodiments, a method of treating metastatic PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, a method of treating metastatic PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin. In some embodiments, a method of treating metastatic PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (e.g., coated) with albumin. In some embodiments, a method of treating metastatic PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a limus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g. , no more than about 120 nm). In some embodiments, a method of treating metastatic PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a limus drug and albumin, wherein the nanoparticles comprise a limus drug bound (e.g., coated ) with albumin and where the nanoparticles have an average particle size of no more than about 150 nm (for example, no more than about 120 nm). In some embodiments, a method of treating metastatic PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (e.g., about 9:1 or approximately 8:1). In some embodiments, a method of treating metastatic PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a Nab-sirolimus-containing composition. In some embodiments, a method of treating metastatic PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of Nab-sirolimus. In some embodiments, the limus drug is administered at a dose of about 10 to about 150 mg/m ² , including, for example, about 45 to about 100 mg/m ² and about 75 to 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 75 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 56 mg/m ² . In some embodiments, the limus drug nanoparticle composition is administered weekly. In some embodiments, the nanoparticle drug composition from the limus group is administered twice every three weeks (for example, on days 1 and 8 of a 21-day cycle). In some embodiments, the limus drug nanoparticle composition is administered three times every four weeks (eg, on days 1, 8, and 15 of a 28-day cycle). In some embodiments, the limus drug nanoparticle composition is administered by intravenous infusion over 30 minutes.

- 14 045904

In some embodiments, a method of treating locally advanced PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, a method of treating locally advanced PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin. In some embodiments, a method of treating locally advanced PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug is bound in the nanoparticles (e.g., coated) with albumin. In some embodiments, a method of treating locally advanced PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a limus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (eg, no more than about 120 nm). In some embodiments, a method of treating locally advanced PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a limus drug and albumin, wherein the nanoparticles comprise a limus drug bound (e.g. , coated) with albumin and where the nanoparticles have an average particle size of no more than about 150 nm (for example, no more than about 120 nm). In some embodiments, a method of treating locally advanced PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (e.g., about 9:1 or approximately 8:1). In some embodiments, a method of treating locally advanced PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a Nab-sirolimus-containing composition. In some embodiments, a method of treating locally advanced PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of Nab-sirolimus. In some embodiments, the limus drug is administered at a dose of about 10 to about 150 mg/m ² , including, for example, about 45 to about 100 mg/m ² and about 75 to 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 75 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 56 mg/m ² . In some embodiments, the limus drug nanoparticle composition is administered weekly. In some embodiments, the nanoparticle drug composition from the limus group is administered twice every three weeks (for example, on days 1 and 8 of a 21-day cycle). In some embodiments, the limus drug nanoparticle composition is administered three times every four weeks (eg, on days 1, 8, and 15 of a 28-day cycle). In some embodiments, the limus drug nanoparticle composition is administered by intravenous infusion over 30 minutes.

In some embodiments, a method of treating advanced malignant PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, a method of treating advanced malignant PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin. In some embodiments, a method of treating advanced malignant PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (e.g., coated ) with albumin. In some embodiments, a method of treating advanced malignant PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a limus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm ( for example, no more than about 120 nm). In some embodiments, a method of treating advanced malignant PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a drug from the group

- 15 045904 limus and albumin, where the nanoparticles contain a drug from the limus group bound (for example, coated) with albumin and where the nanoparticles have an average particle size of no more than about

150 nm (eg, no more than about 120 nm). In some embodiments, a method of treating advanced malignant PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein The nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (e.g., about 9: 1 or approximately 8:1). In some embodiments, a method of treating advanced malignant PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a Nab-sirolimus-containing composition. In some embodiments, a method of treating advanced malignant PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of Nab-sirolimus. In some embodiments, the limus drug is administered at a dose of about 10 to about 150 mg/m ² , including, for example, about 45 to about 100 mg/m ² and about 75 to 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 75 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 56 mg/m ² . In some embodiments, the limus drug nanoparticle composition is administered weekly. In some embodiments, the nanoparticle drug composition from the limus group is administered twice every three weeks (for example, on days 1 and 8 of a 21-day cycle). In some embodiments, the limus drug nanoparticle composition is administered three times every four weeks (eg, on days 1, 8, and 15 of a 28-day cycle). In some embodiments, the limus drug nanoparticle composition is administered by intravenous infusion over 30 minutes.

In some embodiments, a method of treating recurrent PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, a method of treating recurrent PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin. In some embodiments, a method of treating recurrent PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in the nanoparticles is bound (e.g., coated) with albumin. In some embodiments, a method of treating recurrent PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a limus drug and albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g. , no more than about 120 nm). In some embodiments, a method of treating recurrent PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising a limus drug and albumin, wherein the nanoparticles comprise a limus drug bound (e.g., coated ) with albumin and where the nanoparticles have an average particle size of no more than about 150 nm (for example, no more than about 120 nm). In some embodiments, a method of treating recurrent PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (e.g., about 9:1 or approximately 8:1). In some embodiments, a method of treating recurrent PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a Nab-sirolimus-containing composition. In some embodiments, a method of treating recurrent PEComa in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of Nab-sirolimus. In some embodiments, the limus drug is administered at a dose of about 10 to about 150 mg/m ² , including, for example, about 45 to about 100 mg/m ² and about 75 to 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 100 mg/m ² . In some

- 16 045904 embodiments, a drug from the limus group is administered at a dose of approximately 75 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 56 mg/m ² . In some embodiments, the limus drug nanoparticle composition is administered weekly. In some embodiments, the nanoparticle drug composition from the limus group is administered twice every three weeks (for example, on days 1 and 8 of a 21-day cycle). In some embodiments, the limus drug nanoparticle composition is administered three times every four weeks (eg, on days 1, 8, and 15 of a 28-day cycle). In some embodiments, the limus drug nanoparticle composition is administered by intravenous infusion over 30 minutes.

In some embodiments, a method of stabilizing PEComa (e.g., preventing or delaying the exacerbation of the disease) in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, a method of stabilizing PEComa (eg, preventing or delaying the exacerbation of the disease) in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a lymus drug and albumin. In some embodiments, a method is provided for stabilizing PEComa (e.g., preventing or delaying the exacerbation of the disease) in a subject (e.g., a human), comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the limus drug in nanoparticles is bound (eg, coated) with albumin. In some embodiments, a method is provided for stabilizing PEComa (e.g., preventing or delaying the exacerbation of the disease) in a subject (e.g., a human), comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the nanoparticles have a medium particle size no more than about 150 nm (eg, no more than about 120 nm). In some embodiments, a method is provided for stabilizing PEComa (e.g., preventing or delaying the exacerbation of the disease) in a subject (e.g., a human), comprising administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin, wherein the nanoparticles contain a limus drug and albumin, wherein the nanoparticles contain a limus group, bound (eg, coated) with albumin and wherein the nanoparticles have an average particle size of no more than about 150 nm (eg, no more than about 120 nm). In some embodiments, a method of stabilizing PEComa (e.g., preventing or delaying the exacerbation of the disease) in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and human albumin, wherein the nanoparticles comprise sirolimus bound to (e.g., coated) with human albumin, wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (for example, about 9:1 or about 8:1). In some embodiments, a method of stabilizing PEComa (eg, preventing or delaying the exacerbation of the disease) in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of a Nab-sirolimus-containing composition. In some embodiments, a method of stabilizing PEComa (eg, preventing or delaying the exacerbation of the disease) in a subject (eg, a human) is provided, comprising administering to the subject an effective amount of Nab-sirolimus. In some embodiments, the limus drug is administered at a dose of about 10 to about 150 mg/m ² , including, for example, about 45 to about 100 mg/m ² and about 75 to 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 100 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 75 mg/m ² . In some embodiments, the limus drug is administered at a dose of about 56 mg/m ² . In some embodiments, the limus drug nanoparticle composition is administered weekly. In some embodiments, the nanoparticle drug composition from the limus group is administered twice every three weeks (for example, on days 1 and 8 of a 21-day cycle). In some embodiments, the limus drug nanoparticle composition is administered three times every four weeks (eg, on days 1, 8, and 15 of a 28-day cycle). In some embodiments, the limus drug nanoparticle composition is administered by intravenous infusion over 30 minutes.

The methods described herein are suitable for various aspects of the treatment of PEComa. In some embodiments, a method of inhibiting perivascular epithelioid cell proliferation (eg, PEComa tumor growth) in a subject is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin. In some embodiments, cell proliferation is inhibited by at least

- 17 045904 by approximately 10% (including, for example, by at least about 20, 30, 40, 60, 70, 80, 90 or 100%). In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the limus drug in the nanoparticle composition is administered intravenously. In some embodiments, the composition contains nanoparticles containing a limus drug bound (eg, coated) with albumin. In some embodiments, the composition contains nanoparticles having an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles containing a lymus drug bound (eg, coated) with albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles comprising sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (eg, about 9:1 or about 8:1). In some embodiments, the limus drug nanoparticle composition is administered three times every four weeks (eg, on days 1, 8, and 15 of a 28-day cycle). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

In some embodiments, a method of preventing local recurrence (eg, tumor recurrence after resection) in a subject having PEComa is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin. In some embodiments, metastasis is inhibited by at least about 10% (including, for example, at least about 20, 30, 40, 60, 70, 80, 90, or 100%). In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the limus drug in the nanoparticle composition is administered intravenously. In some embodiments, the composition contains nanoparticles containing a limus drug bound (eg, coated) with albumin. In some embodiments, the composition contains nanoparticles having an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles containing a lymus drug bound (eg, coated) with albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles comprising sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (eg, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

In some embodiments, a method of inhibiting metastasis of a PEComa tumor in a subject is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin. In some embodiments, metastasis is inhibited by at least about 10% (including, for example, at least about 20, 30, 40, 60, 70, 80, 90, or 100%). In some embodiments, a method of inhibiting lymph node metastasis is provided. In some embodiments, a method of inhibiting lung metastasis is provided. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the limus drug in the nanoparticle composition is administered intravenously. In some embodiments, the composition contains nanoparticles containing a limus drug bound (eg, coated) with albumin. In some embodiments, the composition contains nanoparticles having an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles containing a lymus drug bound (eg, coated) with albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles comprising sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (eg, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is

- 18 045904

Nab-sirolimus.

In some embodiments, a method of reducing (e.g., eliminating) previously occurring PEComa tumor metastasis (e.g., lung metastasis or lymph node metastasis) in a subject is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin. In some embodiments, metastasis is reduced by at least about 10% (including, for example, at least about 20, 30, 40, 60, 70, 80, 90, or 100%). In some embodiments, a method of reducing lymph node metastasis is provided. In some embodiments, a method of reducing lung metastasis is provided. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the limus drug in the nanoparticle composition is administered intravenously. In some embodiments, the composition contains nanoparticles containing a limus drug bound (eg, coated) with albumin. In some embodiments, the composition contains nanoparticles having an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles containing a lymus drug bound (eg, coated) with albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles comprising sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (eg, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

In some embodiments, a method is provided for reducing the incidence or burden of prior PEComa tumor metastasis (eg, lung metastasis or lymph node metastasis) in a subject, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the limus drug in the nanoparticle composition is administered intravenously. In some embodiments, the composition comprises nanoparticles containing a limus drug bound (eg, coated) with albumin. In some embodiments, the composition contains nanoparticles having an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles containing a lymus drug bound (eg, coated) with albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles comprising sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (eg, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

In some embodiments, a method of reducing the size of a PEComa in a subject is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin. In some embodiments, tumor size is reduced by at least about 10% (including, for example, at least about 20, 30, 40, 60, 70, 80, 90, or 100%). In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the limus drug in the nanoparticle composition is administered intravenously. In some embodiments, the composition contains nanoparticles containing a limus drug bound (eg, coated) with albumin. In some embodiments, the composition contains nanoparticles having an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles containing a lymus drug bound (eg, coated) with albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles comprising sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein mass ratio of human albumin and

- 19 045904 sirolimus in the composition is about 9:1 or lower (for example, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

In some embodiments, a method is provided for prolonging the time until progression of PEComa in a subject, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor and albumin. In some embodiments, the method prolongs the time to disease progression by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the limus drug in the nanoparticle composition is administered intravenously. In some embodiments, the composition contains nanoparticles containing a limus drug bound (eg, coated) with albumin. In some embodiments, the composition contains nanoparticles having an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles containing a lymus drug bound (eg, coated) with albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles comprising sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (eg, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

In some embodiments, a method of prolonging the survival of a subject having PEComa is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor and albumin. In some embodiments, the method prolongs the survival of a subject by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the limus drug in the nanoparticle composition is administered intravenously. In some embodiments, the composition contains nanoparticles containing a limus drug bound (eg, coated) with albumin. In some embodiments, the composition contains nanoparticles having an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles containing a lymus drug bound (eg, coated) with albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles comprising sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (eg, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

In some embodiments, a method of reducing one or more symptoms in a subject having PEComa is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the limus drug in the nanoparticle composition is administered intravenously. In some embodiments, the composition contains nanoparticles containing a limus drug bound (eg, coated) with albumin. In some embodiments, the composition contains nanoparticles having an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles containing a lymus drug bound (eg, coated) with albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles comprising sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (eg, about 9:1 or about 8:1). In some embodiments, the composition contains Nab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

- 20 045904

In some embodiments, a method is provided for inhibiting the progression of CIS (carcinoma in situ) lesions in a subject having PEComa, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the limus drug in the nanoparticle composition is administered intravenously. In some embodiments, the composition contains nanoparticles containing a limus drug bound (eg, coated) with albumin. In some embodiments, the composition contains nanoparticles having an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles containing a lymus drug bound (eg, coated) with albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm. In some embodiments, the composition comprises nanoparticles comprising sirolimus bound (e.g., coated) with human albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), wherein the weight ratio of human albumin to sirolimus in the composition is about 9:1 or lower (eg, about 9:1 or about 8:1). In some embodiments, the average or median diameter of the nanoparticles is from about 10 to about 150 nm. In some embodiments, the average or median diameter of the nanoparticles is from about 40 nm to about 120 nm. In some embodiments, the composition contains Nabsirolimus. In some embodiments, the composition is Nab-sirolimus.

In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nab-sirolimus), wherein the composition is administered at a dose of 100 mg/ ^m2 . In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nab-sirolimus), wherein the composition is administered at a dose of 100 mg/m ² and where the composition is administered weekly, 2 times every 3 weeks (for example, on days 1 and 8 of a 21-day cycle). In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nabsirolimus), wherein the composition is administered at a dose 100 mg/ ^m2 , where the composition is administered weekly, 2 times every 3 weeks (for example, on days 1 and 8 of a 21-day cycle) and where the dose is administered by intravenous infusion over 30 minutes. In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nab-sirolimus), wherein the composition is administered at a dose of 100 mg/m ² and where the composition is administered weekly, 3 times every 4 weeks (for example, on days 1, 8 and 15 of a 28-day cycle). In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nab-sirolimus), wherein the composition is administered at a dose of 100 mg/ ^m2 , where the composition is administered weekly, 3 times every 4 weeks (for example, on days 1, 8 and 15 of a 28-day cycle) and where the dose is administered by intravenous infusion over 30 minutes.

In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nab-sirolimus), wherein the composition is administered at a dose of 75 mg/ ^m2 . In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nab-sirolimus), wherein the composition is administered at a dose of 75 mg/m ² and where the composition is administered weekly, 2 times every 3 weeks (for example, on days 1 and 8 of a 21-day cycle). In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nab-sirolimus), wherein the composition is administered at a dose of 75 mg/ ^m2 , where the composition is administered weekly, 2 times every 3 weeks (for example, on days 1 and 8 of a 21-day cycle) and where the dose is administered by intravenous infusion over 30 minutes. In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nab-sirolimus) , where the composition is administered at a dose of 75 mg/m ² and where the composition is administered weekly, 3 times every 4 weeks (for example, on days 1, 8 and 15 of a 28-day cycle). In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nab-sirolimus), wherein the composition is administered at a dose of 75 mg/ ^m2 , where the composition is administered weekly, 3 times every 4 weeks (for example, on days 1, 8 and 15 of a 28-day cycle) and where the dose is administered by intravenous infusion over 30 minutes.

In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nab-sirolimus), wherein the composition is administered at a dose of 56 mg/ ^m2 . In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nab-sirolimus), wherein the composition is administered at a dose of 56 mg/m ² and where the composition is administered weekly, 2 times every 3 weeks (for example, on days 1 and 8 of a 21-day cycle). In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nabsirolimus), wherein the composition is administered at a dose 56 mg/ ^m2 , where the composition is administered weekly, 2 times every 3 weeks (for example, on days 1 and 8 of a 21-day cycle) and where the dose is administered by intravenous infusion over 30 minutes. In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nab-sirolimus), wherein the composition is administered at a dose of 56 mg/m ² and where the composition is administered weekly, 3 times every 4 weeks (for example, on days 1, 8 and 15 of a 28-day cycle). In some embodiments, a method of treating PEComa in a subject (e.g., a human) is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin (e.g., nanoparticles containing sirolimus coated with albumin, e.g., Nab-sirolimus), wherein the composition is administered at a dose of 56 mg/ ^m2 , where the composition is administered weekly, 3 times every 4 weeks (for example, on days 1, 8 and 15 of a 28-day cycle) and where the dose is administered by intravenous infusion over 30 minutes.

Also provided are pharmaceutical compositions containing nanoparticles containing an mTOR inhibitor (such as a limus drug, eg sirolimus) for use in any of the methods of treating PEComa described herein. In some embodiments, the compositions comprise nanoparticles containing an mTOR inhibitor (such as a lymus drug, such as sirolimus) and albumin (such as human albumin).

Application of biomarkers

The present invention in one aspect provides methods of treating an epithelioid cell tumor, such as PEComa, based on the level of expression or activity, or mutational status of one or more biomarkers.

In some embodiments, a method of treating PEComa (such as malignant PEComa, e.g., metastatic or locally advanced PEComa) in a subject is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug) and albumin where the subject is selected for treatment based on the subject having the mutation status in the gene. In some embodiments, the gene is selected from an ONCOPANEL™ assay (CLIA certified). In some embodiments, the gene is an mTOR pathway gene. In some embodiments, the gene is an mTOR-associated pathway gene.

As used herein, the expression mTOR-associated pathway gene refers to a gene that encodes a molecule, such as a protein, that directly or indirectly interacts with or interacts with the mTOR signaling pathway. In some embodiments, the gene is selected from the group consisting of PIK3CA, TSC1, TSC2, AKT, PTEN, MTOR, and RHEB. In some embodiments, the gene is a TFE3 gene. In some embodiments, mutation status is identified in a sample from a subject using cell-free DNA sequencing. In some embodiments, mutation status is identified in a sample from a subject using exome sequencing. In some embodiments, mutational status is identified in a sample from a subject using mutational analysis of a tumor biopsy sample. In some embodiments, mutation status is identified in a sample from a subject using fluorescence in situ hybridization. In some embodiments, a sample

- 22 045904 is a blood sample. In some embodiments, the sample is a biopsy sample of a tumor. In some embodiments, the sample is obtained prior to the treatment methods described herein. In some embodiments, the sample is obtained after initiation of the treatment methods described herein.

In some embodiments, a method of treating PEComa (such as malignant PEComa, such as metastatic or locally advanced PEComa) in a subject is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug) and albumin, wherein the subject is selected for treatment based on the subject having a protein phosphorylation status associated with the mTOR signaling pathway. In some embodiments, the protein is selected from the group consisting of AKT, S6, S6K, 4EBP1, and SPARC. In some embodiments, the protein is phosphorylated. In some embodiments, the protein is phosphorylated at a specific amino acid site. In some embodiments, the protein is not phosphorylated. In some embodiments, phosphorylation status is identified in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor.

In some embodiments, a method of treating PEComa (such as malignant PEComa, such as metastatic or locally advanced PEComa) in a subject is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug) and albumin, wherein the subject is selected for treatment based on the subject having a proliferation marker expression level. In some embodiments, the proliferation marker is Ki-67. In some embodiments, the expression level of a proliferation marker is measured in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor. In some embodiments, a method of treating PEComa (such as malignant PEComa, e.g., metastatic or locally advanced PEComa) in a subject is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug) and albumin wherein the subject is selected for treatment based on the subject having an expression level of the apoptosis marker. In some embodiments, the apoptosis marker is PARP or a fragment thereof. In some embodiments, the expression level of an apoptosis marker is measured in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor.

In some embodiments, a method of treating PEComa (such as malignant PEComa, e.g., metastatic or locally advanced PEComa) in a subject is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug) and albumin wherein the nanoparticles have an average diameter of no more than about 150 nm and wherein the subject is selected for treatment based on the subject having the mutation status in the gene. In some embodiments, the gene is selected from the ONCOPANEL™ assay. In some embodiments, the gene is selected from an mTOR pathway gene. In some embodiments, the gene is an mTOR-associated pathway gene. In some embodiments, the gene is selected from the group consisting of PIK3CA, TSC1, TSC2, AKT, PTEN, MTOR, and RHEB. In some embodiments, the gene is a TFE3 gene. In some embodiments, mutation status is identified in a sample from a subject using cell-free DNA sequencing. In some embodiments, mutation status is identified in a sample from a subject using exome sequencing. In some embodiments, mutational status is identified in a sample from a subject using mutational analysis of a tumor biopsy sample. In some embodiments, mutation status is identified in a sample from a subject using fluorescence in situ hybridization. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a biopsy sample of a tumor. In some embodiments, the sample is obtained prior to the treatment methods described herein. In some embodiments, the sample is obtained after initiation of the treatment methods described herein.

In some embodiments, a method of treating PEComa (such as malignant PEComa, such as metastatic or locally advanced PEComa) in a subject is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug) and albumin, wherein the nanoparticles have an average diameter of no more than about 150 nm, wherein the subject is selected for treatment based on the subject having a protein phosphorylation status associated with the mTOR signaling pathway. In some embodiments, the protein is selected from the group consisting of AKT, S6, S6K, 4EBP1, and SPARC. In some embodiments, the protein is phosphorylated. In some embodiments, the protein is phosphorylated at a specific amino acid site. In some variants

- 23 045904 implementation of the protein is not phosphorylated. In some embodiments, phosphorylation status is identified in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor.

In some embodiments, a method of treating PEComa (such as malignant PEComa, e.g., metastatic or locally advanced PEComa) in a subject is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug) and albumin wherein the nanoparticles have an average diameter of no more than about 150 nm, wherein the subject is selected for treatment based on the subject having a proliferation marker expression level. In some embodiments, the proliferation marker is Ki-67. In some embodiments, the expression level of a proliferation marker is measured in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor. In some embodiments, a method of treating PEComa (such as malignant PEComa, such as metastatic or locally advanced PEComa) in a subject is provided, comprising administering to the subject an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug) and albumin, wherein the subject is selected for treatment based on the subject having an expression level of the apoptosis marker. In some embodiments, the apoptosis marker is PARP or a fragment thereof. In some embodiments, the expression level of an apoptosis marker is measured in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor.

In some embodiments, a method of treating PEComa (such as malignant PEComa, e.g., metastatic or locally advanced PEComa) in a subject is provided, comprising (a) assessing the mutational status of a gene in the subject, and (b) administering to the subject an effective amount of a composition comprising nanoparticles, containing an mTOR inhibitor (such as a limus drug) and albumin, wherein the subject is selected for treatment based on the subject having the mutation status in the gene. In some embodiments, the gene is selected from the ONCOPANEL™ assay. In some embodiments, the gene is selected from an mTOR pathway gene. In some embodiments, the gene is an mTOR-associated pathway gene. In some embodiments, the gene is selected from the group consisting of PIK3CA, TSC1, TSC2, AKT, PTEN, MTOR, and RHEB. In some embodiments, the gene is a TFE3 gene. In some embodiments, mutation status is identified in a sample from a subject using cell-free DNA sequencing. In some embodiments, mutation status is identified in a sample from a subject using exome sequencing. In some embodiments, mutational status is identified in a sample from a subject using mutational analysis of a tumor biopsy sample. In some embodiments, mutation status is identified in a sample from a subject using fluorescence in-situ hybridization. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a biopsy sample of a tumor. In some embodiments, the sample is obtained prior to the treatment methods described herein. In some embodiments, the sample is obtained after initiation of the treatment methods described herein.

In some embodiments, a method of treating PEComa (such as malignant PEComa, e.g., metastatic or locally advanced PEComa) in a subject is provided, comprising (a) assessing the protein phosphorylation status of the subject and (b) administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a limus drug); and albumin, wherein the subject is selected for treatment based on the subject having a protein phosphorylation status associated with the mTOR signaling pathway. In some embodiments, the protein is selected from the group consisting of AKT, S6, S6K, 4EBP1, and SPARC. In some embodiments, the protein is phosphorylated. In some embodiments, the protein is phosphorylated at a specific amino acid site. In some embodiments, the protein is not phosphorylated. In some embodiments, phosphorylation status is identified in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor.

In some embodiments, a method of treating PEComa (such as malignant PEComa, such as metastatic or locally advanced PEComa) in a subject is provided, comprising (a) assessing the expression level of a proliferation marker in the subject and (b) administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a limus drug); and albumin, wherein the subject is selected for treatment based on the subject having a proliferation marker expression level. In some embodiments, the proliferation marker is Ki-67. In some embodiments, the expression level of a proliferation marker is measured in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor. In some

- 24 045904 embodiments provide a method of treating PEComa (such as malignant PEComa, such as metastatic or locally advanced PEComa) in a subject, comprising (a) assessing the expression level of an apoptotic marker in the subject and (b) administering to the subject an effective amount of a composition comprising nanoparticles, containing an mTOR inhibitor (such as a limus drug) and albumin, wherein the subject is selected for treatment based on the subject having an apoptosis marker expression level. In some embodiments, the apoptosis marker is PARP or a fragment thereof. In some embodiments, the expression level of an apoptosis marker is measured in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor.

In some embodiments, a method of treating PEComa (such as malignant PEComa, such as metastatic or locally advanced PEComa) in a subject is provided, comprising (a) assessing the mutational status of a gene in the subject; (b) selecting (eg, identifying or recommending) a subject for treatment based on the subject having the mutation status in the gene; and (c) administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, the gene is selected from the ONCOPANEL™ assay. In some embodiments, the gene is selected from an mTOR pathway gene. In some embodiments, the gene is an mTOR-associated pathway gene. In some embodiments, the gene is selected from the group consisting of PIK3CA, TSC1, TSC2, AKT, PTEN, MTOR, and RHEB. In some embodiments, the gene is a TFE3 gene. In some embodiments, mutation status is identified in a sample from a subject using cell-free DNA sequencing. In some embodiments, mutation status is identified in a sample from a subject using exome sequencing. In some embodiments, mutational status is identified in a sample from a subject using mutational analysis of a tumor biopsy sample. In some embodiments, mutation status is identified in a sample from a subject using fluorescence in-situ hybridization. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a biopsy sample of a tumor. In some embodiments, the sample is obtained prior to the treatment methods described herein. In some embodiments, the sample is obtained after initiation of the treatment methods described herein.

In some embodiments, a method of treating PEComa (such as malignant PEComa, e.g., metastatic or locally advanced PEComa) in a subject is provided, comprising (a) assessing the protein phosphorylation status of the subject; (b) selecting (eg, identifying or recommending) a subject for treatment based on the subject having a protein phosphorylation status; and (c) administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, the protein is selected from the group consisting of AKT, S6, S6K, 4EBP1, and SPARC. In some embodiments, the protein is phosphorylated. In some embodiments, the protein is phosphorylated at a specific amino acid site. In some embodiments, the protein is not phosphorylated. In some embodiments, phosphorylation status is identified in a human sample using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor.

In some embodiments, a method of treating PEComa (such as malignant PEComa, such as metastatic or locally advanced PEComa) in a subject is provided, comprising (a) assessing the expression level of a proliferation marker in the subject; (b) selecting (eg, identifying or recommending) a subject for treatment based on the subject having a proliferation marker expression level; and (c) administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin, wherein the subject is selected for treatment based on the subject having a proliferation marker expression level. In some embodiments, the proliferation marker is Ki-67. In some embodiments, the expression level of a proliferation marker is measured in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor. In some embodiments, a method of treating PEComa (such as malignant PEComa, e.g., metastatic or locally advanced PEComa) is provided, comprising (a) assessing the expression level of an apoptotic marker in a subject; (b) selecting (eg, identifying or recommending) a subject for treatment based on the subject having an expression level of the apoptotic marker; and (c) administering to the subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin. In some embodiments, the apoptosis marker is PARP or a fragment thereof. In some embodiments, the expression level of an apoptosis marker is measured in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor.

- 25 045904

In some embodiments, a method is provided for selecting (including identifying or recommending) a subject having a PEComa (such as a malignant PEComa, such as metastatic or locally advanced PEComa) for treatment with a composition comprising nanoparticles containing an mTOR inhibitor (such as a limus drug) and albumin, where the method includes (a) assessing the mutation status in a gene in a subject and (b) selecting or recommending a subject for treatment based on the subject having the mutation status in the gene. In some embodiments, the gene is selected from the ONCOPANEL™ assay. In some embodiments, the gene is selected from an mTOR pathway gene. In some embodiments, the gene is an mTOR-associated pathway gene. In some embodiments, the gene is selected from the group consisting of PIK3CA, TSC1, TSC2, AKT, PTEN, MTOR, and RHEB. In some embodiments, the gene is a TFE3 gene. In some embodiments, mutation status is identified in a sample from a subject using cell-free DNA sequencing. In some embodiments, mutation status is identified in a sample from a subject using exome sequencing. In some embodiments, mutational status is identified in a sample from a subject using mutational analysis of a tumor biopsy sample. In some embodiments, mutation status is identified in a sample from a subject using fluorescence in situ hybridization. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a biopsy sample of a tumor. In some embodiments, the sample is obtained prior to the treatment methods described herein. In some embodiments, the sample is obtained after initiation of the treatment methods described herein.

In some embodiments, a method is provided for selecting (including identifying or recommending) a subject having a PEComa (such as a malignant PEComa, such as metastatic or locally advanced PEComa) for treatment with a composition comprising nanoparticles containing an mTOR inhibitor (such as a limus drug) and albumin, where the method includes (a) assessing a protein phosphorylation status of a subject and (b) selecting or recommending a subject for treatment based on the subject having a protein phosphorylation status associated with the mTOR signaling pathway. In some embodiments, the protein is selected from the group consisting of AKT, S6, S6K, 4EBP1, and SPARC. In some embodiments, the protein is phosphorylated. In some embodiments, the protein is phosphorylated at a specific amino acid site. In some embodiments, the protein is not phosphorylated. In some embodiments, phosphorylation status is identified in a human sample using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor.

In some embodiments, a method is provided for selecting (including identifying or recommending) a subject having a PEComa (such as a malignant PEComa, such as metastatic or locally advanced PEComa) for treatment with a composition comprising nanoparticles containing an mTOR inhibitor (such as a limus drug) and albumin, where the method includes (a) assessing the expression level of a proliferation marker in a subject and (b) selecting or recommending a subject for treatment based on the subject having the expression level of a proliferation marker. In some embodiments, the proliferation marker is Ki-67. In some embodiments, the expression level of a proliferation marker is measured in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor. In some embodiments, a method is provided for selecting (including identifying or recommending) a subject having a PEComa (such as a malignant PEComa, such as metastatic or locally advanced PEComa) for treatment with a composition comprising nanoparticles containing an mTOR inhibitor (such as a limus drug) and albumin, where the method includes (a) assessing the expression level of an apoptosis marker in the subject and (b) selecting (eg, identifying or recommending) a subject for treatment based on the subject having the expression level of the apoptosis marker. In some embodiments, the apoptosis marker is PARP or a fragment thereof. In some embodiments, the expression level of an apoptosis marker is measured in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor.

In some embodiments, a method is provided for selecting (including identifying or recommending) a subject having a PEComa (such as a malignant PEComa, such as metastatic or locally advanced PEComa) for treatment with a composition comprising nanoparticles containing an mTOR inhibitor (such as a limus drug) and albumin, where the method includes (a) assessing the mutational status in a gene in a subject; (b) selecting or recommending a subject for treatment based on the subject having a mutation status in the gene; and (c) administering an effective amount of a composition comprising nanoparticles containing a limus drug and albumin to a selected subject. In some embodiments, the gene you

- 26 045904 brane from ONCOPANEL™ analysis. In some embodiments, the gene is selected from an mTOR pathway gene. In some embodiments, the gene is an mTOR-associated pathway gene. In some embodiments, the gene is selected from the group consisting of PIK3CA, TSC1, TSC2, AKT, PTEN, MTOR, and RHEB. In some embodiments, the gene is a TFE3 gene. In some embodiments, mutation status is identified in a sample from a subject using cell-free DNA sequencing. In some embodiments, mutation status is identified in a sample from a subject using exome sequencing. In some embodiments, mutational status is identified in a sample from a subject using mutational analysis of a tumor biopsy sample. In some embodiments, mutation status is identified in a sample from a subject using fluorescence in situ hybridization. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a biopsy sample of a tumor. In some embodiments, the sample is obtained prior to the treatment methods described herein. In some embodiments, the sample is obtained after initiation of the treatment methods described herein.

In some embodiments, a method is provided for selecting (including identifying or recommending) a subject having a PEComa (such as a malignant PEComa, e.g., metastatic or locally advanced PEComa) for treatment with a composition comprising an mTOR inhibitor (such as a limus drug) and albumin. wherein the method includes (a) assessing the phosphorylation status of a protein in a subject; (b) selecting or recommending a subject for treatment based on the subject having a protein phosphorylation status; and (c) administering to the subject an effective amount of a composition comprising nanoparticles containing a limus drug and albumin to the selected subject. In some embodiments, the protein is selected from the group consisting of AKT, S6, S6K, 4EBP1, and SPARC. In some embodiments, the protein is phosphorylated. In some embodiments, the protein is phosphorylated at a specific amino acid site. In some embodiments, the protein is not phosphorylated. In some embodiments, phosphorylation status is identified in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor.

In some embodiments, a method is provided for selecting (including identifying or recommending) a subject having a PEComa (such as a malignant PEComa, such as metastatic or locally advanced PEComa) for treatment with a composition comprising nanoparticles containing an mTOR inhibitor (such as a limus drug) and albumin, where the method includes (a) assessing the expression level of a proliferation marker in the subject; (b) selecting or recommending a subject for treatment based on the subject having a proliferation marker expression level; and (c) administering an effective amount of a composition comprising nanoparticles containing a limus drug and albumin to a selected subject. In some embodiments, the proliferation marker is Ki-67. In some embodiments, the expression level of a proliferation marker is measured in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor. In some embodiments, a method is provided for selecting (including identifying or recommending) a subject having a PEComa (such as a malignant PEComa, such as metastatic or locally advanced PEComa) for treatment with a composition comprising nanoparticles containing an mTOR inhibitor (such as a limus drug) and albumin, where the method includes (a) assessing the expression level of an apoptosis marker in the subject; (b) selecting or recommending a subject for treatment based on the subject having an apoptotic marker expression level; and (c) administering an effective amount of a composition comprising nanoparticles containing a limus drug and albumin to a selected subject. In some embodiments, the apoptosis marker is PARP or a fragment thereof. In some embodiments, the expression level of an apoptosis marker is measured in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor.

Also provided are methods for assessing whether a subject with PEComa (such as malignant PEComa, such as metastatic or locally advanced PEComa) will be more likely to respond or will be less likely to respond to treatment based on the subject having a mutation status in the gene where the treatment includes a composition comprising nanoparticles containing an mTOR inhibitor (such as a limus drug) and albumin, and wherein the method includes assessing the mutational status of a gene in a subject. In some embodiments, the method further comprises administering to a subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin to the subject who is determined to respond to treatment. In some embodiments, the presence of a mutation in a gene indicates that the subject is more likely to respond to treatment, and the absence of a mutation in the gene indicates that the subject is less likely to respond to treatment.

- 27 045904 children respond to treatment. In some embodiments, the gene is selected from the ONCOPANEL™ assay. In some embodiments, the gene is selected from an mTOR pathway gene. In some embodiments, the gene is an mTOR-associated pathway gene.

In some embodiments, the gene is selected from the group consisting of PIK3CA, TSC1, TSC2, AKT, PTEN, MTOR, and RHEB. In some embodiments, the gene is a TFE3 gene. In some embodiments, mutation status is identified in a sample from a subject using cell-free DNA sequencing. In some embodiments, mutation status is identified in a sample from a subject using exome sequencing. In some embodiments, mutational status is identified in a sample from a subject using mutational analysis of a tumor biopsy sample. In some embodiments, mutation status is identified in a sample from a subject using fluorescence in-situ hybridization. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a biopsy sample of a tumor. In some embodiments, the sample is obtained prior to the treatment methods described herein. In some embodiments, the sample is obtained after initiation of the treatment methods described herein.

Also provided are methods for assessing whether a subject with a PEComa (such as a malignant PEComa, e.g., metastatic or locally advanced PEComa) will be more likely to respond or will be less likely to respond to treatment based on the subject having a phosphorylation status on a protein where the treatment includes a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin, and wherein the method includes assessing the protein phosphorylation status of a subject. In some embodiments, the method further comprises administering to a subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin to the subject who is determined to respond to treatment. In some embodiments, the phosphorylated protein indicates that the subject will be more likely to respond to treatment. In some embodiments, the non-phosphorylated protein indicates that the subject will be less likely to respond to treatment.

Methods are also provided for assessing whether a subject with PEComa (such as malignant PEComa, e.g., metastatic or locally advanced PEComa) will be more likely to respond or will be less likely to respond to treatment based on the subject having a proliferation marker expression level where the treatment includes a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin, and wherein the method includes assessing the expression level of a proliferation marker in a subject. In some embodiments, the method further comprises administering to a subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin to the subject who is determined to respond to treatment. In some embodiments, the level of expression of the proliferation marker indicates that the subject is more likely to respond to treatment. In some embodiments, the proliferation marker is Ki-67. In some embodiments, the expression level of a proliferation marker is measured in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor. Methods are also provided for assessing whether a subject with PEComa (such as malignant PEComa, e.g., metastatic or locally advanced PEComa) will be more likely or less likely to respond to treatment based on the subject having an apoptotic marker expression level, wherein the treatment comprises a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin, and wherein the method comprises assessing the expression level of an apoptosis marker in the subject. In some embodiments, the method further comprises administering to a subject an effective amount of a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin to the subject who is determined to respond to treatment. In some embodiments, the level of expression of the apoptosis marker indicates that the subject will be less likely to respond to treatment. In some embodiments, the apoptosis marker is PARP or a fragment thereof. In some embodiments, the expression level of an apoptosis marker is measured in a sample from a subject using immunohistochemistry. In some embodiments, the sample is a biopsy sample of a tumor.

The mutational status of a gene can be assessed in samples from various sources. In some embodiments, the sample is a blood sample. In some embodiments, cell-free DNA is isolated from a blood sample. In some embodiments, the sample is a biopsy sample of a tumor. The mutational status of a gene can be assessed using various methods well known to those skilled in the art. In some embodiments, the mutational status of a gene is assessed using cell-free DNA sequencing techniques. In some embodiments, the mutational status of a gene is assessed using sequencing

- 28 045904 next generation. In some embodiments, the mutational status of a gene isolated from a blood sample is assessed using next generation sequencing. In some embodiments, the mutational status of a gene is assessed using exosome sequencing. In some embodiments, the mutational status of a gene is assessed using a fluorescence in situ hybridization assay. In some embodiments, mutation status is assessed prior to initiation of the treatment methods described herein. In some embodiments, mutation status is assessed after initiation of the treatment methods described herein. In some embodiments, mutation status is assessed before and after initiation of the treatments described herein.

Phosphorylation status can be assessed in samples from various sources. In some embodiments, the sample is a biopsy sample of a tumor. The phosphorylation status of a protein can be assessed using various methods well known to those skilled in the art. In some embodiments, phosphorylation status is assessed using immunohistochemistry. The phosphorylation status of a protein can be site specific. Phosphorylation status can be compared with a control sample. In some embodiments, phosphorylation status is assessed prior to initiation of the treatment methods described herein. In some embodiments, phosphorylation status is assessed after initiation of the treatment methods described herein. In some embodiments, phosphorylation status is assessed before and after initiation of the treatments described herein. In some embodiments, the protein is phosphorylated. In some embodiments, the protein is not phosphorylated. In some embodiments, the site of a particular amino acid is phosphorylated. In some embodiments, the site of a particular amino acid is not phosphorylated.

The level of protein expression (such as a marker of proliferation or apoptosis) may be high or low compared to the control sample. In some embodiments, the subject's protein level is compared to the protein level of a control sample. In some embodiments, the subject's protein level is compared to the protein level of multiple control samples. In some embodiments, multiple control samples are used to generate statistics that are used to classify the protein level of a subject with PEComa. The protein phosphorylation status can be compared with a control sample. In some embodiments, the expression level is assessed prior to the treatment methods described herein. In some embodiments, expression levels are assessed after initiation of the treatments described herein. In some embodiments, expression levels are assessed before and after initiation of the treatments described herein.

Classification or ranking of protein levels (eg, high or low) can be made relative to the statistical distribution of reference levels. In some embodiments, the classification or ranking is performed relative to a control sample obtained from the subject. In some embodiment, protein levels are classified or ranked relative to a statistical distribution of control levels. In some embodiments, the protein level is classified or ranked relative to the level in a control sample obtained from the subject.

Control samples may be obtained using the same sources and methods as non-control samples. In some embodiments, the control sample is obtained from another subject (eg, a subject who does not have PEComa and/or a subject who has a similar ethnic, age, and gender identity). In some embodiments, when the sample is a tumor tissue sample, the control sample may be a non-cancerous tissue sample from the same subject. In some embodiments, multiple control samples (eg, from different subjects) are used to determine a range of biomarker levels in a particular tissue, organ, or cell population. In some embodiments, the control sample is a cultured tissue or cell that has been determined to be an adequate control. In some embodiments, the control is a cell that does not express the biomarker. In some embodiments, a clinically acceptable normal level in a standardized test is used as a reference level to determine the level of a biomarker. In some embodiments, the subject's reference level of the biomarker is classified as high, moderate, or low according to a scoring system, such as an immunohistochemistry-based scoring system. In some embodiments, a subject's biomarker reference level is classified as a low level in the sample when the score is less than or equal to the overall average score.

In some embodiments, the biomarker level is determined by measuring the biomarker level in a subject and comparing it to a control or reference value (eg, the average level for a given patient population or the level in a second subject). For example, if a biomarker level for one subject is determined to be above the average level in a population of patients, then that subject is determined to have a high level of the biomarker. Alternatively, if the biomarker level for

- 29 045904 of one subject is determined to be below the average level in the patient population, then the subject is determined to have a low level of the biomarker. In some embodiments, the subject is compared with a second subject and/or a population of patients who respond to treatment. In some embodiments, the subject is compared with a second subject and/or a population of patients who do not respond to treatment. In any of the embodiments of the present invention, levels can be determined by measuring the level of nucleic acid encoding the biomarker. For example, if the level of mRNA encoding a biomarker in one subject is determined to be above the average level in a population of patients, then that subject is determined to have a high level of mRNA encoding the biomarker. Alternatively, if the level of mRNA encoding a biomarker in one subject is determined to be below the average level in the patient population, then that subject is determined to have a low level of mRNA encoding the biomarker.

In some embodiments, a biomarker reference level is determined by obtaining a statistical distribution of biomarker levels.

In some embodiments, bioinformatics methods are used to determine and classify biomarker levels. Numerous alternative bioinformatics-based approaches have been developed to evaluate gene expression profiles using gene expression profiling data. Methods include, but are not limited to, those described in Segal E. et al., Nat. Genet. 34:66-176 (2003); Segal E. et al., Nat. Genet. 36:1090-1098 (2004); Barry W.T. et al., Bioinformatics, 21:1943–1949 (2005); Tian L. et al., Proc. Nat'l. Acad. Sci. USA, 102:13544-13549 (2005); Novak B.A. and Jain A.N., Bioinformatics, 22:233-41 (2006); Maglietta R. et al., Bioinformatics, 23:2063-72 (2007); Bussemaker H. J., BMC Bioinformatics, 8 Suppl, 6:S6 (2007).

In some embodiments, the level of protein expression is determined, for example, by immunohistochemistry. For example, criteria for interpreting low or high levels can be established based on the number of positively stained cells and/or the intensity of staining, for example, using an antibody that specifically recognizes the biomarker protein. In some embodiments, the level is low if less than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% of the cells stain positive. In some embodiments, the level is low if the staining is 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% lower than the staining intensity of the positive control.

In some embodiments, the level is high if more than about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% of the cells stain positive. In some embodiments, the level is high if the coloring is as intense as the positive control coloring. In some embodiments, the level is high if the staining is 80, 85, or 90% as intense as the positive control staining.

In some embodiments, strong staining, medium staining, and weak staining represent calibrated staining levels where the range and intensity of staining within the range is established. In some embodiments, strong staining represents staining above the 75th percentile of the intensity range, medium staining represents staining from the 25th to 75th percentile of the intensity range, and low staining represents staining below the 25th percentile of the intensity range. In some aspects, one skilled in the art and one familiar with the particular dyeing technology adjusts the binary size and determines the dyeing categories.

In some embodiments, the biomarker is assessed in a blood sample. In some embodiments, the biomarker is assessed in a cell-free DNA sample. In some embodiments, the biomarker is assessed using next generation sequencing. In some embodiments, the biomarker is assessed in a tumor biopsy sample. In some embodiments, the biomarker is assessed using immunohistochemistry.

It also provides pathways for referral for treatment of PEComa by delivering a sample to a diagnostic laboratory to determine biomarker levels; providing a control sample with a known biomarker level; providing an antibody to the biomarker; causing the sample and control sample to bind to the antibody and/or detecting a relative level of antibody binding, wherein the level in the sample is used to provide a determination that the patient should receive treatment by any of the methods described herein.

Also provided herein are methods of referral for treatment of a disease, further including examination or analysis of data regarding the presence (or level) of a biomarker in a sample; and providing an opinion to a subject, such as a health care provider or health care director, about the subject's likelihood or suitability to respond to treatment, where the opinion is based on examination or analysis of data. In one aspect of the invention, the conclusion represents data transmission over a network.

The ONCOPANEL™ assay can be used to probe exonic DNA sequences of cancer-associated genes and intronic regions to detect genetic abnormalities

- 30 045904 tions, including somatic mutations, copy number variations and structural rearrangements in DNA in samples from various sources (such as a tumor biopsy sample or a blood sample), thereby providing a list of candidate genetic aberrations that may represent mTOR-activating aberrations. In some embodiments, the mTOR-associated gene aberration is a genetic aberration or aberrant level (such as expression level or activity level) in a gene selected from the ONCOPANEL™ assay. See, for example, Wagle N. et al. Cancer discovery, 2.1 (2012): 82-93.

A typical version of the ONCOPANEL™ assay includes 300 cancer-associated genes and 113 introns across 35 genes. The 300 genes included in a typical ONCOPANEL™ analysis are ABL1, AKT1, AKT2, AKT3, ALK, ALOX12B, APC, AR, ARAF, ARID1A, ARID1B, ARID2, ASXL1, ATM, ATRX, AURKA, AURKB, AXL, B2M, BAP1 , BCL2, BCL2L1, BCL2L12, BCL6, BCOR, BCORL1, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD4, BRIP1, BUB1B, CADM2, CARD11, CBL, CBLB, CCND1, CCND2, CCND3, CCNE1, CD274, CD58, CD79B , CDC73, CDH1, CDK1, CDK2, CDK4, CDK5, CDK6, CDK9, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, SEVRA, CHEK2, CIITA, CREBBP, CRKL, CRLF2, CRTC1, CRTC2, CSF1R, CSF3R, CTNNB1 , CUX1, CYLD, DDB2, DDR2, DEPDC5, DICER1, DIS3, DMD, DNMT3A, EED, EGFR, EP300, EPHA3, ERNA5, ERNA7, ERBB2, ERBB3, ERBB4, ERCC2, ERCC3, ERCC4, ERCC5, ESR1, ETV1, ETV4 , ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2, FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FAS, FBXW7, FGFR1, FGFR2, FGFR3, FGFR4, FH, FKBP9, FLCN FLT1, FLT3, FLT4, FUS, GATA3, GATA4, GATA6, GLI1, GLI2, GLI3, GNA11, GNAQ, GNAS, GNB2L1, GPC3, GSTM5, H3F3A, HNF1A, HRAS, ID3, IDH1, IDH2, IGF1R, IKZF1, IKZF3, INSIG1, JAK2, JAK3, KCNIP1, KDM5C, KDM6A, KDM6B, KDR, KEAP1, KIT, KRAS, LINC00894, LMO1, LMO2, LMO3, MAP2K1, MAP2K4, MAP3K1, MAPK1, MCL1, MDM2, MDM4, MECOM, MEF2B, MEN1, MET, MITF, MLH1, MLL (KMT2A), MLL2 (KTM2D), MPL, MSH2, MSH6, MTOR, MUTYH, MYB, MYBL1, MYC, MYCL1 (MYCL), MYCN, MYD88, NBN, NEGR1, NF1, NF2, NFE2L2, NFKBIA, NFKBIZ, NKX2 -1, NOTCH1, NOTCH2, NPM1, NPRL2, NPRL3, NRAS, NTRK1, NTRK2, NTRK3, PALB2, PARK2, PAX5, PBRM1, PDCD1LG2, PDGFRA, PDGFRB, PHF6, PHOX2B, PIK3C2B, PIK3CA, PIK3R1, PIM1, PMS1, PMS2 , PNRC1, PRAME, PRDM1, PRF1, PRKAR1A, PRKCI, PRKCZ, PRKDC, PRPF40B, PRPF8, PSMD13, PTCH1, PTEN, PTK2, PTPN11, PTPRD, QKI, RAD21, RAF1, RARA, RB1, RBL2, RECQL4, REL, RET , RFWD2, RHEB, RHPN2, ROS1, RPL26, RUNX1, SBDS, SDHA, SDHAF2, SDHB, SDHC, SDHD, SETBP1, SETD2, SF1, SF3B1, SH2B3, SLITRK6, SMAD2, SMAD4, SMARCA4, SMARCB1, SMC1A, SMC3, SMO , SOCS1, SOX2, SOX9, SQSTM1, SRC, SRSF2, STAG1, STAG2, STAT3, STAT6, STK11, SUFU, SUZ12, SYK, TCF3, TCF7L1, TCF7L2, TERC, TERT, TET2, TLR4, TNFAIP3, TP53, TSC1, TSC2 , U2AF1, VHL, WRN, WT1, XPA, XRS, XPO1, ZNF217, ZNF708, ZRSR2.

Intronic regions examined in a typical ONCOPANEL™ assay occur in the specific introns ABL1, AKT3, ALK, BCL2, BCL6, BRAF, CIITA, EGFR, ERG, ETV1, EWSR1, FGFR1, FGFR2, FGFR3, FUS, IGH, IGL, JAK2 , MLL, MYC, NPM1, NTRK1, PAX5, PDGFRA, PDGFRB, PPARG, RAF1, RARA, RET, ROS1, SS18, TRA, TRB, TRG, TMPRSS2. mTOR-activating aberrations (such as genetic aberration and aberrant levels) of any of the genes included in any embodiment or version of the ONCOPANEL™ assay, including, but not limited to, the genes and intronic regions listed above, are considered as the basis for selecting a subject for treatment with mTOR inhibitor nanoparticle compositions.

Dosing and method of administration of nanoparticle compositions

The dose of mTOR nanoparticles (such as limus nanodrug compositions) administered to a subject (eg, a human) may vary depending on the specific composition, route of administration, and type of PEComa being treated. In some embodiments, the amount of the composition is effective to provide an objective response (eg, partial response or complete response). In some embodiments, the amount of the mTOR nanoparticle composition (eg, the limus drug nanoparticle composition) is sufficient to produce a complete response in the subject. In some embodiments, the amount of an mTOR nanoparticle composition (eg, a limus drug nanoparticle composition) is sufficient to produce a partial response in a subject. In some embodiments, the amount of mTOR nanoparticle composition (e.g., limus drug nanoparticle composition) administered (e.g., self-administered) is sufficient to produce an overall response rate of greater than about 20, 30, 40, 50, 60, or 64% of a population of subjects treated with an mTOR nanoparticle composition (such as a limus drug nanoparticle composition). A subject's responses to treatment by the methods described herein can be determined, for example, based on RECIST levels, cystoscopy (with or without biopsy), biopsy, cytology, and computed tomography.

In some embodiments, the amount of an mTOR nanoparticle composition (eg, a limus drug nanoparticle composition) is sufficient to produce a negative biopsy in a subject.

- 31 045904

In some embodiments, the amount of the composition is sufficient to prolong the subject's survival without disease progression. In some embodiments, the amount of the composition is sufficient to prolong the overall survival of the subject. In some embodiments, the amount of the composition (e.g., when self-administered) is sufficient to produce a clinical effect in greater than 50, 60, 70, or 77% of a population of subjects treated with an mTOR nanoparticle composition (e.g., a limus drug nanoparticle composition).

In some embodiments, the amount of the composition is an amount sufficient to reduce tumor size, reduce the number of malignant cells, or reduce the rate of tumor growth by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 100% compared with the corresponding tumor size or tumor growth rate in the same subject before treatment or compared with the corresponding activity in other subjects not receiving such treatment. To measure the magnitude of this effect, standard methods can be used, such as in vitro tests with purified enzyme, cell tests, animal models or testing in humans.

In some embodiments, the amount of an mTOR inhibitor (such as a lymus drug, e.g., sirolimus) in the composition is below a level that induces a toxic effect (i.e., an effect above a clinically acceptable level of toxicity) or is at a level where a potential adverse effect the effect may be controlled or tolerated when the composition is administered to a subject.

In some embodiments, the amount of the composition is close to the maximum tolerated dose (MTD) of the composition when administered under a similar dosing regimen. In some embodiments, the amount of the composition is greater than about 80, 90, 95, or 98% of the MTD.

In some embodiments, effective amounts of an mTOR inhibitor (e.g., a lymus drug) in the nanoparticle composition include, but are not limited to, at least any of about 25, 30, 50, 60, 75, 80, 90, 100, 120, 160, 175, 180, 200, 210, 220, 250, 260, 300, 350, 400, 500, 540, 750, 1000 or 1080 mg/ ^m2 mTOR inhibitor (eg, sirolimus). In various embodiments, the composition includes any amounts lower than about 350, 300, 250, 200, 150, 120, 100, 90, 50, or 30 mg/m ² of an mTOR inhibitor (eg, sirolimus). In some embodiments, the amount of mTOR inhibitor (e.g., sirolimus) per administration is any amount lower than about 25, 22, 20, 18, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 mg/ ^m2 .

In some embodiments, the effective amount of an mTOR inhibitor (eg, sirolimus) in the composition is in any of the following ranges: about 1 to about 5 mg/ ^m2 , about 5 to about 10 mg/ ^m2 , about 10 to about 25 mg/ ^m2 , about 25 to about 50 mg/ ^m2 , about 50 to about 75 mg/ ^m2 , about 75 to about 100 mg/ ^m2 , about 100 to about 125 mg/ ^m2 , about from 125 to about 150 mg/m ² , from about 150 to about 175 mg/m ² , from about 175 to about 200 mg/m ² , from about 200 to about 225 mg/m ² , from about 225 to about 250 mg /m ² , from about 250 to about 300 mg/m ² , from about 300 to about 350 mg/m ² or from about 350 to about 400 mg/m ² . In some embodiments, the effective amount of mTOR inhibitor (eg, sirolimus) in the composition is from about 5 to about 300 mg/m ² , for example, from about 100 to about 150 mg/m ² , about 120 mg/m ² , about 130 mg /m ² or approximately 140 mg/m ² .

In some embodiments of any of the above-described aspects, an effective amount of an mTOR inhibitor (e.g., sirolimus) in the composition includes at least any of about 1, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 mg/kg. In various embodiments, the effective amount of an mTOR inhibitor (e.g., sirolimus) in the composition includes any amounts lower than about 350, 300, 250, 200, 150, 100, 50, 25, 20, 10, 7.5, 6.5, 5 , 3.5, 2.5, or 1 mg/kg mTOR inhibitor (eg, sirolimus).

In some embodiments, the dosing frequency when administering nanoparticle compositions includes, but is not limited to, once a day, once every two days, once every three days, once every four days, once every five days, once every six days , one weekly without a break, three out of four weeks, once every three weeks; once every two weeks or two out of three weeks. In some embodiments, the composition is administered approximately once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 8 weeks. In some embodiments, the composition is administered at least about 1x, 2x, 3x, 4x, 5x, 6x, or 7x (ie, daily) over the course of a week. In some embodiments, the intervals between each of the administrations are less than about 6 months, 3 months, 1 month, 20 days, 15 days, 12 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day. In some embodiments, the intervals between each of the administrations are greater than about 1, 2, 3, 4, 5, 6, 8, or 12 months. In some embodiments, the dosing regimen is not interrupted. In some embodiments, the interval between each of the administrations is not more than about a week.

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In some embodiments, the dosing frequency is once every two days once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times and eleven times. In some embodiments, the dosing frequency is once every two days, five times. In some embodiments, the mTOR inhibitor (e.g., sirolimus) is administered over at least ten days, where the interval between each administration is no more than two days, and where the dose of the mTOR inhibitor (e.g., sirolimus) in each administration is between about 0. 25 to about 250 mg/ ^m2 , about 0.25 to about 150 mg/ ^m2 , about 0.25 to about 75 mg/ ^m2 , for example, about 0.25 to about 25 mg/ ^m2 or from about 25 to about 50 mg/m ² .

Administration of the composition may be extended over a long period of time, for example, from about a month to about seven years. In some embodiments, the composition is administered for a period of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 48, 60, 72, or 84 months.

In some embodiments, the dose of the mTOR inhibitor (e.g., sirolimus) in the nanoparticle composition may be 5-400 mg/ ^m2 when administered on a 3-week schedule, or 5-250 mg/ ^m2 (e.g., 80-150 mg/ ^m2 , for example 100-120 mg/ ^m2 ) when prescribed on a weekly basis. For example, the amount of an mTOR inhibitor (eg, sirolimus) is from about 60 to 300 mg/m ² (eg, about 260 mg/m ² ) when administered on a 3-week schedule.

In some embodiments, exemplary dosage regimens for administering a nanoparticle composition (eg, a sirolimus/albumin nanoparticle composition) include, but are not limited to, 100 mg/m ² weekly, without interruption; 100 mg/ ^m2 , weekly, 2 of 3 weeks; 100 mg/ ^m2 , weekly, 3 of 4 weeks; 75 mg/ ^m2 , weekly, without interruption; 75 mg/ ^m2 , weekly, 2 of 3 weeks; 75 mg/ ^m2 , weekly, 3 of 4 weeks; 56 mg/ ^m2 , weekly, without interruption; 56 mg/ ^m2 , weekly, 2 of 3 weeks; 56 mg/ ^m2 , weekly, 3 of 4 weeks. The dosing frequency of the composition may be adjusted during treatment based on the judgment of the attending physician.

In some embodiments, the subject is treated in at least one, two, three, four, five, six, seven, eight, nine, or ten treatment cycles.

The compositions described herein allow the composition to be infused into a subject over an infusion time that is less than about 24 hours. For example, in some embodiments, the composition is administered over an infusion time of less than 24, 12, 8, 5, 3, 2, 1 h, 30, 20 or 10 min. In some embodiments, the composition is administered over an infusion time of approximately 30 minutes.

In some embodiments, the approximate dose of an mTOR inhibitor (in some embodiments, a lymus drug, such as sirolimus) in the nanoparticle composition includes, but is not limited to, any dose of about 50, 60, 75, 80, 90, 100, 120, 160 , 175, 200, 210, 220, 260 and 300 mg/ ^m2 . For example, the dose of mTOR inhibitor in the nanoparticle composition may be in the range of about 100-400 mg/m ² when administered on a 3-week schedule, or about 50-250 mg/m ² when administered on a weekly schedule.

An mTOR nanoparticle composition (such as a limus drug nanoparticle composition) can be administered to a subject (e.g., a human) by various routes, including, for example, intravenous, intraarterial, intraperitoneal, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intratracheal, subcutaneous, intraocular , intrathecal or percutaneous. In some embodiments, a sustained release formulation of the composition may be used. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered intravesicularly. In some embodiments, the composition is administered intra-arterially. In some embodiments, the composition is administered intraperitoneally.

In some embodiments, when the nanoparticle limus drug composition is administered intravesicularly, at a dose of mTOR inhibitor (such as a limus drug, e.g., sirolimus) in the nanoparticle composition, which may range from about 30 to about 400 mg by volume from about 20 to about 150 ml, for example, it is retained in the bladder for about 30 minutes to about 4 hours. In some embodiments, the nanoparticle composition is retained in the bladder for about 30 minutes to about 4 hours, including, for example, about 30 minutes to about 1 hour, about 1 to about 2 hours, about 2 to about 3 hours, or about 3 to 4 hours.

In some embodiments, the dose of the mTOR inhibitor (such as a limus drug, such as sirolimus) is from about 100 to 400 mg, such as about 100 mg, about 200 mg, about 300 mg, or about 400 mg. In some embodiments, the limus drug is administered at a dose of about 100 mg per week, about 200 mg per week, about 300 mg per week, about 100 mg twice per week, or about 200 mg twice per week. In some embodiments, administration is then continued at a monthly maintenance dose (which may be the same or different from the weekly doses).

In some embodiments, when the nanoparticle drug composition is made from

- 33 045904 limus group is administered intravenously, the dose of mTOR inhibitor (such as a limus drug, eg sirolimus) in the nanoparticle composition may range from about 30 to about 400 mg. The compositions described herein allow the composition to be infused into a subject over an infusion time of less than about 24 hours. For example, in some embodiments, the composition is administered over an infusion time of less than 24, 12, 8, 5, 3, 2, 1 hour, 30, 20 or 10 min. In some embodiments, the composition is administered over an infusion time of about 30 to about 40 minutes.

Nanoparticle compositions

The nanoparticle compositions described herein contain nanoparticles containing (in various embodiments, consisting primarily of) an mTOR inhibitor (such as a lymus drug, eg sirolimus). The nanoparticles may additionally contain albumin (such as human serum albumin or human albumin). Nanoparticles with poorly water-soluble drugs have been described, for example, in US patents No. 5916596; 6506405; 6749868, 6537579, 7820788, as well as in US patent applications No. 2006/0263434 and 2007/0082838; PCT patent application WO 08/134148, each document is incorporated by reference in its entirety.

In some embodiments, the composition contains nanoparticles with an average or median diameter of no more than about 1000 nm, such as no more than about (or less than about) 900, 800, 700, 600, 500, 400, 300, 200, 150, 120, and 100 nm. In some embodiments, the average or median diameter of the nanoparticles is no more than about 150 nm (eg, no more than about 120 nm). In some embodiments, the average or median diameter of the nanoparticles is no more than about 120 nm. In some embodiments, the average or median diameter of the nanoparticles is from about 10 to about 150 nm. In some embodiments, the average or median diameter of the nanoparticles is from about 40 nm to about 120 nm. In some embodiments, the nanoparticles are sterilized through filtration.

In some embodiments, the nanoparticles in the composition described herein have an average diameter of no more than about 150 nm, including, for example, no more than about 140, 130, 120, 110, 100, 90, 80, 70, or 60 nm. In some embodiments, at least about 50% (e.g., at least about 60, 70, 80, 90, 95, or 99%) of the nanoparticles in the composition have a diameter of no more than about 150 nm, including, for example, no more than than about 140, 130, 120, 110, 100, 90, 80, 70 or 60 nm. In some embodiments, at least about 50% (e.g., at least 60, 70, 80, 90, 95, or 99%) of the nanoparticles in the composition have a diameter in the range of about 20 to about 150 nm, including, for example, from about 40 to about 120 nm.

In some embodiments, albumin contains sulfhydryl groups that can form disulfide bonds. In some embodiments, at least about 5% (including, for example, at least about 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, or 90%) of the albumin in the nanoparticle portion of the composition is cross-linked ( e.g. cross-linked via one or more disulfide bonds).

In some embodiments, nanoparticles containing an mTOR inhibitor (such as a limus drug, such as sirolimus) are bound (eg, coated) with albumin (eg, human albumin or human serum albumin). In some embodiments, the composition contains an mTOR inhibitor (such as a lymus drug, e.g., sirolimus) in both nanoparticle and non-nanoparticle form (e.g., in the form of solutions or in the form of soluble albumin/nanoparticle complexes), wherein at least At least about 50, 60, 70, 80, 90, 95, or 99% of the mTOR inhibitor (such as a lymus drug, eg, sirolimus) in the composition is in nanoparticle form. In some embodiments, the mTOR inhibitor (such as a lymus drug, such as sirolimus) in the nanoparticles constitutes more than about 50, 60, 70, 80, 90, 95, or 99% of the total weight of the nanoparticles. In some embodiments, the nanoparticles comprise a non-polymeric matrix. In some embodiments, the nanoparticles comprise a mTOR inhibitor core (such as a lymus drug, such as sirolimus) that is substantially free of polymeric materials (such as a polymer matrix).

In some embodiments, the composition contains albumin in both the nanoparticle and non-nanoparticle portions of the composition, wherein at least about 50, 60, 70, 80, 90, 95, or 99% of the albumin in the composition is in the non-nanoparticle portion of the composition.

In some embodiments, the weight ratio of albumin (such as human albumin or human serum albumin) to mTOR inhibitor in the nanoparticle composition is about 18:1 or lower, such as about 15:1 or lower, such as about 10:1 or lower. In some embodiments, the weight ratio of albumin (such as human albumin or human serum albumin) to mTOR inhibitor (such as a limus drug, such as sirolimus) in the composition ranges from about 1:1 to about 18:1, about 2:1 to approximately 15:1, approximately 3:1 to approximately 13:1, approximately

- 34 045904 from 4:1 to about 12:1, from about 5:1 to about 10:1. In some embodiments, the weight ratio of albumin to mTOR inhibitor (such as a lymus drug, such as sirolimus) in the nanoparticle portion of the composition is about 1:2, 1:3, 1:4, 1:5, 1:9, 1: 10, 1:15 or lower. In some embodiments, the weight ratio of albumin (such as human albumin or human serum albumin) and mTOR inhibitor (such as a lymus drug, e.g., sirolimus) in the composition is any of the following: from about 1:1 to about 18:1, about 1:1 to about 15:1, about 1:1 to about 7:1, about 1:1 to about 6:1, about 1:1 to about 5:1, about 1:1 to about 4:1, about 1:1 to about 3:1, about 1:1 to about 2:1, about 1:1 to about 1:1.

In some embodiments, the nanoparticle composition has one or more of the above characteristics.

The nanoparticles described herein may be in a dry composition (such as a lyophilized composition) or suspended in a biocompatible medium. Suitable biocompatible media include, but are not limited to, water, buffered aqueous media, saline, buffered saline, optionally buffered amino acid solutions, optionally buffered protein solutions, optionally buffered sugar solutions, optionally buffered vitamin solutions, optionally buffered synthetic polymer solutions, lipid-containing emulsions etc.

Human serum albumin (HSA) is a highly soluble globular protein M _r 65K and consists of 585 amino acids. HSA is the most abundant protein in plasma and accounts for 70–80% of the colloidal osmotic pressure of human plasma. The amino acid sequence of HSA contains a total of 17 disulfide bridges, one free thiol (Cys 34) and one tryptophan (Trp 214). Intravenous HSA solution is indicated for the prevention and treatment of hypovolemic shock (see, for example, Tullis, JAMA, 237:355-360, 460-463, (1977)) and Houser et al., Surgery, Gynecology and Obstetrics, 150:811 -816 (1980)) and in combination with exchange transfusion for the treatment of neonatal hyperbilirubinemia (see, for example, Finlayson, Seminars in Thrombosis and Hemostasis, 6, 85-120, (1980)). Other albumins are contemplated, such as bovine serum albumin. The use of such non-human albumins may be suitable, for example, in the context of use of these compositions in non-human mammals, such as veterinary subjects (including pets and farm animals). Human serum albumin (HSA) has numerous hydrophobic binding sites (a total of eight for fatty acids, the endogenous ligands of HSA) and binds a number of different drugs, especially neutral and negatively charged hydrophobic compounds (Goodman et al., The Pharmacological Basis Therapeutics, 9 ^th ed., McGraw-Hill New York (1996)). The presence of two high-affinity binding sites in subdomains IIA and IIIA of HSA has been proposed, which are highly elongated hydrophobic pockets with charged lysine and arginine residues near the surface that function as attachment points for polar ligands (see, for example, Fehske et al. , Biochem. Pharmcol., 30, 687-92 (198a), Vorum, Dan. Med. Bull., 46, 379-99 (1999), Kragh-Hansen, Dan. Med. Bull., 1441, 131-40 ( 1990), Curry et al., Nat. Struct. Biol., 5, 827-35 (1998), Sugio et al., Protein. Eng., 12, 439-46 (1999), He et al., Nature, 358, 209-15 (199b) and Carter et al., Adv. Protein. Chem., 45, 153-203 (1994)). Sirolimus and propofol have been shown to bind to HSA (see, for example, Paal et al., Eur. J. Biochem., 268(7), 2187-91 (200a), Purcell et al., Biochim. Biophys. Acta , 1478(a), 61-8 (2000), Altmayer et al., Arzneimittelforschung, 45, 1053-6 (1995) and Garrido et al., Rev. Esp. Anestestiol. Reanim., 41, 308-12 (1994 )). In addition, docetaxel has been shown to bind to human plasma proteins (see, for example, Urien et al., Invest. New Drugs, 14(b), 147-51 (1996)).

The albumin (eg, human albumin or human serum albumin) in the composition generally serves as a carrier of the mTOR inhibitor, i.e. the albumin in the composition makes the mTOR inhibitor (such as a limus drug, eg sirolimus) more easily suspended in an aqueous environment or stabilizes the suspension compared to compositions not containing albumin. This helps avoid the use of toxic solvents (or surfactants) to solubilize the mTOR inhibitor and thereby may reduce one or more side effects of administering the mTOR inhibitor (such as a limus drug, eg sirolimus) to a subject (such as a human). Thus, in some embodiments, the composition described herein is substantially free of (eg, free of) surfactants such as Cremophor (or polyoxyethylene castor oil, including Cremophor EL® (BASF)). In some embodiments, the nanoparticle composition is substantially free (eg, free) of surfactants. The composition is substantially free of Cremophor or substantially free of surfactant if the amount of Cremophor or surfactant in the composition is insufficient to cause one or more side effects in a subject when the nanoparticle composition is administered to the subject. In some embodiments, the nanoparticle composition contains less than about 20, 15, 10, 7.5, 5.

- 35 045904

2.5 or 1% organic solvent or surfactant. In some embodiments, the albumin is human albumin or human serum albumin. In some embodiments, the albumin is recombinant albumin.

The amount of albumin in the composition described herein will vary depending on the composition of the other components in the composition. In some embodiments, the composition contains albumin in an amount sufficient to stabilize the mTOR inhibitor (such as a lymus drug, such as sirolimus) in an aqueous suspension, for example, in the form of a stable colloidal suspension (such as a stable nanoparticle suspension). In some embodiments, the albumin is present in an amount that reduces the rate of deposition of an mTOR inhibitor (such as a limus drug, such as sirolimus) in an aqueous environment. For particulate compositions, the amount of albumin also depends on the size and density of the mTOR inhibitor nanoparticles.

An mTOR inhibitor (such as a limus drug, e.g., sirolimus) is stabilized in an aqueous suspension if it remains suspended in the aqueous medium (e.g., without visible precipitation or sedimentation) for an extended period of time, such as at least about 0.1 , 0.2, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, 60 or 72 hours. Suspension usually , but not necessarily suitable for administration to a subject (eg, a human). The stability of the suspension is usually (but not necessarily) assessed at storage temperature (such as room temperature (eg, 20-25°C) or refrigerated conditions (eg, 4°C)). For example, a suspension is stable at storage temperature if it does not exhibit flocculation or agglomeration of particles visible to the naked eye or visible under an optical microscope at 1000x magnification within about 15 minutes of preparation of the suspension. Stability can also be assessed under accelerated aging conditions, such as temperatures above 40°C.

In some embodiments, albumin is present in an amount sufficient to stabilize the mTOR inhibitor (such as a lymus drug, such as sirolimus) in an aqueous suspension at a certain concentration. For example, the concentration of an mTOR inhibitor (such as a lymus drug, e.g., sirolimus) in the composition is from about 0.1 to about 100 mg/mL, including, for example, from about 0.1 to about 50 mg/mL, from about 0.1 to about 20 mg/ml, about 1 to about 10 mg/ml, about 2 to about 8 mg/ml, about 4 to about 6 mg/ml, or about 5 mg/ml. In some embodiments, the concentration of the mTOR inhibitor (such as a lymus drug, such as sirolimus) is at least about 1.3, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10. 15, 20, 25, 30, 40 and 50 mg/ml. In some embodiments, the albumin is present in an amount that avoids the use of surfactants (such as Cremophor), such that the composition contains no or substantially no surfactant (such as Cremophor).

In some embodiments, the composition in liquid form contains from about 0.1 to about 50% (wt./vol.) (for example, about 0.5% (wt./vol.), about 5% (wt./vol.) ), about 10% (w/v), about 15% about, about 20% (w/v), about 30% (w/v), about 40% (w/v) or about 50% (w/v) albumin In some embodiments, the composition in liquid form contains from about 0.5 to about 5% (w/v) albumin.

In some embodiments, the weight ratio of albumin to mTOR inhibitor (such as a lymus drug, such as sirolimus) in the nanoparticle composition is such that a sufficient amount of mTOR inhibitor binds to or is transported into the cell. Although the weight ratio of albumin to mTOR inhibitor must be optimized for different albumin to mTOR inhibitor combinations, typically the weight ratio of albumin to mTOR inhibitor (such as a limus drug such as sirolimus) is approximately 0.01: 1 to about 100:1, about 0.02:1 to about 50:1, about 0.05:1 to about 20:1, about 0.1:1 to about 20:1, about 1: 1 to about 18:1, about 2:1 to about 15:1, about 3:1 to about 12:1, about 4:1 to about 10:1, about 5:1 to about 9:1 or approximately 9:1. In some embodiments, the albumin to mTOR inhibitor weight ratio is about 18:1 or lower, 15:1 or lower, 14:1 or lower, 13:1 or lower, 12:1 or lower, 11:1 or lower, 10 :1 or lower, 9:1 or lower, 8:1 or lower, 7:1 or lower, 6:1 or lower, 5:1 or lower, 4:1 or lower, and 3:1 or lower. In some embodiments, the weight ratio of albumin (such as human albumin or human serum albumin) to mTOR inhibitor in the composition is any of the following: about 1:1 to about 18:1, about 1:1 to about 15:1, about 1:1 to about 12:1, about 1:1 to about 10:1, about 1:1 to about 9:1, about 1:1 to about 8:1, about 1:1 to about 7:1, about 1:1 to about 6:1, about 1:1 to about 5:1, about 1:1 to about 4:1, about 1:1 to about 3:1, about 1:1 to about 2:1, about 1:1 to about 1:1.

- 36 045904

In some embodiments, albumin allows the composition to be administered to a subject (eg, a human) without causing significant side effects. In some embodiments, the albumin (such as human serum albumin or human albumin) is in an amount that is effective to reduce one or more side effects of administering an mTOR inhibitor (such as a lymus drug, such as sirolimus) to a human.

The term reducing one or more side effects of administration of an mTOR inhibitor (such as a drug from the group of drugs, e.g. sirolimus) refers to the reduction, amelioration, elimination or prevention of one or more undesirable effects caused by an mTOR inhibitor, as well as side effects caused by delivery vehicles (such as the solvents that make limus drugs injectable) used to deliver the mTOR inhibitor. Such side effects include, for example, myelosuppression, neurotoxicity, hypersensitivity, inflammation, venous dermatitis, phlebitis, pain, skin irritation, peripheral neuropathy, neutropenic fever, anaphylactic reaction, venous thrombosis, extravasation, and combinations thereof. However, these side effects are purely illustrative, and other side effects or combinations of side effects associated with limus drugs such as sirolimus may be reduced.

In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, such as sirolimus) and albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm. In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, such as sirolimus) and albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm. In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, such as sirolimus) and albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm (for example, approximately 100 nm). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising sirolimus and human albumin (such as human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm (eg, about 100 nm).

In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, such as sirolimus) and albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm, wherein the weight ratio of albumin to mTOR inhibitor in the composition is no more than about 9:1 (for example, about 9:1 or about 8:1). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, such as sirolimus) and albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm, wherein the weight ratio of albumin to mTOR inhibitor in the composition is no more than about 9:1 (for example, about 9:1 or about 8:1). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, such as sirolimus) and albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of about 150 nm, wherein the weight ratio of albumin to mTOR inhibitor in the composition is no more than about 9:1 (for example, about 9:1 or about 8:1). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising sirolimus and human albumin (such as human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., about 100 nm), wherein the weight ratio of albumin to sirolimus in the composition is about 9:1 or about 8:1.

In some embodiments, the nanoparticle compositions described herein comprise nanoparticles containing an mTOR inhibitor (such as a lymus drug, such as sirolimus) bound (eg, coated) with albumin (such as human albumin or human serum albumin). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, e.g., sirolimus) bound (e.g., coated) to albumin (such as human albumin or human serum albumin), wherein nanoparticles have an average diameter of no more than about 150 nm. In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, e.g., sirolimus) bound (e.g., coated) with albumin (such as human albumin or human serum albumin), wherein the nanoparticles

- 37 045904 have an average diameter of no more than about 150 nm. In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, e.g., sirolimus) bound (e.g., coated) with albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm (eg, about 100 nm). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising sirolimus bound (e.g., coated) with human albumin (such as human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., about 100 nm ).

In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, e.g., sirolimus) bound (e.g., coated) to albumin (such as human albumin or human serum albumin), wherein the mass the ratio of albumin to mTOR inhibitor in the composition is no more than about 9:1 (for example, about 9:1 or about 8:1). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, e.g., sirolimus) bound (e.g., coated) with albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm, wherein the weight ratio of albumin to mTOR inhibitor in the composition is no more than about 9:1 (eg, about 9:1 or about 8:1). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, e.g., sirolimus) bound (e.g., coated) with albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm, wherein the weight ratio of albumin to mTOR inhibitor in the composition is no more than about 9:1 (eg, about 9:1 or about 8:1). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, e.g., sirolimus) bound (e.g., coated) with albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of about 150 nm, wherein the weight ratio of albumin to mTOR inhibitor in the composition is no more than about 9:1 (eg, about 9:1 or about 8:1). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising sirolimus bound (e.g., coated) with human albumin (such as human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., about 100 nm ), wherein the weight ratio of albumin to sirolimus in the composition is about 9:1 or about 8:1.

In some embodiments, the nanoparticle compositions described herein comprise nanoparticles containing an mTOR inhibitor (such as a lymus drug, such as sirolimus) stabilized with albumin (such as human albumin or human serum albumin). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, such as sirolimus) stabilized with albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm. In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, such as sirolimus) stabilized with albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of no more than approximately 150 nm. In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, such as sirolimus) stabilized with albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm (for example, about 100 nm). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising sirolimus stabilized with human albumin (such as human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., about 100 nm).

In some embodiments, the nanoparticle compositions described herein comprise nanoparticles containing an mTOR inhibitor (such as a limus drug, such as sirolimus) stabilized with albumin (such as human albumin or human serum albumin), wherein the weight ratio of albumin to mTOR inhibitor is the composition is no more than about 9:1 (for example, about 9:1 or about 8:1). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles containing an mTOR inhibitor (such as a limus drug, such as sirolimus), stabilized

- 38 045904 coated with albumin (such as human albumin or human serum albumin), where the nanoparticles have an average diameter of no more than about 150 nm, where the weight ratio of albumin to mTOR inhibitor in the composition is no more than about 9:1 (for example, about 9 :1 or approximately 8:1). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising an mTOR inhibitor (such as a lymus drug, such as sirolimus) stabilized with albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm, wherein the weight ratio of albumin to mTOR inhibitor in the composition is no more than about 9:1 (for example, about 9:1 or about 8:1). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles containing an mTOR inhibitor (such as a lymus drug, e.g., sirolimus) stabilized with albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of about 150 nm wherein the weight ratio of albumin to mTOR inhibitor in the composition is no more than about 9:1 (for example, about 9:1 or about 8:1). In some embodiments, the nanoparticle compositions described herein comprise nanoparticles comprising sirolimus stabilized with albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of no more than about 150 nm (e.g., about 100 nm), wherein the wt. the ratio of albumin to sirolimus in the composition is about 9:1 or about 8:1.

In some embodiments, the nanoparticle composition contains Nab-sirolimus. In some embodiments, the nanoparticle composition is Nab-sirolimus. Nab-sirolimus is a sirolimus formulation stabilized with human albumin USP that can be dispersed directly into injectable saline. The mass ratio of human albumin and sirolimus ranges from 8:1 to 9:1. When dispersed in a suitable aqueous medium, such as 0.9% sodium chloride injection or 5% dextrose injection, Nab-sirolimus forms a stable colloidal suspension of sirolimus. Nanoparticles have an average particle size in a colloidal suspension of approximately 100 nm. Because HSA is highly soluble in water, Nab-sirolimus can be reconstituted over a wide range of concentrations from dilute (0.1 mg sirolimus/mL) to concentrated (20 mg sirolimus/mL), including, for example, from about 2 mg/mL to about 8 mg/ml or about 5 mg/ml.

Methods for preparing nanoparticle compositions are known in the art. For example, nanoparticles containing an mTOR inhibitor (such as a lymus drug such as sirolimus) and albumin (such as human serum albumin or human albumin) can be prepared under high shear conditions (eg, sonication, high pressure homogenization, or etc.). These methods are disclosed, for example, in US patent No. 5916596; 6506405; 6749868, 6537579 and 7820788, as well as in US patent applications No. 2007/0082838, 2006/0263434, and PCT patent application WO 08/137148.

Briefly, an mTOR inhibitor (such as a limus drug such as sirolimus) is dissolved in an organic solvent and the solution is added to the albumin solution. The mixture is homogenized under high pressure. The organic solvent can then be removed by evaporation. The resulting dispersion can be further lyophilized. Suitable organic solvent includes, for example, ketones, esters, ethers, chlorinated solvents and other solvents known in the art. For example, the organic solvent may be methylene chloride or a chloroform/ethanol mixture (eg, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1 :1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1).

mTOR inhibitor

The methods described herein in some embodiments involve administering nanoparticle compositions of mTOR inhibitors. As used herein, mTOR inhibitor refers to an mTOR inhibitor. mTOR is a serine/threonine-specific protein kinase downstream of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) pathway and is a key regulator of cell survival, proliferation, stress and metabolism. Dysregulation of the mTOR pathway has been found in many human carcinomas, and inhibition of mTOR had a significant suppressive effect on tumor progression. In some embodiments, the mTOR inhibitor is an mTOR kinase inhibitor.

Mammalian target of rapamycin (mTOR) (also known as mechanistic target of rapamycin or FK-506-binding protein 12-rapamycin-associated protein 1 (FRAP1)) is an atypical serine/threonine protein kinase that resides in two distinct complexes, mTOR complex 1 ( mTORC1) and mTOR complex 2 (mTORC2). mTORC1 is composed of mTOR, mTOR regulatory associated protein (Raptor), mammalian lethal protein SEC13 8 (MLST8), PRAS40, and DEPTOR (Kim et al., 2002). Cell, 110:163-75, Fang et al. (2001). Science, 294 (5548): 1942-5). mTORC1 integrates four main input signals: nutrients (such as amino acids and phosphatidic acid), growth factors (insulin), energy and stress (such as hypoxia and

- 39 045904 DNA damage). Amino acid availability is signaled by mTORCl through a pathway involving growth factors Rag and Ragulator (LAMTOR1-3) and hormones (eg, insulin) to mTORC1 via Akt, which inactivates TSC2 to prevent mTORC1 inhibition. Alternatively, low ATP levels lead to AMPK-dependent activation of TSC2 and phosphorylation of Raptor with a decrease in mTORC1 signaling proteins.

Active mTORC1 has a number of downstream biological effects, including mRNA translation through phosphorylation of downstream targets (4E-BP1 and p70 S6 kinase), suppression of autophagy (Atg13, ULK1), ribosome biogenesis, and transcriptional activation leading to mitochondrial metabolism or adipogenesis. Accordingly, mTORC1 activity promotes either cellular growth when conditions are favorable or catabolic processes during stress or when conditions are unfavorable.

mTORC2 is composed of mTOR, mTOR's rapamycin-insensitive partner (RICTOR), GeL, and stress-activated protein kinase interacting protein 1 (mSIN1). In contrast to mTORC1, for which many upstream signals and cellular functions have been determined (see above), relatively little is known about the biology of mTORC2. mTORC2 regulates cytoskeletal organization through stimulation of F-actin stress fibers, paxillin, RhoA, Rac1, Cdc42, and protein kinase Ca (PKCa). It has been noted that knockdown of mTORC2 components affects actin polymerization and disrupts cell morphology (Jacinto et al., 2004). Nat. Cell Biol., 6, 1122-1128, Sarbassov et al. (2004). Curr. Biol., 14, 1296-1302). This suggests that mTORC2 controls the actin cytoskeleton by stimulating protein kinase Ca (PKCa) phosphorylation, paxillin phosphorylation and its relocalization to focal adhesions, and GTP loading of RhoA and Rac1. The molecular mechanism by which mTORC2 regulates these processes has not been determined.

In some embodiments, the mTOR inhibitor is an mTORC1 inhibitor. In some embodiments, the mTOR inhibitor is an mTORC2 inhibitor.

In some embodiments, the mTOR inhibitor is a limus drug, which includes sirolimus and analogs thereof. Examples of limus drugs include, but are not limited to, temsirolimus (CCI-779), everolimus (RAD001), ridaforolimus (AP-23573), deforolimus (MK-8669), zotarolimus (ABT-578), pimecrolimus and tacrolimus (FK506) . In some embodiments, the limus drug is selected from the group consisting of temsirolimus (CCI-779), everolimus (RAD001), ridaforolimus (AP-23573), deforolimus (MK-8669), zotarolimus (ABT-578), pimecrolimus, and tacrolimus (FK-506).

In some embodiments, the mTOR inhibitor is sirolimus. Sirolimus is a macrolide antibiotic that forms a complex with FKBP-12 and inhibits the mTOR pathway by binding to mTORC1.

In some embodiments, the mTOR inhibitor is selected from the group consisting of sirolimus (rapamycin), BEZ235 (NVP-BEZ235), everolimus (also known as RAD001, Zortress, Certican and Afinitor), AZD8055, temsirolimus (also known as CCI-779 and Torisel ), PI-103, Ku-0063794, INK 128, AZD2014, NVP-BGT226, PF-04691502, CH5132799, GDC-0980 (RG7422), Torina 1, WAY-600, WYE-125132, WYE -687, GSK2126458, PF -05212384 (PKI-587), PP-121, OSI-027, Palomida 529, PP242, XL765, GSK1059615, WYE-354 and eforolimus (also known as ridaforolimus or deforolimus).

BEZ235 (NVP-BEZ235) is an imidazoquilonine derivative that is a catalytic inhibitor of mTORC1 (Roper J. et al., PLoS One, 2011, 6 (9), e25132). Everolimus is a 40-O-(2hydroxyethyl) derivative of rapamycin and binds cyclophilin FKBP-12 and also forms a complex with mTORC1. AZD8055 is a small molecule that inhibits the phosphorylation of mTORC1 (p70S6K and 4E-BP1). Temsirolimus is a small molecule that forms a complex with FK506-binding protein and abolishes mTOR activation when it is in the mTORC1 complex. PI-103 is a small molecule that inhibits the activation of the rapamycin-sensitive complex (mTORC1) (Knight et al., 2006), 125:733-47). KU-0063794 is a small molecule that inhibits mTORC1 phosphorylation at Ser2448 in a dose- and time-dependent manner. Each of INK 128, AZD2014, NVP-BGT226, CH5132799, WYE-687 is a small molecule inhibitor of mTORC1. PF-04691502 inhibits mTORC1 activity. GDC-0980 is an orally bioavailable small molecule that inhibits class I kinase PI3 and TORC1. Torin 1 is an effective small molecule inhibitor of mTOR. WAY-600 is an effective, ATP-competitive and selective mTOR inhibitor. WYE-125132 is an ATP-competitive small molecule inhibitor of mTORC1. GSK2126458 is an mTORC1 inhibitor. PKI-587 is a highly potent dual inhibitor of PI3Ka, mTOR asY and mTOR. PP-121 is a multi-target inhibitor of PDGFR, Hck, mTOR, VEGFR2, Src and Abl. OSI-027 is a selective and potent dual inhibitor of mTORC1 and mTORC2 with IC ₅₀ values of 22 and 65 nM, respectively. Palomide 529 is a small molecule mTORC1 inhibitor that has no affinity for ABCB1/ABCG2 and has good penetration into

- 40 045904 brain (Lin et al. (2013)? Int. J. Cancer, DOI: 10.1002/ijc.28126 (e-published journal ahead of print). PP242 is a selective mTOR inhibitor. XL765 is a dual mTOR/PI3k inhibitor for mTOR, p110a, p110b, ρΙΙΟυ and p110δ. GSK1059615 is a novel and dual inhibitor of PI3Ka, PI3Ke, PI3K<5„ PI3Ky and mTOR. WYE-354 inhibits mTORC1 in HEK293 cells (0.2-5 µM) and in HUVEC cells (10 nM-1 µM) WYE-354 is an effective, specific and ATP-competitive mTOR inhibitor.Deforolimus (ridaforolimus, AP23573, MK-8669) is a selective mTOR inhibitor.

In some embodiments, the mTOR kinase inhibitor is selected from the group consisting of CC-115 and CC-223.

Other components in nanoparticle compositions

The nanoparticles described herein may be present in a composition that includes other agents, excipients or stabilizers. For example, some negatively charged components can be added to improve stability by increasing the negative zeta potential of the nanoparticles. Such negatively charged components include, but are not limited to, bile salts and bile acids consisting of glycocholic acid, cholic acid, chenodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, dehydrocholic acid, and others; phospholipids, including lecithin (egg yolk)-based phospholipids, which include the following phosphatidylcholines: palmitoyloleoylphosphatidylcholine, palmitoyllinoleoylphosphatidylcholine, stearoyllinoleoylphosphatidylcholine, stearoyloleoylphosphatidylcholine, stearoylarachidoylphosphatidylcholine and dipalmitoylphosphatidylcholine. Other phospholipids include L-dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), hydrogenated salt phosphatidylcholine (HSPC) and other related compounds. Negatively charged surfactants or emulsifiers are also useful as additives, for example sodium cholesteryl sulfate and the like.

In some embodiments, the composition is suitable for administration to a human. In some embodiments, the composition is suitable for administration to a mammal, such as veterinary animals, pets, and farm animals. There is a wide range of suitable formulations based on nanoparticle compositions (see, for example, US patent No. 5916596 and 6096331). The following preparations and methods are purely illustrative and not limiting in any way. Formulations suitable for oral administration include (a) liquid solutions, such as an effective amount of the compound dissolved in diluents such as water, saline or orange juice, (b) capsules, sachets or tablets, each containing a predetermined amount the active ingredient in the form of solids or granules, (c) suspensions in a suitable liquid and (d) suitable emulsions. Tablets may include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, gum acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid and other excipients, coloring agents, diluents, buffering agents, humectants, preservatives, flavoring agents and pharmacologically compatible excipients. Lozenges may comprise the active ingredient in a flavoring agent, typically sucrose and gum acacia or tragacanth, or may be in the form of lozenges containing the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like. ., containing, in addition to the active ingredient, excipients that are known in the art.

Examples of suitable carriers, excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methyl and propyl hydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations may further include lubricants, wetting agents, emulsifying and suspending agents, preservatives, sweetening agents or flavoring agents.

Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injectable solutions, which may contain antioxidants, buffers, bacteriostats and solutes that make the composition compatible with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may include suspending agents. agents, solubilizers, thickeners, stabilizers and preservatives. The formulations may be presented in single-dose or multi-dose containers, such as ampoules and vials, and may be stored in a lyophilized (freeze-dried) state, requiring only the addition of a sterile liquid excipient, such as water for injection, immediately before use. Prepared extemporaneous injection solutions and suspensions can be prepared from the sterile powders, granules and tablets described previously. Injectable preparations are preferred.

- 41 045904

In some embodiments, the composition is formulated to have a pH in the range of about 4.5 to about 9.0, including, for example, a pH in the range of about 5.0 to about 8.0, about 6.5 to about 7.5 and from about 6.5 to about 7.0. In some embodiments, the pH of the composition is no less than about 6, including, for example, no less than about 6.5, 7, or 8 (eg, about 8). The composition can also be made isotonic with blood by adding a suitable tonicity modifier such as glycerol.

Kits, drugs, compositions and single dosage forms.

The invention also provides kits, formulations, compositions and unit dosage forms for use in any of the methods described herein.

The kits of the invention include one or more containers containing nanoparticle compositions containing a limus drug (or unit dosage forms and/or articles) and also contain instructions for use in accordance with any of the methods described herein. The kit may further comprise a description of selecting a subject suitable for treatment. The instructions included in the kits of the invention are typically written instructions on a label or on a package insert (for example, on a piece of paper included in the kit), but machine-readable instructions (for example, instructions that are stored on a magnetic or optical disk) are also acceptable. .

For example, in some embodiments, the kit comprises a) a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin (such as human serum albumin); and b) instructions for administration of the nanoparticle composition for the treatment of PEComa. In some embodiments, the kit includes a) a composition comprising nanoparticles containing an mTOR inhibitor (such as a lymus drug) and albumin (such as human serum albumin); and b) instructions for administration (eg, subcutaneous or intravenous administration) of the nanoparticle composition and other agents for the treatment of PEComa. Nanoparticles and other agents may be in separate containers or in the same container. For example, the kit may contain one single composition or two or more compositions, where one composition contains nanoparticles and one composition contains another agent.

The kits according to the invention are provided in suitable packaging. Suitable packaging includes, but is not limited to, vials, carboys, flasks, flexible packaging (eg, Mylar seal bags or plastic bags), and the like. Sets may optionally provide additional components such as buffers and interpretive information. Thus, the present application also provides products that include vials (eg, sealed vials), bottles, bottles, flexible packaging, and the like.

Instructions regarding the use of nanoparticles typically include information about the dose, dosage regimen, and route of administration for the intended treatment. Containers may be single dose containers, bulk packs (eg, multi-dose bags), or subunit dose containers. For example, kits may be provided that contain sufficient doses of an mTOR inhibitor (e.g., such as a limus drug such as sirolimus) described herein to provide effective treatment to a subject over an extended period of time, such as a week, 8, 9 , 10, 11, 12, 13 days, 2, 3, 4, 6, 8 weeks, 3, 4, 5, 7, 8, 9 months or more. The kits may also include multiple unit doses of an mTOR inhibitor (eg, such as a limus drug) and pharmaceutical compositions and instructions for use, and are packaged in quantities sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies. prescription products.

Also provided are drugs, compositions and unit dosage forms suitable for the methods described herein. In some embodiments, a drug (or composition) for use in the treatment of PEComa is provided, comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug) and albumin (such as human serum albumin).

Those skilled in the art will understand that other embodiments are possible without departing from the scope and spirit of the present invention. The invention will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the invention, but are of course not to be construed as limiting the invention in any way.

Examples

Example 1.

Multicenter phase II study in patients treated with ABI-009.

Patients with advanced malignant PEComa who had not previously received mTOR inhibitor treatment were enrolled in a phase II, single-arm, open-label, multicenter study to evaluate the efficacy and safety profile of intravenous ABI-009 (also referred to herein as Nab-sirolimus or Nab-rapamycin). If the inappropriateness criteria were not met, then stage 2 should be open for inclusion.

- 42 045904

At least 30 patients will be included in the study. The histology of malignant PEComa can be examined locally at each institution for inclusion, but must be retrospectively confirmed by central review for each patient after enrollment to meet the specified criteria for malignant PEComa as specified in the study inclusion criteria. If the patient does not retrospectively meet these criteria, then replacement should be considered.

A patient will be eligible for inclusion in this study only if all of the following criteria are met: (i) patients must have a histologically confirmed diagnosis of advanced malignant PEComa; (ii) patients should have an available block of tumor specimen along with an appropriate pathology report (or approximately 30 unstained slides with a minimum of 16 slides required) and/or results of a recent biopsy to provide retrospective central confirmation of the presence of malignant PEComa and for mTOR pathway analysis and analysis biomarkers; (iii) patients must have one or more measurable target lesions on CT or MRI or evaluable disease according to RECIST v1.1; (iv) patients should not have previously been treated with an mTOR inhibitor; (v) prior treatment (diagnostic or otherwise), chemotherapy, radiation therapy, surgery or other therapeutic agents (except mTOR inhibitors) is acceptable if completed within a period greater than five times the half-life or >28 days before enrollment in the study, whichever is less; (vi) the study included patients aged 18 years or older with Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1; (vii) patients must have the following blood chemistry levels at screening (obtained <14 days prior to study entry (at a local laboratory)): (a) total bilirubin <1.5x upper limit of normal (ULN) mg/dL, (b ) ACT <2.5x ULN (<5x ULN if due to liver metastases), (c) serum creatinine <1.5x ULN; (viii) adequate biological parameters, as evidenced by the following blood parameters at screening (obtained <14 days before study entry, in a local laboratory): (a) absolute neutrophil count (ANC) >1.5 x 10 ⁹ /L; (b) platelet count >100,000/mm ³ (100x10 ⁹ /l); (c) hemoglobin >9 g/dL; (ix) urine protein <2 g proteinuria/24 h; (x) serum triglycerides <300 mg/dL; serum cholesterol <350 mg/dL, (xi) men or non-pregnant and non-breastfeeding women (women of childbearing potential must agree to use effective contraception without interruption, 28 days before starting IP, and also during the drug trial drug and have a negative serum pregnancy test (β-hCG) at screening and consent to pregnancy testing during the study and after completion of treatment, and male patients must practice abstinence or agree to use a condom during sexual intercourse with a pregnant woman or woman childbearing age while participating in the study, even if he or she has undergone a successful vasectomy); (xii) likely life expectancy greater than 3 months, as determined by the investigator; (xiii) ability to understand and sign informed consent; (xiv) Willingness and ability to perform scheduled visits, laboratory tests, and other procedures during the study.

A patient will not be eligible to participate in this study if one of the following criteria applies: (i) patients with lymphangioleiomyomatosis (LAM) are excluded; (ii) presence of known active uncontrolled or symptomatic central nervous system (CNS) metastases (patients with controlled and asymptomatic CNS metastases are eligible to participate in this study). However, the patient must have completed any prior treatment for CNS metastases >28 days (including radiation therapy and/or surgery) before starting treatment in this study and must not have received long-term corticosteroid therapy for CNS metastases); (iii) presence of active gastrointestinal bleeding if transfusion dependent; (iv) pre-existing thyroid disease is permitted provided that thyroid function can be controlled with medications; (v) presence of uncontrolled severe physical or mental illness. Patients with a current active second malignancy other than nonmelanoma skin cancer, cervical carcinoma in situ, resected incidental prostate cancer (stage pT2 with Gleason score <6 and postoperative PSA <0.5 ng/mL), or other appropriate treated carcinoma in situ are not included in the study (patients are not considered to have a current active second malignancy if they have completed treatment and have not had disease for >1 year); (vi) treatment of liver disease up to 2 months prior to study entry (including treatment with radiotherapy (including radiolabeled beads and/or cyberknife, hepatic artery embolization (with or without chemotherapy) or cryotherapy/ablation) is acceptable if these treatments are not influence the areas of disease being assessed used for this protocol); (vii) recent infection requiring systemic treatment that was completed 14 days before study entry (excluding uncomplicated urinary tract infection or upper respiratory tract infection); (viii) uncontrolled diabetes mellitus with HbA1c >8%, despite adequate therapy,

- 43 045904 unstable angina or myocardial infarction during the previous 6 months; (ix) receiving any concomitant anticancer therapy; (x) patients with a history of interstitial lung disease and/or pneumonitis or pulmonary hypertension; (xi) the use of certain drugs and prohibited drugs is not permitted for a period of more than five times the half-life or 28 days, whichever is less, before the first dose of the study drug and throughout the study; (xii) use of strong CYP3A4 inhibitors and inducers 14 days before the first dose of ABI-009. In addition, use of any known CYP3A4 substrates with a narrow therapeutic window (eg, fentanyl, alfentanil, astemizole, cisapride, dihydroergotamine, pimozide, quinidine, terfanide) within 14 days before the first dose of ABI-009.

The study is expected to take approximately 32 months from first patient enrollment to patient follow-up, including approximately 24 months of study enrollment period, approximately 6 months of treatment (or until treatment is no longer tolerated), and an end-of-treatment visit through 4 weeks (±7 days) after the last treatment.

The following assessments will be performed on Day 1 of each cycle unless otherwise noted: (i) physical examination; (ii) determination of body weight; (iii) BSA calculation; (iv) assessment of concomitant medications and procedures; (v) vital signs (such as temperature, systolic and diastolic blood pressure and pulse); (vi) ECOG performance status; (vii) ECG; (viii) a clinical chemistry panel (including, but not limited to, sodium, potassium, chloride, glucose, alkaline phosphatase (ALP), AST/SGOT, ALT/SGPT, serum albumin); (ix) CBC, differential and platelet counts; (x) thyroid function; (xi) screening for hepatitis and HIV infection (every odd-numbered cycle, starting with C3); (xii) fasting lipids (every even-numbered cycle starting from C2); (xiii) assessment of adverse events; (xiv) determination of pharmacokinetic parameters (only on day 1 of cycle 1).

The following assessments will be performed on day 8 of each cycle unless otherwise noted: (i) assessment of concomitant medications and procedures; (ii) vital signs; (iii) CBC, differential and platelet counts; (iv) thyroid function; (v) assessment of adverse events; (vi) determination of pharmacokinetic parameters (only on day 8 of cycle 1).

Tumor response to treatment will be assessed using CT or MRI of the chest, abdomen and pelvis; acquisition and evaluation of images will conform to the specifications specified in RECIST 1.1. The same methods (CT or MRI) should be used at screening and throughout the study.

End of study (EOS) is defined by the date of the last patient visit to complete the study or the date of the last data point from the last patient required for primary, secondary, and/or exploratory analyses, as prespecified in the protocol.

The end of treatment (EOT) for a patient is determined by the date of the last dose of ABI-009. The patient's end-of-treatment visit occurs when safety assessments and procedures are completed after the last treatment, which should occur at least 4 weeks (±7 days) after the last dose of ABI-009.

The follow-up period represents the period of time in the study after the EOT visit. All patients who discontinued participation in the drug study and did not withdraw full consent to participate in the study will continue to participate in the follow-up phase for survival and initiation of anticancer therapy. Follow-up continues approximately every 12 weeks (±3 weeks) until death, withdrawal of consent, or study closure, whichever occurs first. This assessment can be made by examination of the recording and/or telephone contact.

The rapamycin PK study will be conducted with limited PK sampling for all patients in the phase 2 study. Blood samples will be obtained only on day 1 of cycle 1 (C1D1) and will be collected immediately before drug administration (before infusion), after 0.5 hours (immediately after the end of infusion), after 1, 2, 4 and 168 hours (immediately before dosing at C1D8). Note: T=0 is defined as the start of the infusion, i.e. all sample collection time points refer to the start of the infusion. The end-of-infusion sample (0.5 h) is collected immediately before the end of the infusion. If the duration of the infusion is changed, the sample should be collected immediately before the end of the infusion. Whole blood samples will be collected in EDTA tubes to determine rapamycin concentrations in a central laboratory.

Patients will receive ABI-009 at a dose of 100 mg/m ² 2 times every 3 weeks by intravenous infusion over 30 minutes. Two dose reduction levels are allowed: 75 and 56 mg/ ^m2 . Patients will continue treatment until disease progression, unacceptable toxicity, until, in the opinion of the investigator, the patient no longer benefits from therapy, or at the discretion of the patients.

Patients will be assessed for complete response (CR), partial response (PR), stable

- 44 045904 disease (SD) or progressive disease (PD) using CT. Contrast-enhanced MRI may also be used if the study uses this technique. Pre-treatment scan results may be accepted from external institutions but must be completed within 4 weeks of initiation of therapy and should include (as clinically indicated) CT or MRI of the chest, abdomen and pelvis. The first response assessment with CT or MRI documenting the target lesions will be performed 6 weeks after the first treatment and should be repeated every 6 weeks for the first year, then every 12 weeks until disease progression. If initial observation of an objective response (CR or PR) is made, then a confirmatory scan should be performed 6 weeks after the initial observation.

The primary endpoint, ORR, will be determined by independent radiologist(s). The independent radiology review will follow separate imaging protocols.

After disease progression, patients will be followed for survival every 12 weeks, or more frequently as appropriate, until death, withdrawal of consent, or study closure, whichever occurs first.

Safety and tolerability will be monitored through ongoing reporting of treatment and treatment-emergent adverse events (AEs), AEs of special interest, laboratory abnormalities, and the proportion of patients with dose changes, dose delays/dose withdrawals, dose interruptions, and/or premature cessation of IP administration due to AE. All AEs are recorded by the investigator from the time the patient signs the informed consent until 28 days after the last IP dose. Adverse events will be assessed according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v4.03.

Physical status, vital signs, laboratory tests (eg, serum chemistry, hematology), and ECOG performance status will be monitored. All SAEs (regardless of IP relationship) will be observed prior to analysis. Laboratory tests will be carried out in accordance with the study schedule.

The primary analysis will address all study objectives and will occur when all patients have been treated for at least 6 months. All primary, secondary and exploratory efficacy and safety analyzes will be conducted at the time of the primary analysis, with the exception of biomarkers, which may be analyzed at a later date.

The primary endpoint is ORR assessed by independent radiology review and defined as the proportion of patients who achieve a confirmed PR or CR according to RECIST 1.1.

This study is designed to test whether the confirmed ORR in patients treated with ABI-009 is greater than 5%, as determined using an exact binomial one-sided test with a type 1 error of 0.025. With approximately 30 patients, the study will provide greater than 95% power to reject the null hypothesis of an ORR of <5% given a true response rate of 30%. In addition to the hypothesis test, the number and percentage of patients achieving ORR will be summed and an exact 95% confidence interval will be provided. Assuming that the observed ORR is 30%, the lower bound of the 95% confidence interval (CI) for the estimated ORR would exclude values below 14.7%.

Analysis of the secondary efficacy endpoints of PFS, PFS rate at 6 months, DOR, and OS will be performed separately for two subgroups of patients: 1) patients with metastatic disease and 2) patients with unresectable locally advanced disease or resectable disease requiring multiple resections. Some of the patients in the subgroup with locally advanced disease may be given a clinical recommendation for surgery if there is sufficient reduction in tumor size to result in an outlier.

For patients with metastatic disease, 6-month PFS, median PFS, DOR, and OS will be summarized using Kaplan-Meier (KM) analysis. Quartiles with 95% CIs will be summed. The number of patients with unresectable locally advanced disease or resectable disease requiring multiple resections is expected to be small; therefore, PFS (median and at 6 months), DOR, and OS for these patients will be summarized by descriptive statistics.

The main goal of molecular profiling of tumors is to identify specific markers that are predictive of response to treatment with AB1-009.

Blood samples will be collected to obtain a collection of cell-free plasma DNA (pre- and post-treatment samples are required): molecular analysis of cell-free plasma DNA using next generation sequencing to assess the prevalence of mutations identified in the primary tumor sample over time as an indicator of response to therapy.

Analysis of mutations or epigenetic changes in the biopsy specimen will also be performed.

- 45 045904 Holi. Archival or recent biopsy samples of tumor tissue are required from patients participating in this study (mandatory).

In addition, biopsies will be performed during treatment to evaluate pharmacodynamic effects, if any. After the last treatment and during progression, if a biopsy is performed, a sample of the tumor will be taken. Tumor biopsies are collected and pharmacodynamic changes analyzed to determine the effect of the drug on the target(s) in the tumor, as well as to potentially analyze the molecular mechanisms associated with acquired resistance. To evaluate mutations in all known mTOR pathway genes, including but not limited to PIK3CA, TSC1, TSC2, AKT, PTEN, MTOR, exome sequencing will be performed using the ONCOPANEL™ assay (BHH Pathology Department, CLIA certified) of approximately 300 genes, and RHEB. Correlates of clinical response to therapy will be assessed, as well as correlation testing between biopsy and circulating DNA analysis will be performed. Analysis of translocations in TFE3 using FISH (fluorescence in situ hybridization) will be performed. These studies will be conducted at the Center for Molecular Diagnostics Laboratory at Brigham and Women's Hospital, Boston, Massachusetts, which is also CLIA certified.

Immunohistochemical studies will be performed for relevant pathway markers including, but not limited to, phosphoproteins p-AKT, p-S6, p-S6K, p-4EBP1, and p-SPARC; proliferation markers such as Ki-67; and markers of apoptosis such as PARP or fragments thereof. Post-treatment (progression) samples will be analyzed using similar exome sequencing as above to investigate causes of resistance, including secondary mutations and genomic amplification or deletion events.

Example 2.

Clinical pilot study of Nab-sirolimus in malignant tumors with aberrant mTOR pathway.

A single-arm, phase II clinical trial is designed to evaluate the efficacy of Nab-sirolimus (also referred to as ABI-009) in a subject with PEComa and mTOR pathway gene mutation status, particularly subjects with mutation status of a gene that confers sensitivity to mTOR inhibitors (such as as a composition containing nanoparticles containing a drug from the limus group and albumin). The mutational status of genes is identified in subjects in a clinical trial using next generation sequencing. The primary objective of the study is to evaluate the response rate to Nab-sirolimus in subjects with PEComa and mTOR-activating gene mutation status. Secondary objectives are (1) to establish time to progression and overall survival of selected subjects and (2) to evaluate the adverse event profile associated with Nab-sirolimus in selected subjects. In addition, a correlation study is being conducted to evaluate the frequency of a subject's mTOR-activating gene mutational status and to evaluate the association between a subject's mTOR-activating gene mutational status and clinical outcome in subjects with PEComa.

The clinical trial includes one group of subjects. Prior to enrollment, subjects are assessed in a CLIA-certified laboratory for mTOR-activating gene mutations in at least one of the mTOR-associated genes selected from the group consisting of, for example, AKT1, MTOR, PIK3CA, PIK3CG, TSC1, TSC2, RHEB , STK11, NF1, NF2, PTEN, TP53, FGFR4 and VAP1. Subjects who have at least one mTOR-activating gene mutation and meet all inclusion criteria are selected for treatment. An archival paraffin-embedded (PPFE) tumor tissue specimen may not necessarily be obtained from each subject. Selected subjects are administered Nab-sirolimus intravenously at a dose of approximately 75 mg/m ² on days 1, 8 and 15 of a 28-day cycle or approximately 100 mg/m ² on days 1 and 8 of a 21-day cycle. The infusion of Nab-sirolimus is carried out over approximately 30 minutes with each injection. Subjects continue to receive treatment with Nab-sirolimus and are actively monitored until disease progression and/or unacceptable adverse events occur, or the subject refuses treatment. If multiple adverse events are observed, Nabsirolimus administration may be interrupted or the dose reduced to ensure drug-related toxicity is controlled. For example, the dose of Nab-sirolimus can be first reduced to 56 mg/ ^m2 IV on days 1, 8 and 15 of a 28-day cycle, and then reduced a second time to 45 mg/ ^m2 IV on days 1, 8 and day 15 of a 28-day cycle. Only two dose reductions are allowed for each subject. Adjuvant medications such as antiemetics, growth factors (G-CSFs), bisphosphonates or denosumab for preexisting, painful bone metastases, blood and blood products, warfarin or LMWH, and/or loperamide for diarrhea may be authorized at the physician's discretion. Subjects must visit the authorizing institution for treatment and evaluation at least once every 28 days (or approximately 25 to 31 days) during treatment. Biomarkers are determined (sequences and levels of AKT1, MTOR, PIK3CA, TSC1, TSC2, RHEB, STK11, NF1, NF2 and PTEN and the level of phosphorylated 4EBP1 and S6) for each subject on day 1 of cycle 1, day 1 (±3 days) of cycle 2 and day 1 (±3 days) of cycle 3 and then every 2 cycles. A blood sample is collected from each subject for analysis.

- 46 045904 for circulating (eg cell-free) DNA before and after the full course of treatment.

Various biological samples are collected from each subject during the course of the study (eg, before treatment, during treatment, and after treatment), and the biological samples are used to assess mutational status and levels of relevant biomarkers. Biological samples during treatment may be collected from the subject, for example, on day 1 of cycle 1, day 1 (±3 days) of cycle 2 and day 1 (±3 days) of cycle 3 and every 2 cycles thereafter. A blood sample is collected from each subject before and after treatment. A sample of cell-free plasma DNA is obtained from each blood sample to assess circulating DNA. Cell-free plasma DNA samples are analyzed using next-generation sequencing techniques to assess the prevalence of mTOR-activating gene mutations identified in the primary tumor sample over time as an indicator of response to treatment. In addition, fresh or archival (eg, PPFE) tumor biopsies are collected from each subject prior to treatment and, optionally, during the course of treatment (ie, during treatment). During treatment, tumor biopsies are used to evaluate the pharmacodynamic effects of Nab-sirolimus in subjects. Post-treatment tumor biopsies are collected from each subject at the time of disease progression after response to treatment to assess mechanisms of resistance, including secondary mutations, genomic amplification events, or gene deletions. Exome sequencing experiments using the approximately 300-gene ONCOPANEL™ assay are performed to evaluate mutations in mTOR pathway genes including, but not limited to, PIK3CA, TSC1, TSC2, AKT, PTEN, MTOR, and RHEB. In addition, mTOR-activating aberrations (sequences and levels of biomarkers including, but not limited to, AKT1, MTOR, PIK3CA, PIK3CG, TSC1, TSC2, RHEB, STK11, NF1, NF2, PTEN, TP53, FGFR4, and BAP1) are assessed, and levels of phosphorylated AKT (i.e., p-AKT), 4EBP1 (i.e., p-4EBP1), and S6K (i.e., p-S6K) using tumor biopsies. Markers of proliferation (such as Ki-67) and markers of apoptosis (such as PARP) can be assessed using immunohistochemical techniques. Translocations in TFE3 are analyzed using FISH (fluorescence in situ hybridization). The assay results are used to evaluate the correlation of mTOR-activating gene mutations with clinical response to treatment and to test the correlation between mTOR-activating gene mutations identified in tumor biopsy specimens and circulating DNA.

The primary endpoint of this study is the confirmed response rate. For PEComas, a confirmed response is defined as a CR or PR noted as objective status at two consecutive assessments at least week 8. A confirmed response will be assessed during all treatment cycles. An exact binomial confidence interval for the true endorsed response rate is calculated. Secondary endpoints of this study include survival time, time to disease progression, and adverse events. The distribution of survival time and the distribution of time to disease progression were estimated using the Kaplan-Meier method. For all primary and secondary endpoints, statistical analyzes were performed for the entire patient population and within each group.

A correlation study is conducted to determine the relationship of treatment with quality of life and individual mutational status of mTOR-activating genes for a general group of patients. Quality of life is assessed before analyzing the response to treatment and discussing the patient's general health since the last treatment assessment. Quality of life is assessed using the EORTC QLQ-C30, a 30-item patient questionnaire about the patient's ability to function, symptoms associated with cancer and its treatment, general health and quality of life, and the perceived financial impact of cancer and its treatment. Scaled quality of life score curves over time are examined using stream plots and mean plots with standard deviations as bars. Changes from baseline in each cycle are statistically analyzed using paired t tests, and the standardized response is interpreted after applying Middel's (2002) correction using Cohen's (1988) cutoffs: <0.20 = trivial; 0.20-<0.50 = small; 0.5-<0.8 = medium and >0.8 = large. The frequency of mTOR-activating aberrations per subject is described and the relationship with confirmed response is examined using Fisher's exact test. Associations with time to progression and overall survival were examined using log-rank tests. In all cases, one-sided values at the <0.10 level are considered statistically significant.

Subjects eligible for inclusion in the study must meet all of the following inclusion criteria: (a) have histological evidence of PEComa (eg, lymphangioleiomyomatosis); (b) have advanced stage cancer; (c) have a mutation in at least one mTOR pathway gene confirmed by a CLIA certified laboratory, and the mTOR pathway mutation is selected from genetic mutations in AKT1, MTOR, PIK3CA, TSC1, TSC2, RHEB, STK11, NF1, NF2, PTEN (eg, deletion of PTEN), TP53, FGFR4 and BAP1; (d) did not undergo any of the following treatments: (1) chemotherapy within 4 weeks before treatment with Nab-sirolimus; (2) hormonal therapy for 4 weeks before treatment with Nab-sirolimus; (3) radiation therapy within 4 weeks before

- 47 045904 treatment with Nab-sirolimus; (4) treatment with nitrosoureas, mitomycin, or intense radiation therapy within 6 weeks before treatment with Nab-sirolimus; (5) immunosuppressants for 3 weeks before treatment with Nab-sirolimus (except corticosteroids used as antiemetics); (6) use of prior mTOR pathway inhibitor therapy; (d) have the following laboratory parameters obtained no more than 14 days before inclusion in the study: (1) absolute neutrophil count (ANK) >1.5x10 ⁹ /l, platelet count >100,000/mm ³ (100x10 ⁹ /l) , (2) hemoglobin >9 g/dL, (3) total bilirubin <1.5x established upper limit of normal (ULN); (4) aspartate transaminase (AST); alanine aminotransferase (ALT) <3x ULN or <5x ULN if the subject has liver involvement; (5) serum cholesterol <350 mg/dL; (6) serum triglycerides <300 mg/dL; (7) serum creatinine <1.5x ULN; (f) subjects had previously failed to respond to, were intolerant of, or had refused other available active treatments; (f) have adequate blood coagulation function, defined by one of the following criteria: (1) !NoI<1.5x ULN; (2) for subjects receiving warfarin or LMWH: subjects must, in the judgment of the investigator, be clinically stable with no evidence of active bleeding while on anticoagulant therapy.

Exclusion criteria: (a) pregnant or lactating women or women of childbearing potential who are biologically capable of becoming pregnant, or men who are fertile without using two forms of highly effective contraception; (b) patients with a history of interstitial lung disease and/or pneumonia; (c) receiving any concomitant anticancer therapy or CYP3A4 inhibitors; (d) a history of allergic reactions associated with compounds of similar chemical or biological composition, including macrolide (eg, azithromycin, clarithromycin, dirithromycin, and erythromycin) and ketolide antibiotics; (f) major surgery (eg, intrathoracic, abdominal, or pelvic surgery) <4 weeks prior to study entry or failure to recover from side effects of such surgery except for port placement, nephrectomy, tumor biopsies, and minor surgery ; (f) concomitant use of any other approved or investigational anticancer agents that will be considered for treatment of the primary neoplasm; (g) uncontrolled diabetes mellitus, defined by HbA1c >8%, despite adequate therapy; (h) unstable angina or myocardial infarction within the previous 6 months and (i) hypertension not controlled by medications.

Example 3.

Treatment of metastatic PEComa with Nab-sirolimus.

A patient with PEComa of the lung (previously treated with adjuvant radiation therapy and then undergoing surgical resection of PEComa of the lung approximately 5 months ago) was suspected of developing a new suspicious metastatic lesion on CT examination. The new precarinal (mediastinal) lymph node had a diameter of approximately 1.5 cm. Because the size of the new lesion was borderline relative to the inclusion criteria for PEC001 (RECIST, requiring the presence of the disease being evaluated), the patient underwent another CT scan approximately 1 month later. At this time, the lesion was measurable, and the patient also had a new lesion in the pleural cavity >2 cm. Given the presence of a new lesion identified on the last CT scan, metastatic PEComa was considered to be an aggressive tumor that was rapidly progressing. The patient consented to participate in the PEC001 study and treatment with Nab-sirolimus (also related to ABI-009) was started at a dose of 100 mg/ ^m2 , IV infusion over 30 minutes and repeated on the second of a 3-week schedule. The next CT scan during follow-up was scheduled after 6 weeks according to clinical protocol PEC001. During this period, the patient received Nab-sirolimus as per protocol, tolerated the drug well, and reported no significant adverse events. At CT scan at week 6 of the follow-up period, the lesions were unchanged from the previous scan and the disease was believed to have stabilized as a result of treatment with Nab-sirolimus. Further treatment and follow-up continue as per protocol, and the patient continues to tolerate treatment without significant adverse events.

Claims (18)

1. A method of treating a malignant perivascular epithelioid cell tumor (PEComa) in a subject, comprising administering to the subject an effective amount of a composition comprising nanoparticles containing sirolimus and albumin, wherein the nanoparticle composition is administered intravenously, wherein the subject is at least about 50 years of age. 2. The method according to claim 1, where PEComa is selected from the group consisting of clear cell sugar lung tumor or PEComa of other localizations (PEComa-NOS). 3. The method according to claim 1 or 2, where the epithelioid tumor is locally advanced, metastatic or recurrent. - 48 045904 4. The method of any one of claims 1 to 3, wherein the effective amount of sirolimus in the nanoparticle composition is from about 10 to about 150 mg/m ² . 5. The method according to any one of claims 1 to 4, wherein the effective amount of sirolimus in the nanoparticle composition is about 100 mg/m ² . 6. The method according to any one of claims 1 to 4, wherein the effective amount of sirolimus in the nanoparticle composition is approximately 75 mg/m ² . 7. Method according to any one of claims 1 to 6, wherein the nanoparticle composition is administered weekly. 8. Method according to any one of claims 1-7, where the composition of nanoparticles is administered weekly, 2 times every 3 weeks. 9. A method according to any one of claims 1 to 8, wherein the nanoparticles in the composition have an average diameter of no more than about 150 nm. 10. Method according to any one of claims 1 to 9, wherein sirolimus in nanoparticles is associated with albumin. 11. The method according to any one of claims 1 to 10, wherein the weight ratio of albumin to sirolimus in the nanoparticle composition is from about 1:1 to about 9:1. 12. The method according to any one of claims 1 to 11, wherein the albumin is human albumin or wherein the albumin is human serum albumin. 13. The method according to any one of claims 1 to 12, where the subject is a human. 14. The method according to any one of claims 1 to 13, wherein the subject is selected for treatment based on the level of melanocyte marker and smooth muscle marker. 15. The method of any one of claims 1 to 14, wherein the subject is selected for treatment based on the mutational status of a gene selected from the group consisting of TSC1, TSC2, TFE3, RHEB, MTOR, AKT, PIK3CA and PTEN. 16. The method of any one of claims 1 to 15, wherein the subject is selected for treatment based on the phosphorylation status of a protein selected from the group consisting of AKT, S6, S6K and 4EBP1. 17. The method according to any one of claims 1 to 16, wherein the subject is selected for treatment based on the level of a proliferation marker or an apoptosis marker. 18. The method according to any one of claims 1 to 17, wherein the subject has not previously been treated with an mTOR inhibitor.