Methods of treating a cancer in an individual

Abstract

The present application, in certain aspects, pertains to methods of treating a cancer in an individual using a composition comprising nanoparticles comprising an mTOR inhibitor (such as sirolimus) and an albumin. In some aspects, the methods are directed to an individual subjected to two or more prior treatments. In other aspects, the methods are directed to a cancer of the adrenal cortex. In certain aspects, provided are methods of reducing the size of a tumor and / or reducing a tumor-associated fluid accumulation using a composition comprising nanoparticles comprising an mTOR inhibitor (such as sirolimus) and an albumin.

Description

Attorney Reference: 638772023240 METHODS OF TREATING A CANCER IN AN INDIVIDUALCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial. No. 63 / 610,351, filed on December 14, 2023, the entire content of which is incorporated herein by reference for all purposes. FIELD OF THE INVENTION

[0002] The present application, in certain aspects, pertains to methods of treating a cancer in an individual using a composition comprising nanoparticles comprising an mTOR inhibitor (such as sirolimus) and an albumin. BACKGROUND OF THE INVENTION

[0003] Certain cancers are known to be especially difficult to treat, including cancers in patients having been subjected to prior cancer treatments. Moreover, there is a need in the field to not only treat a tumor mass, such as by reducing the tumor volume, but also address certain tumor-associated complications such as a tumor-associated fluid accumulation. While complete surgical resection of a tumor has been demonstrated as an effective treatment, many patients are not candidates for surgery due to, e.g., advancement of the tumor into or around healthy tissue and / or the positioning of the tumor. At this time, there remains a continuing need in the field for advanced cancer treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] FIG. 1 shows the study design of PRECISION 1 discussed in Example 1.

[0005] FIG. 2 shows demographics of efficacy evaluable population in this study.

[0006] FIG. 3 shows tumor types of patients enrolled in each arm of the PRECISION 1 study.

[0007] FIG. 4 shows results of various efficacy measurements in the TSC1 arm.sf-6235590Attorney Reference: 638772023240

[0008] FIG. 5 shows patient time on treatment in the TSC1 arm.

[0009] FIG. 6 shows best overall response and maximal percentage of target tumor reduction (%) in the TSC1 arm.

[0010] FIG. 7 shows a summary of efficacy conclusions for the TSC1 arm.

[0011] FIG. 8 shows results of various efficacy measurements in the TSC2 arm.

[0012] FIG. 9 shows patient time on treatment in the TSC2 arm.

[0013] FIG. 10 shows best overall response and maximal percentage of target tumor reduction (%) in the TSC2 arm.

[0014] FIG. 11 shows a summary of efficacy conclusions for the TSC2 arm.

[0015] FIG. 12 shows a summary of safety conclusions for the PRECISION1 study. BRIEF SUMMARY OF THE INVENTION

[0016] The present application provides novel methods of treating cancer. In some aspects, provided herein is a method of treating a cancer in an individual in need thereof, wherein the individual has been subjected to two or more prior treatments for the cancer, the method comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin. In some embodiments, the individual has failed the two or more prior treatments for the cancer. In some embodiments, the individual has progressed on the two or more prior treatments for cancer.

[0017] In some embodiments, the individual has been subjected to three or more prior treatments for the cancer. In some embodiments, the individual has failed the three or more prior treatments for the cancer. In some embodiments, the individual has progressed on the three or more prior treatments for cancer. In some embodiments, the individual has been subjected to five or more prior treatments for the cancer. In some embodiments, the individual has failed the five or more prior treatments for the cancer. In some embodiments, the individual has progressed on the five or more prior treatments for cancer.

[0018] In some embodiments, the cancer is refractory, relapsed, or recurrent to the prior treatments.sf-6235590Attorney Reference: 638772023240

[0019] In some embodiments, the prior treatments do not comprise treatment with an mTOR inhibitor. In some embodiments, the prior treatments do not comprise treatment with a composition comprising nanoparticles comprising an mTOR inhibitor and an albumin.

[0020] In other aspects, provided herein is a method of reducing the size of a tumor in an individual having a cancer, the method comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin.

[0021] In some embodiments, the cancer is unresectable prior to the administering to the individual the composition comprising nanoparticles comprising sirolimus and the albumin. In some embodiments, the reducing the size of the tumor is a reduction of at least about 30% of the tumor as compared to tumor size prior to the administering to the individual the composition comprising nanoparticles comprising sirolimus and the albumin. In some embodiments, the reducing the size of the tumor results in the tumor being suitable for surgical resection.

[0022] In some embodiments, the method further comprises surgically resecting the tumor following the administering to the individual the composition comprising nanoparticles comprising sirolimus and the albumin.

[0023] In other aspects, provided herein is a method of reducing a fluid accumulation associated with a tumor (e.g., pleural effusion, e.g., ascites) in an individual having a cancer, the method comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin.

[0024] In some embodiments, the reducing the fluid accumulation associated with a tumor (e.g., pleural effusion, e.g., ascites) is a reduction of at least about 30% of the fluid volume as compared to the fluid volume prior to the administering to the individual the composition comprising nanoparticles comprising sirolimus and the albumin.

[0025] In some embodiments, the cancer is selected from the group consisting of a bladder cancer, ovarian cancer, endometrial cancer, colon cancer, leiomyosarcoma, breast cancer, cervical cancer, adrenocortical carcinoma, hepatocellular carcinoma, sarcoma, soft tissue sarcoma, gastrointestinal stromal tumor (GIST), primitive neuroectodermal tumor (PNET), vaginal cancer, osteosarcoma, mesothelioma, head and neck cancer, hepatobiliary cancer,sf-6235590Attorney Reference: 638772023240 esophagogastric cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, gynecological cancer, and genito-urinary cancer.

[0026] In other aspects, provided herein is a method of treating a cancer of the adrenal cortex in an individual in need thereof, the method comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin.

[0027] In some embodiments according to any of the methods described above, the cancer is locally advanced, advanced, malignant, advanced malignant, or metastatic.

[0028] In some embodiments according to any of the methods described above, the individual is selected for treatment on the basis of having a TSC1 or TSC2 inactivating mutation. In some embodiments, the individual is selected for treatment on the basis of having the TSC1 inactivating mutation. In some embodiments, the individual is selected for treatment on the basis of having the TSC2 inactivating mutation.

[0029] In some embodiments according to any of the methods described above, the inactivating mutation in TSC1 or TSC2 comprises a homozygous deletion, bi-allelic mutations, splice site mutation, frameshift mutation, somatic mutation, truncation, deletion, nonsense mutation in coding region, missense mutation with confirmed impact, or a loss or deletion of TSC1 or TSC2. In some embodiments, the inactivating mutation in TSC1 or TSC2 is a somatic mutation. In some embodiments, the inactivating mutation in TSC1 or TSC2 is a truncation, deletion, or frameshift mutation. In some embodiments, the inactivating mutation in TSC1 or TSC2 comprises bi-allelic mutations.

[0030] In some embodiments according to any of the methods described above, the method further comprises assessing for the inactivating mutation in TSC1 or TSC2. In some embodiments, the method further comprises assessing if the inactivating mutation in TSC1 or TSC2 is pathogenic.

[0031] In some embodiments according to any of the methods described above, sirolimus in the sirolimus nanoparticle composition is administered at an amount of about 10 mg / m2to about 150 mg / m2. In some embodiments, sirolimus in the sirolimus nanoparticle composition is administered at an amount of about 30 mg / m2to about 100 mg / m2. In some embodiments, sirolimus in the sirolimus nanoparticle composition is administered at an amount of about 100sf-6235590Attorney Reference: 638772023240 mg / m2. In some embodiments, sirolimus in the sirolimus nanoparticle composition is administered at an amount of about 75 mg / m2. In some embodiments, sirolimus in the sirolimus nanoparticle composition is administered at an amount of about 56 mg / m2. In some embodiments, sirolimus in the sirolimus nanoparticle composition is administered at an amount of about 45 mg / m2. In some embodiments, sirolimus in the sirolimus nanoparticle composition is administered at an amount of about 35 mg / m2.

[0032] In some embodiments according to any of the methods described above, the sirolimus nanoparticle composition is administered twice out of every 3 weeks. In some embodiments, the sirolimus nanoparticle composition is administered on days 1 and 8 of a 21-day cycle. In some embodiments, the average diameter of the nanoparticles in the composition is no greater than about 150 nm. In some embodiments, the average diameter of the nanoparticles in the composition is no greater than about 120 nm. In some embodiments, the sirolimus nanoparticle composition is administered intravenously.

[0033] In some embodiments, the individual is human.

[0034] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present application provides, in certain aspects, methods of treating a cancer in an individual in need thereof, the methods comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin. In some embodiments, the methods of treating a cancer are directed to method of treating a caner in an individual having been subjected to two or more (including three or more, four or more, or five or more) prior treatments for the cancer. In some embodiments, the methods of treating a cancer are directed to treating a cancer of the adrenal cortex in an individual in need thereof, the method comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin. In certain other aspects, the methods provided herein are directed to reducing the size of a tumor and / or reducing a tumor-associated fluid accumulation in ansf-6235590Attorney Reference: 638772023240 individual having a cancer, the methods comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin.

[0036] The subject matter of the present application is based, at least in part, on the inventors’ findings from a study demonstrating that nab-sirolimus (a composition comprising nanoparticles comprising sirolimus and an albumin) produces rapid treatment response with meaningful decreases in tumor volume within, e.g., 6 weeks after the first dose of nab-sirolimus, with clinically meaningful durations of response (e.g., a patient with greater than 9 months duration of response) in patients that are heavily pretreated for said cancer (e.g., subjected to two or more prior treatments). Furthermore, such treatment responses were exhibited in patients also having inactivating mutations in TSC1 and TSC2. It has also been demonstrated that the taught methods result in a reduction of fluid accumulation associated with a tumor (e.g., pleural effusion, e.g., ascites) in an individual having a cancer. These findings open the door to additional cancer treatments such as surgical resection, that may not be available at the start of treatment due to highly advanced disease states or tumor position. In summary, these findings demonstrate the clinical significance of the methods taught herein.

[0037] In some embodiments, there is provided a method of treating a cancer in an individual (such as a human), wherein the individual has been subjected to two or more (such as any of three or more, four or more, five or more, or six or more) prior treatments for the cancer, comprising administering to the individual an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) and an albumin (e.g., nab-sirolimus). In some embodiments, the method comprises administering to the individual an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) and an albumin (e.g., nab-sirolimus), wherein the nanoparticles have an average particle size of no greater than about 150 nm (such as no greater than about 120 nm). In some embodiments, the method comprises administering to the individual an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) and an albumin (e.g., nab-sirolimus), wherein the nanoparticles have an average particle size of no greater than about 150 nm (such as no greater than about 120 nm, for example about 100 nm), wherein the weight ratio of albumin and thesf-6235590Attorney Reference: 638772023240 mTOR inhibitor in the mTOR inhibitor nanoparticle composition is about 9:1 or less (such as about 9:1 or about 8:1). In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the mTOR inhibitor is sirolimus or a derivative thereof. In some embodiments, the mTOR inhibitor nanoparticle composition comprises nab-sirolimus. In some embodiments, the mTOR inhibitor nanoparticle composition is nab-sirolimus. In some embodiments, the mTOR inhibitor nanoparticle composition is administered 2 out of every 3 weeks, such as on days 8 and 15 of a 21-day cycle. In some embodiments, the amount of the mTOR inhibitor in the mTOR inhibitor nanoparticle composition is from about 10 mg / m2to about 150 mg / m2, such as about any of 100 mg / m2, 75 mg / m2, 56 mg / m2, 45 mg / m2, or 35 mg / m2. In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., nab-sirolimus) is administered intravenously. In some embodiments, the individual has progressed on one or more prior treatments. In some embodiments, the individual has not been treated with an mTOR inhibitor (such as nab-sirolimus) prior to commencing a method described herein. In some embodiments, the cancer is selected from the group consisting of a bladder cancer, ovarian cancer, endometrial cancer, colon cancer, leiomyosarcoma, breast cancer, cervical cancer, adrenocortical carcinoma, hepatocellular carcinoma, sarcoma, soft tissue sarcoma, gastrointestinal stromal tumor (GIST), primitive neuroectodermal tumor (PNET), vaginal cancer, osteosarcoma, mesothelioma, head and neck cancer, hepatobiliary cancer, esophagogastric cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, gynecological cancer, and genito-urinary cancer. In some embodiments, the cancer is metastatic or locally advanced. In some embodiments, the individual is selected for treatment on the basis of having a TSC1 or TSC2 inactivating mutation.

[0038] In some embodiments, there is provided a method of reducing the size of a tumor in an individual (such as a human) having a cancer in, comprising administering to the individual an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) and an albumin (e.g., nab- sirolimus). In some embodiments, the method comprises administering to the individual an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) and an albumin (e.g., nab- sirolimus), wherein the nanoparticles have an average particle size of no greater than about 150 nm (such as no greater than about 120 nm). In some embodiments, the method comprises administering to the individual an effective amount of a composition comprising nanoparticlessf-6235590Attorney Reference: 638772023240 comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) and an albumin (e.g., nab-sirolimus), wherein the nanoparticles have an average particle size of no greater than about 150 nm (such as no greater than about 120 nm, for example about 100 nm), wherein the weight ratio of albumin and the mTOR inhibitor in the mTOR inhibitor nanoparticle composition is about 9:1 or less (such as about 9:1 or about 8:1). In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the mTOR inhibitor is sirolimus or a derivative thereof. In some embodiments, the mTOR inhibitor nanoparticle composition comprises nab-sirolimus. In some embodiments, the mTOR inhibitor nanoparticle composition is nab-sirolimus. In some embodiments, the mTOR inhibitor nanoparticle composition is administered 2 out of every 3 weeks, such as on days 8 and 15 of a 21-day cycle. In some embodiments, the amount of the mTOR inhibitor in the mTOR inhibitor nanoparticle composition is from about 10 mg / m2to about 150 mg / m2, such as about any of 100 mg / m2, 75 mg / m2, 56 mg / m2, 45 mg / m2, or 35 mg / m2. In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., nab-sirolimus) is administered intravenously. In some embodiments, the cancer is unresectable prior to the administering to the individual the composition comprising nanoparticles comprising sirolimus and the albumin. In some embodiments, the reducing the size of the tumor is a reduction of at least about 30% (such as at least about any of 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the tumor as compared to tumor size prior to the administering to the individual the composition comprising nanoparticles comprising sirolimus and the albumin. In some embodiments, the reducing the size of the tumor results in the tumor being suitable for surgical resection. In some embodiments, the method further comprises surgically resecting the tumor following the administering to the individual the composition comprising nanoparticles comprising sirolimus and the albumin. In some embodiments, the individual has been subjected to two or more prior treatments. In some embodiments, the individual has progressed on one or more prior treatments. In some embodiments, the individual has not been treated with an mTOR inhibitor (such as nab-sirolimus) prior to commencing a method described herein. In some embodiments, the cancer is selected from the group consisting of a bladder cancer, ovarian cancer, endometrial cancer, colon cancer, leiomyosarcoma, breast cancer, cervical cancer, adrenocortical carcinoma, hepatocellular carcinoma, sarcoma, soft tissue sarcoma, gastrointestinal stromal tumor (GIST), primitive neuroectodermal tumor (PNET), vaginalsf-6235590Attorney Reference: 638772023240 cancer, osteosarcoma, mesothelioma, head and neck cancer, hepatobiliary cancer, esophagogastric cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, gynecological cancer, and genito-urinary cancer. In some embodiments, the cancer is metastatic or locally advanced. In some embodiments, the individual is selected for treatment on the basis of having a TSC1 or TSC2 inactivating mutation.

[0039] In some embodiments, there is provided a method of treating a cancer in the adrenal cortex of in an individual (such as a human), comprising administering to the individual an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) and an albumin (e.g., nab- sirolimus). In some embodiments, the method comprises administering to the individual an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) and an albumin (e.g., nab- sirolimus), wherein the nanoparticles have an average particle size of no greater than about 150 nm (such as no greater than about 120 nm). In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the mTOR inhibitor is sirolimus or a derivative thereof. In some embodiments, the mTOR inhibitor nanoparticle composition comprises nab-sirolimus. In some embodiments, the mTOR inhibitor nanoparticle composition is nab-sirolimus. In some embodiments, the mTOR inhibitor nanoparticle composition is administered 2 out of every 3 weeks, such as on days 8 and 15 of a 21-day cycle. In some embodiments, the amount of the mTOR inhibitor in the mTOR inhibitor nanoparticle composition is from about 10 mg / m2to about 150 mg / m2, such as about any of 100 mg / m2, 75 mg / m2, 56 mg / m2, 45 mg / m2, or 35 mg / m2. In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., nab- sirolimus) is administered intravenously. In some embodiments, the individual has been subjected to two or more prior treatments. In some embodiments, the individual has progressed on one or more prior treatments. In some embodiments, the individual has not been treated with an mTOR inhibitor (such as nab-sirolimus) prior to commencing a method described herein. In some embodiments, the cancer is metastatic or locally advanced. In some embodiments, the individual is selected for treatment on the basis of having a TSC1 or TSC2 inactivating mutation.sf-6235590Attorney Reference: 638772023240 I. Definitions

[0040] As used herein “nab” stands for nanoparticle albumin-bound, and “nab-sirolimus” is an albumin stabilized nanoparticle formulation of sirolimus. nab-sirolimus is also known as nab- rapamycin, which has been previously described. See, for example, U.S. Patent Nos. 8,911,786 and 11,497,737, each of which is incorporated herein by reference in their entirety.

[0041] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, reducing recurrence rate of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival. In some embodiments, the treatment reduces the severity of one or more symptoms associated with cancer by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to the corresponding symptom in the same subject prior to treatment or compared to the corresponding symptom in other subjects not receiving the treatment. Also encompassed by "treatment" is a reduction of pathological consequence of cancer. The methods of the invention contemplate any one or more of these aspects of treatment.

[0042] The terms “recurrence,” “relapse” or “relapsed” refers to the return of a cancer or disease after clinical assessment of the disappearance of disease. A diagnosis of distant metastasis or local recurrence can be considered a relapse.

[0043] The term “refractory” or “resistant” refers to a cancer or disease that has not responded to treatment. In some embodiments, the cancer does not respond to the treatment. In some embodiments, the cancer become resistant during treatment, e.g., there may be an initial response (to some degree) followed by disease progression.

[0044] As used herein, “delaying” the development of cancer means to defer, hinder, slow, retard, stabilize, and / or postpone development of the disease. This delay can be of varyingsf-6235590Attorney Reference: 638772023240 lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. A method that “delays” development of cancer is a method that reduces probability of disease development in a given time frame and / or reduces the extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of subjects. Cancer development can be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT scan), Magnetic Resonance Imaging (MRI), ultrasound, clotting tests, arteriography, biopsy, urine cytology, and cystoscopy. Development may also refer to cancer progression that may be initially undetectable and includes occurrence, recurrence, and onset.

[0045] The term “effective amount” used herein refers to an amount of a compound or composition sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and / or delay one or more of its symptoms. In reference to cancer, an effective amount comprises an amount sufficient to cause a tumor to shrink and / or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation in cancer. In some embodiments, an effective amount is an amount sufficient to delay development of cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. In some embodiments, an effective amount is an amount sufficient to reduce recurrence rate in the individual. An effective amount can be administered in one or more administrations. The effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; (vii) reduce recurrence rate of tumor, and / or (viii) relieve to some extent one or more of the symptoms associated with the cancer.

[0046] As is understood in the art, an “effective amount” or “amount” may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and a nanoparticle composition (e.g., a composition including sirolimus andsf-6235590Attorney Reference: 638772023240 an albumin) may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable or beneficial result may be or is achieved. The components (e.g., the first and second therapies) in a combination therapy of the invention may be administered sequentially, simultaneously, or concurrently using the same or different routes of administration for each component. Thus, an effective amount of a combination therapy includes an amount of the first therapy and an amount of the second therapy that when administered sequentially, simultaneously, or concurrently produces a desired outcome.

[0047] As used herein, by “pharmaceutically acceptable” or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and / or are included on the Inactive Ingredient Guide prepared by the U. S. Food and Drug administration.

[0048] As used herein, the term “individual” refers to a mammal and includes, but is not limited to, human, bovine, horse, feline, canine, rodent, rat, mouse, dog, or primate. In some embodiments, the individual is a human individual.

[0049] The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”

[0050] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range,sf-6235590Attorney Reference: 638772023240 is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0051] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

[0052] As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

[0053] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present disclosure. The following description illustrates the disclosure and, of course, should not be construed in any way as limiting the scope of the inventions described herein. II. Treatment methods

[0054] Provided herein are methods for the treatment of certain individuals in need thereof, including individuals having been subjected to two or more prior treatments for the cancer or an individual having adrenocortical carcinoma. Also provided herein are methods for reducing the size of a tumor and / or reducing a tumor-associated fluid accumulation in an individual having a cancer, the methods comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin. In some embodiments described herein, the individual is selected, at least in part, for treatment on the basis of having a TSC1 or TSC2 inactivating mutation.

[0055] Further discussion of the methods, and aspects thereof, taught herein is included in the sections below. The modular discussion of such components does not limit the scope of the invention and one of ordinary skill in the art will readily appreciate how certain features from the sections below can be combined.sf-6235590Attorney Reference: 638772023240 A. Methods of treating a cancer in an individual having been subjected to prior treatments for the cancer

[0056] Provided herein in certain aspects are methods of treating a cancer in an individual in need thereof, wherein the individual has been subjected to two or more prior treatments for the cancer, the method comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin. In some embodiments, the individual has failed (e.g., progressed on) the two or more prior treatments for the cancer. In some embodiments, the individual has progressed on the two or more (such as any of three or more, four or more, five or more, or six or more) prior treatment for cancer. In some embodiments, the individual having cancer has malignant cancer and is refractory, relapses, or recurs in certain tumor locations (and not necessarily all prior tumor locations).

[0057] In some embodiments, the individual achieves a complete response or partial response. In some embodiments, the response to the treatment as described herein is durable. In some embodiments, a durable response lasts for more than about 9 months. In some embodiments, a durable response lasts for more than 9 months, and the patient has a TSC1 inactivating mutation. In some embodiments, a durable response lasts for more than any of about 4 months, 5 months or 6 months. In some embodiments, a durable response lasts for more than any of about 4 months, 5 months or 6 months, and the patient has a TSC2 inactivating mutation.

[0058] In some embodiments, the individual has been subjected to three or more prior treatments for the cancer. In some embodiments, the individual has failed (e.g., progressed on) the three or more prior treatments for the cancer. In some embodiments, the individual has progressed on the three or more prior treatments for cancer. In some embodiments, the individual has been subjected to four or more prior treatments for the cancer. In some embodiments, the individual has failed (e.g., progressed on) the four or more prior treatments for the cancer. In some embodiments, the individual has progressed on the four or more prior treatments for cancer. In some embodiments, the individual has been subjected to five or more prior treatments for the cancer. In some embodiments, the individual has failed (e.g., progressed on) the five or more prior treatments for the cancer. In some embodiments, the individual has progressed on the five or more prior treatments for cancer. In some embodiments, the individual has been subjected to six or more prior treatments for the cancer. In some embodiments, thesf-6235590Attorney Reference: 638772023240 individual has failed (e.g., progressed on) the six or more prior treatments for the cancer. In some embodiments, the individual has progressed on the six or more prior treatments for cancer. In some embodiments, the individual has been subjected to seven or more prior treatments for the cancer. In some embodiments, the individual has failed (e.g., progressed on) the seven or more prior treatments for the cancer. In some embodiments, the individual has progressed on the seven or more prior treatments for cancer. In some embodiments, the individual has been subjected to eight or more prior treatments for the cancer. In some embodiments, the individual has failed (e.g., progressed on) the eight or more prior treatments for the cancer. In some embodiments, the individual has progressed on the eight or more prior treatments for cancer.

[0059] In some embodiments, the cancer is refractory, relapsed, or recurrent to one or more prior treatments. In some embodiments, the individual progressed on a prior treatment and / or stopped the prior treatment due to toxicity-related issues (such as an unacceptable level of an adverse event). In some embodiments, the individual stopped a prior treatment due to withdrawal of consent or at a doctor’s discretion. In some embodiments, the cancer initially responded to a prior treatment but later the prior treatment failed due to, e.g., the cancer becoming non-responsive and / or progressing and / or the individual experience one or more adverse events, such as unacceptable treatment toxicity.

[0060] In some embodiments, the prior treatment directly before use of the methods taught herein completed at least about 1 month, such as at least about any of 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months, before administration of a composition comprising nanoparticles comprising sirolimus and an albumin according the description here. In some embodiments, progression following administration of a prior treatment is within about 24 months, such as within about any of 18 months, 12 months, 6 months, 5 months, 4 months, 3 months, 2 months, or 1 months.

[0061] In some embodiments, the prior treatments do not comprise treatment with an mTOR inhibitor. In some embodiments, the prior treatments do not comprise treatment with a composition comprising nanoparticles comprising an mTOR inhibitor and an albumin. In some embodiments, the individual has not been previously treated with an mTOR inhibitor. In some embodiments, the individual has not been previously treated with a composition comprising ansf-6235590Attorney Reference: 638772023240 mTOR inhibitor (e.g., sirolimus) and an albumin. In some embodiments, the individual has not been previously treated with nab-sirolimus.

[0062] In some embodiments, the prior treatments comprise a series of different treatments or treatment regimen. In some embodiments, the prior treatments comprise a series of treatment regimen where there is some overlap between at least two of the treatments, e.g., a drug administered to a patient used in two different treatments treatment regimen.

[0063] In some embodiments, the cancer is selected from the group consisting of a bladder cancer, ovarian cancer, endometrial cancer, colon cancer, leiomyosarcoma, breast cancer, cervical cancer, adrenocortical carcinoma, hepatocellular carcinoma, sarcoma, soft tissue sarcoma, gastrointestinal stromal tumor (GIST), primitive neuroectodermal tumor (PNET), vaginal cancer, osteosarcoma, mesothelioma, head and neck cancer, hepatobiliary cancer, esophagogastric cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, gynecological cancer, and genito-urinary cancer. In some embodiments, the cancer is an epithelial carcinoma. In some embodiments, the cancer is a sarcoma.

[0064] In some embodiments, the cancer is locally advanced, advanced, malignant, advanced malignant, or metastatic. In some embodiments, the cancer is unresectable or not resectable without risk of serious injury or death prior to starting the methods described herein.

[0065] In some embodiments, the individual is selected for treatment on the basis of having a TSC1 or TSC2 inactivating mutation. In some embodiments, the individual is selected for treatment on the basis of having the TSC1 inactivating mutation. In some embodiments, the individual is selected for treatment on the basis of having the TSC2 inactivating mutation. B. Methods of reducing the size of a tumor and / or reducing a fluid accumulation

[0066] In certain aspects, provided herein are methods of reducing the size of a tumor in an individual having a cancer, the methods comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin.

[0067] In some embodiments, the cancer is unresectable prior to the administering to the individual the composition comprising nanoparticles comprising sirolimus and the albumin. In some embodiments, the reducing the size of the tumor is a reduction of at least about 30%, suchsf-6235590Attorney Reference: 638772023240 as at least about any of 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, of the tumor as compared to tumor size prior to the administering to the individual the composition comprising nanoparticles comprising sirolimus and the albumin. Methods for assessing tumor size are well known in the field and include imaging techniques such as a computed tomography (CT) scan.

[0068] In some embodiments, the reducing the size of the tumor results in the tumor being suitable for surgical resection. In some embodiments, the method further comprises surgically resecting the tumor following the administering to the individual the composition comprising nanoparticles comprising sirolimus and the albumin.

[0069] In some aspects, provided herein is a method of reducing a fluid accumulation associated with a tumor (e.g., pleural effusion, e.g., ascites) in an individual having a cancer (such as an advanced cancer), the method comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin. In some embodiments, the reducing the fluid accumulation associated with a tumor (e.g., pleural effusion, e.g., ascites) is a reduction of at least about 30%, such as at least about any of 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, of the fluid volume as compared to the fluid volume prior to (including directly prior to) the administering to the individual the composition comprising nanoparticles comprising sirolimus and the albumin. In some embodiments, the individual has malignant pleural effusion. In some embodiments, the individual has malignant ascites. In some embodiments, the malignant pleural effusion or malignant ascites is associated with the individual having a lung cancer, breast cancer, ovarian cancer (e.g., an individual having ovarian cancer and a TSC1 inactivating mutation), or colorectal cancer. In some embodiments, the cancer is an epithelial carcinoma. In some embodiments, the cancer is a sarcoma. In some embodiments, the individual has a TSC1 inactivating mutation and an epithelial carcinoma. In some embodiments, the individual has a TSC2 inactivating mutation and an epithelial carcinoma. In some embodiments, the cancer is a sarcoma. In some embodiments, the individual has a TSC2 inactivating mutation and a sarcoma.

[0070] In some embodiments, the individual has been subjected to two or more prior treatments for adrenocortical carcinoma. In some embodiments, the individual has failed (e.g., progressed on) the two or more prior treatments for adrenocortical carcinoma. In somesf-6235590Attorney Reference: 638772023240 embodiments, the individual has progressed on the two or more prior treatments for cancer. In some embodiments, the cancer is refractory, relapsed, or recurrent to prior treatments.

[0071] In some embodiments, the prior treatments do not comprise treatment with an mTOR inhibitor. In some embodiments, the prior treatments do not comprise treatment with a composition comprising nanoparticles comprising an mTOR inhibitor and an albumin. In some embodiments, the individual has not been previously treated with an mTOR inhibitor. In some embodiments, the individual has not been previously treated with a composition comprising an mTOR inhibitor (e.g., sirolimus) and an albumin. In some embodiments, the individual has not been previously treated with nab-sirolimus.

[0072] In some embodiments, the cancer is selected from the group consisting of a bladder cancer, ovarian cancer, endometrial cancer, colon cancer, leiomyosarcoma, breast cancer, cervical cancer, adrenocortical carcinoma, hepatocellular carcinoma, sarcoma, soft tissue sarcoma, gastrointestinal stromal tumor (GIST), primitive neuroectodermal tumor (PNET), vaginal cancer, osteosarcoma, mesothelioma, head and neck cancer, hepatobiliary cancer, esophagogastric cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, gynecological cancer, and genito-urinary cancer. In some embodiments, the cancer is an epithelial carcinoma. In some embodiments, the cancer is a sarcoma.

[0073] In some embodiments, the cancer is locally advanced, advanced, malignant, advanced malignant, or metastatic. In some embodiments, the cancer is unresectable or not resectable without risk of serious injury or death prior to starting the methods described herein.

[0074] In some embodiments, the individual is selected for treatment on the basis of having a TSC1 or TSC2 inactivating mutation. In some embodiments, the individual is selected for treatment on the basis of having the TSC1 inactivating mutation. In some embodiments, the individual is selected for treatment on the basis of having the TSC2 inactivating mutation. C. Methods of treating a cancer of the adrenal cortex

[0075] In certain aspects, provided herein are methods of treating a cancer of the adrenal cortex (also known as an adrenocortical carcinoma or ACC) in an individual in need thereof, thesf-6235590Attorney Reference: 638772023240 methods comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin.

[0076] Adrenocortical carcinoma is a rare cancer in which cancer cells form in the outer layer of the adrenal gland. Adrenocortical carcinoma can form in one or both adrenal glands, which are each located above a kidney. Symptoms of adrenocortical carcinoma include pain in the abdomen, and further imaging studies, blood and urine tests, and histopathology can be used to diagnose adrenocortical carcinoma.

[0077] In some embodiments, the individual has been subjected to two or more prior treatments for adrenocortical carcinoma. In some embodiments, the individual has failed (e.g., progressed on) the two or more prior treatments for adrenocortical carcinoma. In some embodiments, the individual has progressed on the two or more prior treatments for adrenocortical carcinoma. In some embodiments, the cancer is refractory, relapsed, or recurrent to prior treatments.

[0078] In some embodiments, the prior treatments do not comprise treatment with an mTOR inhibitor. In some embodiments, the prior treatments do not comprise treatment with a composition comprising nanoparticles comprising an mTOR inhibitor and an albumin. In some embodiments, the individual has not been previously treated with an mTOR inhibitor. In some embodiments, the individual has not been previously treated with a composition comprising an mTOR inhibitor (e.g., sirolimus) and an albumin. In some embodiments, the individual has not been previously treated with nab-sirolimus.

[0079] In some embodiments, the adrenocortical carcinoma is locally advanced, advanced, malignant, advanced malignant, or metastatic. In some embodiments, the adrenocortical carcinoma is unresectable or not resectable without risk of serious injury or death prior to starting the methods described herein.

[0080] In some embodiments, the individual is selected for treatment on the basis of having a TSC1 or TSC2 inactivating mutation. In some embodiments, the individual is selected for treatment on the basis of having the TSC1 inactivating mutation. In some embodiments, the individual is selected for treatment on the basis of having the TSC2 inactivating mutation.sf-6235590Attorney Reference: 638772023240 D. Individuals selected for treatment based on having a TSC1 or TSC2 inactivating mutation

[0081] As taught herein, in some embodiments, the individual is selected for treatment on the basis of having a TSC1 or TSC2 inactivating mutation. In some embodiments, the individual is selected for treatment on the basis of having the TSC1 inactivating mutation. In some embodiments, the individual is selected for treatment on the basis of having the TSC2 inactivating mutation. In some embodiments, the inactivating mutation in TSC1 or TSC2 comprises a homozygous deletion, bi-allelic mutations, splice site mutation, frameshift mutation, somatic mutation, truncation, deletion (such as an in-frame deletion), nonsense mutation in coding region, missense mutation with confirmed impact, or a loss or deletion of TSC1 or TSC2. In some embodiments, the inactivating mutation in TSC1 or TSC2 is a somatic mutation. In some embodiments, the inactivating mutation in TSC1 or TSC2 is a truncation, deletion, or frameshift mutation. In some embodiments, the inactivating mutation in TSC1 or TSC2 comprises bi-allelic mutations. In some embodiments, the method further comprises assessing for the inactivating mutation in TSC1 or TSC2. In some embodiments, the method further comprises assessing if the inactivating mutation in TSC1 or TSC2 is pathogenic.

[0082] In some embodiments, the individual has a pathogenic (i.e., inactivating) mutation in TSC1 or TSC2. Pathogenic inactivating mutations (loss-of-function) of certain gene (e.g., TSC1 or TSC2) can be determined by review of experimental evidence within the published scientific literature and review of critical regions that may be disrupted, including but not limited to frameshift, missense mutations, truncating mutations, deletions, copy number variations, nonsense mutations, and loss or deletion of the gene. A pathogenic mutation is inferred as inactivating.

[0083] Pathogenic or inactivating mutations include but are not limited to homozygous deletions, bi-allelic (double hit) mutations, splice site mutations (e.g., a 2ndor an additional splice site mutation), frameshift mutations, and nonsense mutations in coding region, missense mutations with confirmed impact.

[0084] In some embodiments, the methods described herein comprises a step of determining if a mutation in TSC1 or TSC2 is a pathogenic mutation. Methods are described herein and also include those taught in US20230000844, which is hereby incorporated by reference in its entirety. Certain TSC1and TSC2 mutation are disclosed in Gulati et al., Landscape of TSC1 andsf-6235590Attorney Reference: 638772023240 TSC2 alterations in patients with advanced solid cancers, (<https: / / www.tessellon.com / expertise / DownloadLandscapeTSC1TSC2alterationsinpressPdf>), which is hereby incorporated herein by reference in its entirety.

[0085] In some embodiments, the inactivating mutation comprises a nonsense mutation, an out-of-frame insertion, a deletion mutation, or a mutation that affects canonical splice site in TSC1 or TSC2. In some embodiments, the allele frequency of mutated TSC1 or TSC2 is similar to or higher than a reference cancer gene in the tumor sample. In some embodiments, there is a second hit or loss of the other allele of mutated TSC1. In some embodiments, there is a mutation occurring in the last nucleotide position of an exon (i.e., 3’ end of an exon, e.g., a G).

[0086] In some embodiments, the inactivating mutation in TSC1 or TSC2 comprises an in- frame deletion mutation in TSC1 or TSC2. In some embodiments, the in-frame deletion mutation has been reported in the LOVD database (e.g., <https: / / databases.lovd.nl / shared / genes / TSC2>). In some embodiments, the in-frame deletion mutation in TSC1 or TSC2 deletes a size of more than one amino acids.

[0087] In some embodiments, the inactivating mutation comprises a missense mutation in TSC1. In some embodiments, the missense mutation in TSC1 comprises a non-conservative substitution within amino acids 34-224 or exons 4-8 of TSC1.

[0088] In some embodiments, the inactivating mutation comprises a missense mutation in TSC2. In some embodiments, the missense mutation in TSC2 comprises a non-conservative substitution and / or has been reported in the LOVD database (<https: / / databases.lovd.nl / shared / genes / TSC2>).

[0089] In some embodiments, the inactivating mutation in TSC1 or TSC2 comprises a homozygous deletion mutation. In some embodiments, the homozygous deletion mutation affects one or more exons of TSC1 or TSC2.

[0090] In some embodiments, there is provided a method of assessing if an inactivating mutation in TSC1 or TSC2 is pathogenic, comprising determining if the mutation is i) a nonsense mutation, an out-of-frame insertion, a deletion mutation, or a mutation that affects canonical splice site in TSC1 or TSC2, ii) an in-frame deletion mutation in TSC1 or TSC2, iii) a missense mutation in TSC1 or TSC2, or iv) a homozygous deletion in TSC1 or TSC2.sf-6235590Attorney Reference: 638772023240

[0091] In some embodiments, the mutation in TSC1 or TSC2 is a nonsense mutation, an out- of-frame insertion, a deletion mutation, or a mutation that affects canonical splice site in TSC1 or TSC2, and the method further comprises determining if: a) the allele frequency of mutated TSC1 or TSC2 is similar to or higher than a reference cancer gene in the tumor sample, b) there is a second hit or loss of the other allele of mutated TSC1, or c) there is a mutation occurring in the last nucleotide position of an exon (i.e., 3’ end of an exon, e.g., a G); wherein the method further comprises determining that the mutation is a pathogenic mutation if the answer to any of a)-c) above is yes.

[0092] In some embodiments, the mutation in TSC1 or TSC2 is a nonsense mutation, an out- of-frame insertion, a deletion mutation, or a mutation that affects canonical splice site in TSC1 or TSC2, and the method further comprises determining if: a) the allele frequency of mutated TSC1 or TSC2 is significantly lower (e.g., at least about 10%, 20%, 30%, 40%, 50% lower) than a reference cancer gene examined in the tumor sample, b) the mutation is in 3’ half of exon 22 and all of exon 23 of TSC1; c) the mutation affects i) amino acids 947-989 of exon 26 of TSC2 or ii) amino acids 1272-1295 of exon 32 of TSC2, or d) the individual has a tumor mutation burden of more than 10 / Mb; wherein the method further comprises determining that the mutation is not pathogenic if the answer is yes to any of a) - d) above is yes.

[0093] In some embodiments, the mutation in TSC1 or TSC2 is an in-frame deletion mutation in TSC1 or TSC2, and the method further comprises determining if: a) the deletion mutation is previously seen and / or reported in LOVD database (e.g.., <https: / / databases.lovd.nl / shared / genes / TSC2>); or b) the if the deletion mutation comprises a deletion of size more than one amino acid; wherein the method further comprises determining that the mutation is pathogenic if the answer is yes to a) or b).

[0094] In some embodiments, the mutation in TSC1 or TSC2 is an in-frame deletion mutation in TSC1 or TSC2, and the method further comprises determining if a) the deletion mutation affects a single amino acid and b) the deletion mutation has not been reported in LOVD database (e.g., <https: / / databases.lovd.nl / shared / genes / TSC2>); and the method further comprises determining that the mutation is not pathogenic if the answer is yes to both a) and b).

[0095] In some embodiments, the mutation is a missense mutation in TSC1, and the method further comprises determining if a) the missense mutation comprises a mutation in amino acidssf-6235590Attorney Reference: 638772023240missense mutation comprises a mutation in amino acids 34-224 of exons 4-8 of TSC1 and the mutation is a conservative substitute (e.g., L->V), wherein the method further comprises determining that 1) the mutation is pathogenic if answer is yes to a), or 2) the mutation is not pathogenic if the answer is yes to b).

[0096] In some embodiments, the mutation is a missense mutation in TSC2, and the method further comprises determining if a) the missense mutation is a non-conservative substitution and / or is confirmed in LOVD database, b) the missense mutation is a conservative substitution; wherein optionally the method further comprises determining that 1) the mutation is pathogenic if answer is yes to a), or 2) the mutation is not pathogenic if the answer is yes to b).

[0097] In some embodiments, the mutation is a homozygous deletion in TSC1 or TSC2, wherein the method further comprises determining if the homozygous deletion affects one or more than one exons, wherein optionally the method further comprises determining that the mutation is pathogenic if answer is yes to the above question.

[0098] TSC2 is also known as Tuberin, Tuberous sclerosis 2 protein, protein phosphatase 1 regulatory subunit 160, TSC4, PPP1R160, and LAM. TSC2 protein functions as part of a complex with TSC1 by negatively regulating mTORC1 signaling. In some embodiments, the nucleic acid sequence of a wildtype TSC2 gene is identified by the Genbank accession number NC_ 000016.10, from nucleotide 2047936 to nucleotide 2088712 on the forward strand of chromosome 16 according to the GRCh38.p2 assembly of the human genome. The wildtype TSC2 gene comprises 42 exons. A mutation of the TSC2 gene may occur in any one or any combination of the 42 exons, or in any intron or noncoding regions of the TSC2 gene.

[0099] In some embodiments, the amino acid sequence of a wildtype TSC2 protein is identified by the Genbank accession number NP_ 000539.2. In some embodiments, the amino acid sequence of a wildtype TSC2 protein is identified by the Genbank accession number NP_001070651.1. In some embodiments, the amino acid sequence of a wildtype TSC2 protein is identified by the Genbank accession number NP_001107854.1.

[0100] In some embodiments, the nucleic acid sequence of a cDNA encoding a wildtype TSC2 protein is identified by the Genbank accession number NM_000548.3. In some embodiments, the nucleic acid sequence of a cDNA encoding a wildtype TSC2 protein issf-6235590Attorney Reference: 638772023240 identified by the Genbank accession number NM_001077183.1. In some embodiments, the nucleic acid sequence of a cDNA encoding a wildtype TSC2 protein is identified by the Genbank accession number NM_001114382.1.

[0101] In some embodiments, the mutation is a two-point mutation (i.e., bi-allelic mutations). In some embodiments, the mutation comprises three-point mutation or four-point mutation. In some embodiments, the mutation at TSC2 is a loss of function mutation. In some embodiments, the mutation at TSC2 comprises a homozygous deletion. In some embodiments, the mutation at TSC2 comprises a copy number variation of TSC2. In some embodiments, the mutation at TSC2 comprises an aberrant expression level of TSC2. In some embodiments, the mutation at TSC2 comprises an aberrant activity level of a protein encoded by TSC2.

[0102] In some embodiments, the individual has a mutation (e.g., inactivating mutation) in any one or more of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44 according to Genbank accession number NM_000548. In some embodiments, the individual has bi-allelic mutations (e.g., bi-allelic inactivating mutation) in two of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44 according to Genbank accession number NM_000548. In some embodiments, the individual has an inactivating mutation in any of exons 18, 22, 27, 30, and 42 of TSC2. In some embodiments, the individual has bi-allelic mutations in any two of exons 18, 22, 27, 30, and 42 of TSC2. In some embodiments, the individual has bi-allelic mutations in exons 18 and 30 of TSC2. In some embodiments, the individual has bi-allelic mutations in exons 22 and 27 of TSC2.

[0103] In some embodiments, the mutation is not within amino acids 947-989 or exon 26. In some embodiments, the mutation is not within amino acids 1272-1295 or exon 32.

[0104] In some embodiments, the mutation comprises a non-conservative substitution.

[0105] In some embodiments, the mutation has been reported by the LOVD database (https: / / databases.lovd.nl / shared / genes / TSC2 ).

[0106] TSC1 and TSC2 gene mutations were described in e.g., Rosset et al., Genetics and Molecular Biolegy, 40, 1, 69-79 (2017), which is incorporated herein by its entirety. In somesf-6235590Attorney Reference: 638772023240 embodiments, the individual has a continuous deletion (e.g., TSC2-PKD1 deletion). See e.g., Boronat et al., Brain Dev. 36:801-806. In some embodiments, the individual has a c.5238-5255 del in TSC2. See e.g., Rok et al. Med Sci Monit 11:230-234. In some embodiments, the individual has a proximal region mutation (e.g., in any of exons 1-22) and / or a distal region mutation (e.g., in any of exons 23-41). See e.g., van Eeghena et al. Epilepsy Res 103:83-87.

[0107] TSC1 is also known as Hamartin, Tuberous sclerosis 1 protein, TSC, KIAA0243, and LAM. TSC1 protein functions as part of a complex with TSC2 by negatively regulating mTORC1 signaling. In some embodiments, the nucleic acid sequence of a wildtype TSC1 gene is identified by the Genbank accession number NC_ 000009.12, from nucleotide 132891348 to nucleotide 132945370 on the reverse strand of chromosome 9 according to the GRCh38.p2 assembly of the human genome. The wildtype TSC1 gene comprises 25 exons. A mutation of the TSC1 gene may occur in any one or any combination of the 25 exons, or in any intron or noncoding regions of the TSC1 gene.

[0108] In some embodiments, the amino acid sequence of a wildtype TSC1 protein is identified by the Genbank accession number NP_ 000359.1. In some embodiments, the amino acid sequence of a wildtype TSC1 protein is identified by the Genbank accession number NP_ 001155898.1. In some embodiments, the amino acid sequence of a wildtype TSC1 protein is identified by the Genbank accession number NP_ 001155899.1.

[0109] In some embodiments, the nucleic acid sequence of a cDNA encoding a wildtype TSC1 protein is identified by the Genbank accession number NM_000368.4. In some embodiments, the nucleic acid sequence of a cDNA encoding a wildtype TSC1 protein is identified by the Genbank accession number NM_001162426.1. In some embodiments, the nucleic acid sequence of a cDNA encoding a wildtype TSC1 protein is identified by the Genbank accession number NM_001162427.1.

[0110] In some embodiments, the individual is selected for treatment on the basis of having an aberration at TSC1. In some embodiments, the aberration at TSC1 comprises a mutation (e.g., an inactivating mutation) in TSC1. In some embodiments, the mutation is selected from the group consisting of a splice site mutation, a nonsense mutation, a frameshift mutation, a missense mutation and a loss or deletion of the gene. In some embodiments, the mutation at TSC1 comprises a single-nucleotide variant (SNV). In some embodiments, the mutation is a two-sf-6235590Attorney Reference: 638772023240 point mutation. In some embodiments, the mutation at TSC1 is a loss of function mutation. In some embodiments, the mutation at TSC1 comprises a homozygous deletion. In some embodiments, the mutation at TSC1 comprises a copy number variation of TSC1. In some embodiments, the mutation at TSC1 comprises an aberrant expression level of TSC1. In some embodiments, the mutation at TSC1 comprises an aberrant activity level of a protein encoded by TSC1.

[0111] In some embodiments, the individual has a mutation (e.g., inactivating mutation) in any one or more of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 according to Genbank accession number NM_000368. In some embodiments, the individual has bi-allelic mutations (e.g., bi-allelic inactivating mutation) in two of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 according to Genbank accession number NM_000368. In some embodiments, the mutation is not in exon 23. In some embodiments, the mutation is not in 3’ half of exon 22.

[0112] In some embodiments, the mutation comprises a non-conservative substitution.

[0113] In some embodiments, the mutation has been reported by the LOVD database (<https: / / databases.lovd.nl / shared / genes / TSC1>).

[0114] In some embodiments, the individual has a TSC1 loss or deletion. E. Dosing and Methods of Administration

[0115] The dose of the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) administered to an individual (e.g., a human) may vary with the particular composition, the method of administration, the cancer being treated, and the particular stage or size of tumor being treated. The amount should be sufficient to produce a desirable response, such as a therapeutic or prophylactic response against the tumor. In some embodiments, the amount of mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) in the composition is below the level that induces a toxicological effect (e.g., an effect above a clinically acceptable level of toxicity) or is at a level where a potential side effect can be controlled or tolerated when the mTOR inhibitor nanoparticle composition is administered to the individual.sf-6235590Attorney Reference: 638772023240

[0116] As described herein, in some embodiments, reference to amounts of a composition comprising nanoparticles comprising an mTOR inhibitor and an albumin are based on the amount of the mTOR inhibitor therein. In some embodiments, the amount of an mTOR inhibitor (such as a limus drug, e.g., sirolimus) in the mTOR inhibitor nanoparticle composition is about any of 10 mg / m2, 15 mg / m2, 20 mg / m2, 25 mg / m2, 30 mg / m2, 45 mg / m2, 50 mg / m2, 56 mg / m2, 60 mg / m2, 75 mg / m2, 80 mg / m2, 90 mg / m2, 100 mg / m2, 120 mg / m2, 160 mg / m2, 175 mg / m2, 180 mg / m2, 200 mg / m2, 210 mg / m2, 220 mg / m2, 250 mg / m2, 260 mg / m2, 300 mg / m2, 350 mg / m2, 400 mg / m2, 500 mg / m2, 540 mg / m2, 750 mg / m2, 1000 mg / m2, or 1080 mg / m2mTOR inhibitor. In some embodiments, the mTOR inhibitor nanoparticle composition includes less than about any of 350 mg / m2, 300 mg / m2, 250 mg / m2, 200 mg / m2, 150 mg / m2, 120 mg / m2, 100 mg / m2, 90 mg / m2, 50 mg / m2, or 30 mg / m2mTOR inhibitor (such as a limus drug, e.g., sirolimus). In some embodiments, the amount of the mTOR inhibitor (such as a limus drug, e.g., sirolimus) per administration is less than about any of 40 mg / m2, 39 mg / m2, 38 mg / m2, 37 mg / m2, 36 mg / m2, 35 mg / m2, 34 mg / m2, 33 mg / m2, 32 mg / m2, 31 mg / m2, 30 mg / m2, 29 mg / m2, 28 mg / m2, 27 mg / m2, 26 mg / m2, 25 mg / m2, 24 mg / m2, 23 mg / m2, 22 mg / m2, 21 mg / m2, 20 mg / m2, 19 mg / m2, 18 mg / m2, 17 mg / m2, 16 mg / m2, 15 mg / m2, 14 mg / m2, 13 mg / m2, 12 mg / m2, 11 mg / m2, or 10 mg / m2. In some embodiments, the mTOR inhibitor (such as a limus drug, e.g., sirolimus) in the mTOR inhibitor nanoparticle composition is included 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 125 to about150 mg / m2, about150 to about 175 mg / m2, about175 to about 200 mg / m2, about 200 to about 225 mg / m2, about 225 to about 250 mg / m2, about 250 to about 300 mg / m2, about 300 to about 350 mg / m2, or about 350 to about 400 mg / m2. In some embodiments, the mTOR inhibitor (such as a limus drug, e.g., sirolimus) in the mTOR inhibitor nanoparticle composition is about 30 to about 300 mg / m2, such as about 100 to about 150 mg / m2, about 120 mg / m2, about 130 mg / m2, or about 140 mg / m2. In some embodiments, the amount of the mTOR inhibitor nanoparticle composition is administered every four weeks (e.g., day 1 of a 28-day cycle). In some embodiments, the amount of the mTOR inhibitor nanoparticle composition is administered every three weeks (e.g., day 1 of a 21-day cycle). In some embodiments, the amount of the mTOR inhibitor nanoparticle composition is administered every two weeks (e.g., day 1 of a 14-day cycle). In some embodiments, the amount of the mTOR inhibitor nanoparticle 27sf-6235590Attorney Reference: 638772023240 composition is administered weekly. In some embodiments, the amount of the mTOR inhibitor nanoparticle composition is administered weekly every 2 out of 3 weeks. In some embodiments, the amount of the mTOR inhibitor nanoparticle composition is on days 8 and 15 of a 21-day cycle, days 1 or 8 of a 21-day cycle, days 15 and 21 or a 21-day cycle, days 1 and 15 of a 21-day cycle, or days 1 and 21 of a 21-day cycle.

[0117] In some embodiments, the dosing frequencies for the administration of the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) include, but are not limited to, daily, every two days, every three days, every four days, every five days, every six days, weekly without break, three out of four weeks (such as on days 1, 8, and 15 of a 28-day cycle), once every three weeks, once every two weeks, or two out of three weeks. In some embodiments, the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) is administered about 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 mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) is administered at least about any of 1x, 2x, 3x, 4x, 5x, 6x, or 7x (i.e., daily) a week. In some embodiments, the intervals between each administration are less than about any of 6 months, 3 months, 1 month, 20 days, 15, days, 14 days, 13 days, 12 days, 11 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 administration are more than about any of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, or 12 months. In some embodiments, there is no break in the dosing schedule. In some embodiments, the interval between each administration is no more than about a week.

[0118] In some embodiments, the dosing frequency is once every two days for one time, two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or eleven times. In some embodiments, the dosing frequency is once every two days for five times. In some embodiments, the mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) is administered over a period of at least ten days, wherein the interval between each administration is no more than about two days, and wherein the dose of the mTOR inhibitor at each administration is about 0.25 mg / m2to about 250 mg / m2, about 0.25 mg / m2tosf-6235590Attorney Reference: 638772023240 about 150 mg / m2, about 0.25 mg / m2to about 75 mg / m2, such as about 0.25 mg / m2to about 25 mg / m2, or about 25 mg / m2to about 50 mg / m2.

[0119] The administration of the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) can be extended over an extended period of time, such as from about a month up to about seven years. In some embodiments, the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) is administered over a period of at least about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 48, 60, 72, or 84 months.

[0120] In some embodiments, the dosage of an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) in a nanoparticle composition can be in the range of 5-400 mg / m2when given on a 3-week schedule, or 5-250 mg / m2(such as 80-150 mg / m2, for example 100-120 mg / m2) when given on a weekly schedule. For example, the amount of an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) is about 60 to about 300 mg / m2(e.g., about 260 mg / m2) on a 3-week schedule.

[0121] In some embodiments, the exemplary dosing schedules for the administration of the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) include, but are not limited to, 100 mg / m2, weekly, without break; 10 mg / m2weekly, 3 out of four weeks (such as on days 1, 8, and 15 of a 28-day cycle); 45 mg / m2weekly, 3 out of four weeks (such as on days 1, 8, and 15 of a 28-day cycle); 75 mg / m2weekly, 3 out of four weeks (such as on days 1, 8, and 15 of a 28-day cycle); 100 mg / m2,weekly, 3 out of 4 weeks; 125 mg / m2, weekly, 3 out of 4 weeks; 125 mg / m2, weekly, 2 out of 3 weeks; 130 mg / m2, weekly, without break; 175 mg / m2, once every 2 weeks; 260 mg / m2, once every 2 weeks; 260 mg / m2, once every 3 weeks; 180-300 mg / m2, every three weeks; 60-175 mg / m2, weekly, without break; 20-150 mg / m2twice a week; and 150-250 mg / m2twice a week. The dosing frequency of the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) may be adjusted over the course of the treatment based on the judgment of the administering physician.

[0122] In some embodiments, the individual is treated for at least about any of one, two, three, four, five, six, seven, eight, nine, or ten treatment cycles.sf-6235590Attorney Reference: 638772023240

[0123] The mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) described herein allow infusion of the mTOR inhibitor nanoparticle composition to an individual over an infusion time that is shorter than about 24 hours. For example, in some embodiments, the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) is administered over an infusion period of less than about any of 24 hours, 12 hours, 8 hours, 5 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, or 10 minutes. In some embodiments, the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) is administered over an infusion period of about 30 minutes.

[0124] In some embodiments, the exemplary dose of the mTOR inhibitor (in some embodiments a limus drug, e.g., sirolimus) in the mTOR inhibitor nanoparticle composition includes, but is not limited to, about any of 10 mg / m2, 20 mg / m2, 30 mg / m2, 35 mg / m2, 40 mg / m2, 45 mg / m2, 50 mg / m2, 56 mg / m2, 60 mg / m2, 75 mg / m2, 80 mg / m2, 90 mg / m2, 100 mg / m2, 120 mg / m2, 160 mg / m2, 175 mg / m2, 200 mg / m2, 210 mg / m2, 220 mg / m2, 260 mg / m2, and 300 mg / m2. For example, the dosage of an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) in a nanoparticle composition can be in the range of about 20- 400 mg / m2when given on a 3-week schedule, or about 10-250 mg / m2when given on a weekly schedule.

[0125] In some embodiments, the dosage of an mTOR inhibitor (such as a limus drug, e.g., sirolimus) is about 100 mg to about 400 mg, for example about 100 mg, about 200 mg, about 300 mg, or about 400 mg. In some embodiments, the limus drug is administered at about 100 mg weekly, about 200 mg weekly, about 300 mg weekly, about 100 mg twice weekly, or about 200 mg twice weekly. In some embodiments, the administration is further followed by a monthly maintenance dose (which can be the same or different from the weekly doses).

[0126] In some embodiments when the mTOR nanoparticle composition is administered intravenously, the dosage of an mTOR inhibitor (such as a limus drug, e.g., sirolimus) in a nanoparticle composition can be in the range of about 30 mg to about 400 mg. The mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) described herein allow infusion of the mTOR inhibitor nanoparticle composition to an individual over an infusion time that is shorter than about 24 hours. For example, in some embodiments,sf-6235590Attorney Reference: 638772023240 the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) is administered over an infusion period of less than about any of 24 hours, 12 hours, 8 hours, 5 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, or 10 minutes. In some embodiments, the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) is administered over an infusion period of about 30 minutes to about 40 minutes.

[0127] An mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) in pure form or in an appropriate pharmaceutical composition, can be administered via any of the accepted modes of administration or agents known in the art. The compositions and / or agents can be administered, for example, parenterally (such as intravenous). The dosage form can be, for example, a solid, semi-solid, lyophilized powder, or liquid dosage form, such as tablets, pills, soft elastic or hard gelatin capsules, powders, solutions, suspensions, suppositories, aerosols, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.

[0128] As discussed herein, the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) can be administered in a single unit dose or separate dosage forms. Auxiliary and adjuvant agents may include, for example, preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents. Prevention of the action of microorganisms is generally provided by various antibacterial and antifungal agents, such as, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, such as sugars, sodium chloride, and the like, may also be included. Prolonged absorption of an injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. The auxiliary agents also can include wetting agents, emulsifying agents, pH buffering agents, and antioxidants, such as citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, and the like.

[0129] Solid dosage forms can be prepared with coatings and shells, such as enteric coatings and others well-known in the art. They can contain pacifying agents and can be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedded compositions that can be used aresf-6235590Attorney Reference: 638772023240 polymeric substances and waxes. The active compounds also can be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0130] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Such dosage forms are prepared, for example, by dissolving, or dispersing, the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) described herein, or a pharmaceutically acceptable salt thereof, and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like; solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethyl formamide; oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan; or mixtures of these substances, and the like, to thereby form a solution or suspension.

[0131] In some embodiments, depending on the intended mode of administration, the pharmaceutically acceptable compositions will contain about 1% to about 99% by weight of the compounds described herein, or a pharmaceutically acceptable salt thereof, and 99% to 1% by weight of a pharmaceutically acceptable excipient. In one example, the composition will be between about 5% and about 75% by weight of a compound described herein, or a pharmaceutically acceptable salt thereof, with the rest being suitable pharmaceutical excipients.

[0132] Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art. Reference is made, for example, to Remington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company, Easton, Pa., 1990).

[0133] The mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) can be administered to an individual (such as a human) via various routes, including, for example, via intravenous administration. In some embodiments, sustained continuous release formulation of the composition may be used. In some embodiments, the composition is administered intravenously.sf-6235590Attorney Reference: 638772023240 F. Further steps of the described methods

[0134] As described herein, the taught methods reduce tumor volume such that a treated induvial who was previously not a candidate for surgical resection of the cancer can now receive such a surgery.

[0135] Thus, in certain aspects, the methods described herein further comprise surgically resecting the tumor following administering to an individual a composition comprising nanoparticles comprising sirolimus and an albumin. III. Compositions comprising nanoparticles comprising an mTOR inhibitor

[0136] The mTOR inhibitor nanoparticle compositions described herein comprise nanoparticles comprising (in various embodiments consisting essentially of or consisting of) an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) and an albumin (such as human serum albumin). It is noted that the terms sirolimus and rapamycin are used interchangeably herein. Nanoparticles of poorly water soluble drugs (such as macrolides) have been disclosed in, for example, U. S. Pat. Nos.5,916,596; 6,506,405; 6,749,868, 6,537,579, 7,820,788, and 8,911,786, 11,497,737, and also in U. S. Pat. Pub. Nos. 2006 / 0263434, and 2007 / 0082838; PCT Patent Application W008 / 137148, U.S. Patent Application No.: 62 / 927,047, each of which is incorporated herein by reference in their entirety.

[0137] Albumin-based nanoparticle compositions have been developed as a drug delivery system for delivering substantially water insoluble drugs. See, for example, U. S. Pat. Nos.5,916,596; 6,506,405; 6,749,868, and 6,537,579, 7,820,788, and 7,923,536. Abraxane®, an albumin stabilized nanoparticle formulation of paclitaxel, was approved in the United States in 2005 and subsequently in various other countries for treating metastatic breast cancer. It was recently approved for treating non-small cell lung cancer in the United States, and has also shown therapeutic efficacy in various clinical trials for treating difficult-to-treat cancers such as bladder cancer and melanoma. Albumin derived from human blood has been used for the manufacture of Abraxane® as well as various other albumin-based nanoparticle compositions. Albumin-based nanoparticle composition comprising sirolimus, e.g., nab-sirolimus or Fyarrao®, are known, e.g., US. Pat. No. 8,911,786 and US Pat. No. 11,497,737.sf-6235590Attorney Reference: 638772023240

[0138] In some embodiments, the composition comprises nanoparticles with an average or mean diameter of no greater than about 1000 nanometers (nm), such as no greater than about any of 900, 800, 700, 600, 500, 400, 300, 200, and 100 nm. In some embodiments, the average or mean diameters of the nanoparticles is no greater than about 200 nm. In some embodiments, the average or mean diameters of the nanoparticles is no greater than about 150 nm. In some embodiments, the average or mean diameters of the nanoparticles is no greater than about 100 nm. In some embodiments, the average or mean diameter of the nanoparticles is about 10 to about 400 nm. In some embodiments, the average or mean diameter of the nanoparticles is about 10 to about 150 nm. In some embodiments, the average or mean diameter of the nanoparticles is about 40 to about 120 nm. In some embodiments, the average or mean diameter of the nanoparticles are no less than about 50 nm. In some embodiments, the nanoparticles are sterile- filterable.

[0139] Methods of determining average particle sizes are known in the art, for example, dynamic light scattering (DLS) has been routinely used in determining the size of submicrometre-sized particles based. International Standard ISO22412 Particle Size Analysis – Dynamic Light Scattering, International Organisation for Standardisation (ISO) 2008 and Dynamic Light Scattering Common Terms Defined, Malvern Instruments Limited, 2011. In some embodiments, the particle size is measured as the volume-weighted mean particle size (Dv50) of the nanoparticles in the composition.

[0140] In some embodiments, the albumin has sulfhydryl groups that can form disulfide bonds. In some embodiments, at least about 5% (including for example at least about any one of 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the albumin in the nanoparticle portion of the composition are crosslinked (for example crosslinked through one or more disulfide bonds).

[0141] In some embodiments, the composition comprises an mTOR inhibitor (such as a limus drug, e.g., rapamycin or a derivative thereof) in both nanoparticle and non-nanoparticle forms (e.g., in the form of solutions or in the form of soluble albumin / nanoparticle complexes), wherein at least about any one of 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the mTOR inhibitor in the composition are in nanoparticle form. In some embodiments, the mTOR inhibitor (such as a limus drug, e.g., rapamycin or a derivative thereof) in the nanoparticles constitutessf-6235590Attorney Reference: 638772023240 more than about any one of 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the nanoparticles by weight. In some embodiments, the nanoparticles have a non-polymeric matrix. In some embodiments, the nanoparticles comprise a core of an mTOR inhibitor (such as a limus drug, e.g., rapamycin or a derivative thereof) that is substantially free of polymeric materials (such as polymeric matrix).

[0142] In some embodiments, the composition comprises an albumin in both nanoparticle and non-nanoparticle portions of the composition, wherein at least about any one of 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the albumin in the composition are in non-nanoparticle portion of the composition.

[0143] In some embodiments, the weight ratio of the albumin to the mTOR inhibitor (such as a limus drug, e.g., rapamycin or a derivative thereof) in the mTOR inhibitor nanoparticle composition is such that a sufficient amount of mTOR inhibitor binds to, or is transported by, the cell. While the weight ratio of an albumin to an mTOR inhibitor (such as a limus drug, e.g., rapamycin or a derivative thereof) will have to be optimized for different albumin and mTOR inhibitor combinations, generally the weight ratio of an albumin to an mTOR inhibitor (such as a limus drug, e.g., rapamycin or a derivative thereof) (w / w) is about 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 about 9:1. In some embodiments, the albumin to mTOR inhibitor (such as a limus drug, e.g., rapamycin or a derivative thereof) weight ratio is about any of 18:1 or less, 15:1 or less, 14:1 or less, 13:1 or less, 12:1 or less, 11:1 or less, 10:1 or less, 9:1 or less, 8:1 or less, 7:1 or less, 6:1 or less, 5:1 or less, 4:1 or less, and 3:1 or less. In some embodiments, the weight ratio of the albumin (such as human albumin or human serum albumin) to the mTOR inhibitor (such as a limus drug, e.g., rapamycin or a derivative thereof) in the composition is any one 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.

[0144] In some embodiments, the composition comprises nanoparticles comprising an mTOR inhibitor and an albumin, wherein the weight ratio of the albumin to the mTOR inhibitorsf-6235590Attorney Reference: 638772023240 in the composition is about 0.01:1 to about 100:1. In some embodiments, the composition comprises nanoparticles comprising an mTOR inhibitor (such as rapamycin) and an albumin, wherein the weight ratio of the albumin to the mTOR inhibitor (such as rapamycin) in the composition is about 18:1 or less (including for example any of 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, and about 9:1). In some embodiments, the composition comprises nanoparticles comprising rapamycin, or a derivative thereof, and an albumin, wherein the weight ratio of the albumin to the rapamycin or derivative thereof in the composition is about 18:1 or less (including for example any of 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, and about 9:1).

[0145] In some embodiments, the mTOR inhibitor nanoparticle composition (such as rapamycin / albumin nanoparticle composition) comprises one or more of the above characteristics.

[0146] The nanoparticles described herein may be present in a dry formulation (such as 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 solutions of amino acids, optionally buffered solutions of proteins, optionally buffered solutions of sugars, optionally buffered solutions of vitamins, optionally buffered solutions of synthetic polymers, lipid-containing emulsions, and the like.

[0147] In some embodiments, the pharmaceutically acceptable carrier comprises an albumin (such as human albumin or human serum albumin). The albumin may either be natural in origin or synthetically prepared. In some embodiments, the albumin is human albumin or human serum albumin. In some embodiments, the albumin is a recombinant albumin.

[0148] Human serum albumin (HSA) is a highly soluble globular protein of Mr 65K and consists of 585 amino acids. HSA is the most abundant protein in the plasma and accounts for 70-80 % of the colloid osmotic pressure of human plasma. The amino acid sequence of HSA contains a total of 17 disulfide bridges, one free thiol (Cys 34), and a single tryptophan (Trp 214). Intravenous use of HSA solution has been indicated for the prevention and treatment of hypovolemic shock (see, e.g., Tullis, JAMA, 237: 355-360, 460-463, (1977)) and Houser et al., Surgery, Gynecology and Obstetrics, 150: 811-816 (1980)) and in conjunction with exchangesf-6235590Attorney Reference: 638772023240 transfusion in the treatment of neonatal hyperbilirubinemia (see, e.g., Finlayson, Seminars in Thrombosis and Hemostasis, 6, 85-120, (1980)). Other albumins are contemplated, such as bovine serum albumin. Use of such non-human albumins could be appropriate, for example, in the context of use of these compositions in non-human mammals, such as the veterinary (including domestic pets and agricultural context). Human serum albumin (HSA) has multiple hydrophobic binding sites (a total of eight for fatty acids, an endogenous ligand of HSA) and binds a diverse set of drugs, especially neutral and negatively charged hydrophobic compounds (Goodman et al., The Pharmacological Basis of Therapeutics, 9thed, McGraw-Hill New York (1996)). Two high affinity binding sites have been proposed in subdomains IIA and IIIA of HSA, which are highly elongated hydrophobic pockets with charged lysine and arginine residues near the surface which function as attachment points for polar ligand features (see, e.g., 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)). Rapamycin and propofol have been shown to bind HSA (see, e.g., Paal et al., Eur. J. Biochem., 268(7), 2187-91 (200a), Purcell et al., Biochem. 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, e.g., Urien et al., Invest. New Drugs, 14(b), 147-51 (1996)).

[0149] An mTOR inhibitor (such as a limus drug, e.g., rapamycin or a derivative thereof) is “stabilized” in an aqueous suspension if it remains suspended in an aqueous medium (such as without visible precipitation or sedimentation) for an extended period of time, such as for at least about any of 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. The suspension is generally, but not necessarily, suitable for administration to an individual (such as a human). Stability of the suspension is generally (but not necessarily) evaluated at a storage temperature (such as room temperature (such as 20-25 ºC) or refrigerated conditions (such as 4 ºC)). For example, a suspension is stable at a storage temperature if it exhibits no flocculation or particle agglomeration visible to the naked eye or when viewed using an optical microscope at 1000 times, at about fifteen minutes after preparation of the suspension. Stability can also besf-6235590Attorney Reference: 638772023240 evaluated under accelerated testing conditions, such as at a temperature that is about 40 ºC or higher.

[0150] The compositions described herein may be a stable aqueous suspension of the mTOR inhibitor, such as a stable aqueous suspension of the mTOR inhibitor at a concentration of any of about 0.1 to about 200 mg / ml, about 0.1 to about 150 mg / ml, about 0.1 to about 100 mg / ml, about 0.1 to about 50 mg / ml, about 0.1 to about 20 mg / ml, about 1 to about 10 mg / ml, about 2 mg / ml to about 8 mg / ml, about 4 to about 6 mg / ml, and about 5 mg / ml. In some embodiments, the concentration of the mTOR inhibitor is at least about any of 0.2 mg / ml, 1.3 mg / ml, 1.5 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 100 mg / ml, 150 mg / ml, or 200 mg / ml.

[0151] In some embodiments, the albumin is present in an amount that is sufficient to stabilize the mTOR inhibitor (such as a limus drug, e.g., rapamycin or a derivative thereof) in an aqueous suspension at a certain concentration. For example, the concentration of the mTOR inhibitor (such as a limus drug, e.g., rapamycin or a derivative thereof) in the composition is about 0.1 to about 100 mg / ml, including for example about any of 0.1 to about 50 mg / ml, about 0.1 to about 20 mg / ml, about 1 to about 10 mg / ml, about 2 mg / ml 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 limus drug, e.g., rapamycin or a derivative thereof) is at least about any of 1.3 mg / ml, 1.5 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 40 mg / ml, and 50 mg / ml. In some embodiments, the albumin is present in an amount that avoids use of surfactants (such as Cremophor), so that the composition is free or substantially free of surfactant (such as Cremophor).

[0152] In some embodiments, the composition, in liquid form, comprises from about 0.1% to about 50% (w / v) (e.g., about 0.5% (w / v), about 5% (w / v), about 10% (w / v), about 15% (w / v), about 20% (w / v), about 30% (w / v), about 40% (w / v), or about 50% (w / v)) of an albumin. In some embodiments, the composition, in liquid form, comprises about 0.5% to about 5% (w / v) of albumin.sf-6235590Attorney Reference: 638772023240

[0153] In some embodiments, the albumin allows the composition to be administered to an individual (such as a human) without 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 administration of the mTOR inhibitor (such as a limus drug, e.g., rapamycin or a derivative thereof) to a human. The term “reducing one or more side effects” of administration of the mTOR inhibitor (such as a limus drug, e.g., rapamycin or a derivative thereof) refers to reduction, alleviation, elimination, or avoidance of one or more undesirable effects caused by the mTOR inhibitor, as well as side effects caused by delivery vehicles (such as solvents that render the limus drugs suitable for injection) used to deliver the mTOR inhibitor. Such side effects include, for example, myelosuppression, neurotoxicity, hypersensitivity, inflammation, venous irritation, phlebitis, pain, skin irritation, peripheral neuropathy, neutropenic fever, anaphylactic reaction, venous thrombosis, extravasation, and combinations thereof. These side effects, however, are merely exemplary and other side effects, or combination of side effects, associated with limus drugs (such as a limus drug, e.g., rapamycin or a derivative thereof) can be reduced.

[0154] In some embodiments, the composition is a dry (such as lyophilized) composition that can be reconstituted, resuspended, or rehydrated to form generally a stable aqueous suspension of the nanoparticles comprising an mTOR inhibitor and an albumin. In some embodiments, the composition is a liquid (such as aqueous) composition obtained by reconstituting or resuspending a dry composition. In some embodiments, the composition is an intermediate liquid (such as aqueous) composition that can be dried (such as lyophilized). A. mTOR inhibitors

[0155] The methods described herein in some embodiments comprise administration of nanoparticle compositions of mTOR inhibitors. “mTOR inhibitor” used herein refers to an inhibitor of mTOR. mTOR is a serine / threonine-specific protein kinase downstream of the phosphatidylinositol 3-kinase (PI3K) / Akt (protein kinase B) pathway, and a key regulator of cell survival, proliferation, stress, and metabolism. mTOR pathway dysregulation has been found in many human carcinomas, and mTOR inhibition produced substantial inhibitory effects on tumor progression.sf-6235590Attorney Reference: 638772023240

[0156] The mammalian target of rapamycin (mTOR) (also known as mechanistic target of rapamycin or FK506 binding protein 12-rapamycin associated protein 1 (FRAP1)) is an atypical serine / threonine protein kinase that is present in two distinct complexes, mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). mTORC1 is composed of mTOR, regulatory- associated protein of mTOR (Raptor), mammalian lethal with SEC13 protein 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 major signal inputs: nutrients (such as amino acids and phosphatidic acid), growth factors (insulin), energy and stress (such as hypoxia and DNA damage). Amino acid availability is signaled to mTORC1 via a pathway involving the Rag and Ragulator (LAMTOR1-3) Growth factors and hormones (e.g., insulin) signal to mTORC1 via Akt, which inactivates TSC2 to prevent inhibition of mTORC1. Alternatively, low ATP levels lead to the AMPK-dependent activation of TSC2 and phosphorylation of raptor to reduce mTORC1 signaling proteins.

[0157] Active mTORC1 has a number of downstream biological effects including translation of mRNA via the phosphorylation of downstream targets (4E-BP1 and p70 S6 Kinase), suppression of autophagy (Atg13, ULK1), ribosome biogenesis, and activation of transcription 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.

[0158] mTORC2 is composed of mTOR, rapamycin-insensitive companion of mTOR (RICTOR), GβL, and mammalian stress-activated protein kinase interacting protein 1 (mSIN1). In contrast to mTORC1, for which many upstream signals and cellular functions have been defined (see above), relatively little is known about mTORC2 biology. mTORC2 regulates cytoskeletal organization through its stimulation of F-actin stress fibers, paxillin, RhoA, Rac1, Cdc42, and protein kinase C α (PKCα). It had been observed that knocking down mTORC2 components affects actin polymerization and perturbs 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 promoting protein kinase Cα (PKCα) phosphorylation, phosphorylation of paxillin and its relocalization to focal adhesions, and thesf-6235590Attorney Reference: 638772023240 GTP loading of RhoA and Rac1. The molecular mechanism by which mTORC2 regulates these processes has not been determined.

[0159] In some embodiments, the mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) is an inhibitor of mTORC1. In some embodiments, the mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) is an inhibitor of mTORC2. In some embodiments, the mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) is an inhibitor of both mTORC1 and mTORC2.

[0160] In some embodiments, the mTOR inhibitor is a limus drug, which includes sirolimus and its analogs. 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-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, the mTOR inhibitor is an mTOR kinase inhibitor, such as CC-115 or CC-223.

[0161] In some embodiments, the mTOR inhibitor is sirolimus. Sirolimus is macrolide antibiotic that complexes with FKBP-12 and inhibits the mTOR pathway by binding mTORC1.

[0162] 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), CC-115, CC-223, 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 ridaforolimus (also known as deforolimus).

[0163] BEZ235 (NVP-BEZ235) is an imidazoquilonine derivative that is an mTORC1 catalytic inhibitor (Roper J, et al. PLoS One, 2011, 6(9), e25132). Everolimus is the 40-O-(2- hydroxyethyl) derivative of sirolimus and binds the cyclophilin FKBP-12, and this complex also 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 the FK506-binding protein and prohibits the activation of mTOR when it resides in the mTORC1complex. PI-103 issf-6235590Attorney Reference: 638772023240 a small molecule that inhibits the activation of the rapamycin-sensitive (mTORC1) complex (Knight et al. (2006) Cell. 125: 733-47). KU-0063794 is a small molecule that inhibits the phosphorylation of mTORC1 at Ser2448 in a dose-dependent and time-dependent manner. INK 128, AZD2014, NVP-BGT226, CH5132799, WYE-687, and are each small molecule inhibitors of mTORC1. PF-04691502 inhibits mTORC1 activity. GDC-0980 is an orally bioavailable small molecule that inhibits Class I PI3 Kinase and TORC1. Torin 1 is a potent small molecule inhibitor of mTOR. WAY-600 is a potent, ATP-competitive and selective inhibitor of mTOR. WYE-125132 is an ATP-competitive small molecule inhibitor of mTORC1. GSK2126458 is an inhibitor of mTORC1. PKI-587 is a highly potent dual inhibitor of PI3Kα, PI3Kγ 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 IC50 of 22 nM and 65 nM, respectively. Palomid 529 is a small molecule inhibitor of mTORC1 that lacks affinity for ABCB1 / ABCG2 and has good brain penetration (Lin et al. (2013) Int J Cancer DOI: 10.1002 / ijc. 28126 (e-published ahead of print). PP242 is a selective mTOR inhibitor. XL765 is a dual inhibitor of mTOR / PI3k for mTOR, p110α, p110β, p110γ and p110δ. GSK1059615 is a novel and dual inhibitor of PI3Kα, PI3Kβ, PI3Kδ, PI3Kγ and mTOR. WYE-354 inhibits mTORC1 in HEK293 cells (0.2 μM–5 μM) and in HUVEC cells (10 nM-1μM). WYE-354 is a potent, specific, and ATP-competitive inhibitor of mTOR. Deforolimus (Ridaforolimus, AP23573, MK-8669) is a selective mTOR inhibitor. B. Other components in the Nanoparticle Composition

[0164] 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, in the veterinary context, domestic pets and agricultural animals. The following formulations and methods are merely exemplary and are in no way limiting. Formulations suitable for oral administration can consist of (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 of the active ingredient, as solids or granules, (c) suspensions in an appropriate liquid, and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin,sf-6235590Attorney Reference: 638772023240 colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising 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, such excipients as are known in the art.

[0165] 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 propylhydroxybenzoates, talc, magnesium stearate, and mineral oil. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.

[0166] Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation compatible with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Injectable formulations are preferred.

[0167] In some embodiments, the composition is formulated to have a pH range of about 4.5 to about 9.0, including for example pH ranges of about any of 5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0. In some embodiments, the pH of the composition is formulated to no less than about 6, including for example no less than about any of 6.5, 7, or 8 (such as about 8). The composition can also be made to be isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol.sf-6235590Attorney Reference: 638772023240 C. Albumin-based nanoparticle compositions of sirolimus

[0168] The methods described herein are particularly suitable for albumin-based nanoparticle compositions described herein in more details. The nanoparticle composition in some embodiments includes (a) nanoparticles that include rapamycin and albumin, and (b) a non-nanoparticle portion that includes rapamycin and albumin. As used herein, “in the nanoparticles” is used synonymously with “in the nanoparticle portion.” The albumin of the nanoparticles may be further distinguishable from the albumin in the non-nanoparticle portion of the composition; for example, the oligomeric profile of the albumin in the nanoparticles may differ from the oligomeric profile of the albumin in the non-nanoparticle portion of the composition. The oligomer profile means the percentage of various albumin species compared with the total albumin in the composition. The types of albumin species includes albumin monomers, dimers, trimers, oligomers, and polymers. As used herein, “albumin monomers” or “monomeric albumin” refers to an albumin species having one, and only one, albumin unit; “albumin dimers” or “dimeric albumin” refers to an albumin species having two, and only two, albumin units; “albumin trimers” or “trimeric albumin” refers to albumin species having three, and only three, albumin units; “albumin polymers” refers to albumin species having a higher molecular weight than albumin monomers and albumin dimers; “albumin oligomers” or “oligomeric albumin” refers to lower molecular weight polymeric albumin species associated with a UV-based size-exclusion chromatography peak observed between a peak associated with albumin dimers and higher molecular weight polymeric albumin species.

[0169] The albumin of the nanoparticles associates with the rapamycin of the nanoparticles so that a nanoparticle suspension has a high concentration of rapamycin, which allows the composition to be used as a pharmaceutical composition for treating certain diseases, such as cancer. Manufactured nanoparticles (which may be made, for example, using the methods described herein) may be formulated, filtered, or otherwise processed to obtain the pharmaceutical composition, which may be suitable for medical use in a human individual.

[0170] Generally, to make the rapamycin pharmaceutical compositions described herein, rapamycin is dissolved in an organic solvent. Suitable organic solvents include, for example, ketones, esters, ethers, chlorinated solvents, and other solvents known in the art. For example,sf-6235590Attorney Reference: 638772023240 the organic solvent can be a mixture of methylene chloride / ethanol, chloroform / ethanol, or chloroform / tert-butanol (for example with a ratio of about any one of 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 or with a ratio of about any one of 3:7, 5:7, 4:6, 5:5, 6:5, 8:5, 9:5, 9.5:5, 5:3, 7:3, 6:4, or 9.5:0.5). In some embodiments, the organic solvent comprises between about 10% and about 50% tert-butanol by volume. In some embodiments, the organic solvent comprises about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% tert-butanol by volume. In some embodiments, the organic solvent comprises about any of 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, or 45-50%, or any combination of such ranges, of tert-butanol by volume. In some embodiments, the organic solvent comprises between about 50% and about 90% chloroform by volume. In some embodiments, the organic solvent comprises about any of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% chloroform by volume. In some embodiments, the organic solvent comprises about any of 50- 55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, or 85-90%, or any combination of such ranges, of chloroform by volume. In some embodiments, the organic solvent comprises between about 10% and about 50% tert-butanol by volume and between about 50% and about 90% chloroform by volume. In some embodiments, the organic solvent comprises chloroform and tert-butanol at a volumetric ratio of about 1:1 to about 1:9, such as about any of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, and 9:1.

[0171] Albumin (such as recombinant albumin, for example NOVOZYMETMrecombinant albumin or INTRIVIATMrecombinant albumin disclosed herein) is dissolved in an aqueous solution (such as water) and combined with the rapamycin solution to form a crude emulsion. The mixture is subjected to high pressure homogenization (e.g., using an Avestin, APV Gaulin, MICROFLUIDIZER™ such as a MICROFLUIDIZER™ Processor M-110EH from Microfluidics, Stansted, or Ultra Turrax homogenizer). The emulsion may be cycled through the high pressure homogenizer for between about 2 to about 100 cycles, such as about 5 to about 50 cycles or about 6 to about 20 cycles (e.g., about any one of 6, 8, 10, 12, 14, 16, 18 or 20 cycles). The organic solvent can then be removed by evaporation utilizing suitable equipment known for this purpose, including, but not limited to, rotary evaporators, falling film evaporators, wiped film evaporators, spray driers, and the like that can be operated in batch mode or in continuous operation. In some embodiments, the evaporator is a wiped film evaporator. The solvent may be removed at reduced pressure (such as at about any one of 25 mm Hg, 30 mm Hg, 40 mm Hg, 50sf-6235590Attorney Reference: 638772023240 mm Hg, 100 mm Hg, 200 mm Hg, or 300 mm Hg). The amount of time used to remove the solvent under reduced pressure may be adjusted based on the volume of the formulation. For example, for a formulation produced on a 300 mL scale, the solvent can be removed at about 1 to about 300 mm Hg (e.g., about any one of 5-100 mm Hg, 10-50 mm Hg, 20-40 mm Hg, or 25 mm Hg) for about 5 to about 60 minutes (e.g., about any one of 7, 8, 9, 10, 11, 12, 13, 14, 1516, 18, 20, 25, or 30 minutes). The dispersion obtained can be further lyophilized.

[0172] The nanoparticle compositions described herein (such a pharmaceutical composition) may have distinct characteristics for any one or more (in any combination) of the following: (1) the oligomeric status of the albumin associated with (such as in) the nanoparticles, such as the percentage of albumin monomers, dimers, and / or polymers (or trimers) of the albumin associated with (such as in) the nanoparticles; (2) the oligomeric status of the albumin associated with (such as in) the non-nanoparticle portion of the composition, such as the percentage of albumin monomers, dimers, and / or polymers (or trimers) of the albumin associated with (such as in) the non-nanoparticle portion of the composition; (3) the oligomeric status of the total albumin in the composition, such as the percentage of albumin monomers, dimers, and / or polymers (or trimers) of the total albumin in the composition; (4) the particle size profile of the nanoparticles, such as the average particle size, polydispersity index, and / or size distribution; (5) the portion (e.g., weight percentage) of the nanoparticles that is albumin and / or the portion (e.g., weight percentage) of the nanoparticles that is rapamycin; (6) the weight ratio of the albumin to the rapamycin in the nanoparticles; (7) the weight ratio of the albumin to the rapamycin in the non-nanoparticle portion of the composition; (8) the weight ratio of the albumin to the rapamycin in the non-nanoparticle portion of the composition (9) the weight ratio of the total albumin to the total rapamycin in the composition; (10) the portion (e.g., weight percentage) of rapamycin that is in the nanoparticles (or the non-nanoparticle portion of the composition) compared to the total rapamycin in the composition; (11) the portion (e.g., weight percentage) of albumin that is in the non-nanoparticle portion (or in the nanoparticles) compared to the total albumin in the composition; (12) the concentration of albumin in the composition; (13) the concentration of albumin in the non-nanoparticle portion of the composition; (14) the concentration of albumin in the composition that is associated with (such as in) the nanoparticles; (15) the concentration of rapamycin in the composition; (16) the concentration of rapamycin in the non-nanoparticle portion of the composition; (17) the concentration ofsf-6235590Attorney Reference: 638772023240 rapamycin in the composition that is associated with (such as in) the nanoparticles; (18) the osmolality of the composition; (19) the viscosity of the composition; (20) the pH of the composition; (21) the stability of the nanoparticles in the composition; (22) the amount of residual solvent in the composition; (23) the zeta potential of the nanoparticles in the composition; (24) the crystalline status of the rapamycin in the nanoparticles; (25) the particle morphology of the nanoparticles, such as the shape, sphericity, thickness of the coating, and / or surface-to-volume ratio; (26) the weight percentage of seco-rapamycin in the nanoparticles, as compared to the sum of seco-rapamycin and rapamycin, by weight; (27) the presence, percentage, or concentration of albumin stabilizer (such as sodium caprylate and / or N-acetyltryptophanate) in the composition; (28) the recovery of rapamycin following filtration; (29) in vitro release kinetics of the nanoparticles; (30) the portion of total rapamycin in the composition that is both in the non-nanoparticle portion of the composition and not bound to albumin; and / or (31) the weight percentage of seco-rapamycin in the composition, as compared to the sum of seco-rapamycin and rapamycin, by weight. In some embodiments, the oligomeric status (such as the percentage of albumin monomers, dimers, or polymers (or trimers)) of the nanoparticles, the non-nanoparticles portion, or the total composition is assessed by size- exclusion chromatography using a saline mobile phase coupled with a multiple angle light scattering (MALS) detector).

[0173] The nanoparticle compositions described herein (such a pharmaceutical composition) may have distinct characteristics for any one or more (in any combination) of the following: (1) the oligomeric status of the albumin associated with (such as in) the nanoparticles, such as the percentage of albumin monomers, dimers, oligomers, and / or polymers (other than oligomers) of the albumin associated with (such as in) the nanoparticles; (2) the oligomeric status of the albumin associated with (such as in) the non-nanoparticle portion of the composition, such as the percentage of albumin monomers, dimers, oligomers, and / or polymers (other than oligomers) of the albumin associated with (such as in) the non-nanoparticle portion of the composition; (3) the oligomeric status of the total albumin in the composition, such as the percentage of albumin monomers, dimers, oligomers, and / or polymers (other than oligomers) of the total albumin in the composition; (4) the particle size profile of the nanoparticles, such as the average particle size, polydispersity index, and / or size distribution; (5) the portion (e.g., weight percentage) of the nanoparticles that is albumin and / or the portion (e.g., weight percentage) of the nanoparticlessf-6235590Attorney Reference: 638772023240 that is rapamycin; (6) the weight ratio of the albumin to the rapamycin in the nanoparticles; (7) the weight ratio of the albumin to the rapamycin in the non-nanoparticle portion of the composition; (8) the weight ratio of the albumin to the rapamycin in the non-nanoparticle portion of the composition (9) the weight ratio of the total albumin to the total rapamycin in the composition; (10) the portion (e.g., weight percentage) of rapamycin that is in the nanoparticles (or the non-nanoparticle portion of the composition) compared to the total rapamycin in the composition; (11) the portion (e.g., weight percentage) of albumin that is in the non-nanoparticle portion (or in the nanoparticles) compared to the total albumin in the composition; (12) the concentration of albumin in the composition; (13) the concentration of albumin in the non- nanoparticle portion of the composition; (14) the concentration of albumin in the composition that is associated with (such as in) the nanoparticles; (15) the concentration of rapamycin in the composition; (16) the concentration of rapamycin in the non-nanoparticle portion of the composition; (17) the concentration of rapamycin in the composition that is associated with (such as in) the nanoparticles; (18) the osmolality of the composition; (19) the viscosity of the composition; (20) the pH of the composition; (21) the stability of the nanoparticles in the composition; (22) the amount of residual solvent in the composition; (23) the zeta potential of the nanoparticles in the composition; (24) the crystalline status of the rapamycin in the nanoparticles; (25) the particle morphology of the nanoparticles, such as the shape, sphericity, thickness of the coating, and / or surface-to-volume ratio; (26) the weight percentage of seco- rapamycin in the nanoparticles, as compared to the sum of seco-rapamycin and rapamycin, by weight; (27) the presence, percentage, or concentration of albumin stabilizer (such as sodium caprylate and / or N-acetyltryptophanate) in the composition; (28) the recovery of rapamycin following filtration; (29) in vitro release kinetics of the nanoparticles; (30) the portion of total rapamycin in the composition that is both in the non-nanoparticle portion of the composition and not bound to albumin; and / or (31) the weight percentage of seco-rapamycin in the composition, as compared to the sum of seco-rapamycin and rapamycin, by weight. As used herein, “albumin oligomers” or “oligomeric albumin” refers to lower molecular weight polymeric albumin species associated with a UV-absorbance-based size-exclusion chromatography peak observed between a peak associated with albumin dimers and higher molecular weight polymeric albumin species. In some embodiments, the oligomeric status (such as the percentage of albumin monomers, dimers, oligomers, or polymers (other than oligomers)) of the nanoparticles, the non-sf-6235590Attorney Reference: 638772023240 nanoparticle portion, or the total composition is assessed by size-exclusion chromatography using a mobile phase containing an aqueous portion and a miscible organic portion (such as an aqueous buffer containing 7.5% methanol) coupled with a UV detector. In some embodiments, the percentage of albumin in the nanoparticle portion that is in the form of monomeric, dimeric, oligomeric, or polymeric albumin (other than oligomeric albumin) is determined by separating the nanoparticles from the non-nanoparticle portion, dissolving the nanoparticles, and subjecting the dissolved nanoparticles to size-exclusion chromatography. In some embodiments, the size- exclusion chromatography uses a mobile phase containing an aqueous portion and a miscible organic portion (such as an aqueous buffer containing 7.5% methanol) coupled with a UV detector.

[0174] In some embodiments, the nanoparticle composition has one or more of the following distinct characteristics: (1) about 80% to about 95% (or as further provided herein) of the total albumin in the composition is in the form of monomeric albumin; (2) about 4% to about 15% (or as further provided herein) of the total albumin in the composition is in the form of dimeric albumin; (3) about 0.5% to about 5% (or as further provided herein) of the total albumin in the composition is in the form of polymeric albumin (or trimeric albumin); (4) the weight ratio of the total albumin to the total rapamycin in the composition is about 1:1 to about 10:1 (or as further provided herein); (5) about 90% or more (or as further provided herein) of the total rapamycin in the composition is in the nanoparticles; (6) about 90% or more (or as further provided herein) of the total albumin in the composition is in the non-nanoparticle portion of the nanoparticles; (7) the composition comprises tert-butanol at a concentration of less than about 10 µg / mL or less than about 10 ppm (or as further provided herein); (8) the composition comprises chloroform at a concentration of less than about 5 µg / mL or less than about 5 ppm (or as further provided herein); (9) the composition comprises an albumin stabilizer (such as sodium caprylate and / or N-acetyltryptophanate); (10) at least about 80% or more (or as further provided herein) of the rapamycin in the composition is recoverable after filtering the composition with a 0.2 micron filter; (11) the composition is stable for at least 24 hours; and / or (12) less than about 5% of the total rapamycin in the composition is both in the non-nanoparticle portion of the composition and unbound to albumin in the non-nanoparticle portion of the composition. In some embodiments, the nanoparticle composition may be a nanoparticle suspension, and the nanoparticle composition may have one or more of the following distinct characteristics (insf-6235590Attorney Reference: 638772023240 addition to or in alternative to any one of the previously described district characteristics): (1) the concentration of albumin in the composition is about 30 mg / mL to about 100 mg / mL (or as further provided herein); (2) the concentration of rapamycin in the composition is about 1 mg / mL to about 15 mg / mL (or as further provided herein, such as about 1 mg / mL to about 7 mg / mL); (3) the osmolality of the composition is about 300 mOsm / kg to about 350 mOsm / kg (or as otherwise provided herein); (4) the viscosity of the composition is about 1.2 cP to about 1.5 cP (or as otherwise provided herein); and / or (5) the pH of the composition is about 6.0 to about 7.5 (or as otherwise provided herein).

[0175] In some embodiments, the nanoparticles of the composition have one or more of the following distinct characteristics: (1) about 70% to about 85% (or as otherwise provided herein) of the albumin in the nanoparticles is in the form of albumin monomers; (2) about 9% to about 20% (or as otherwise provided herein) of the albumin in the nanoparticles is in the form of albumin dimers; (3) about 5% to about 15% (or as otherwise provided herein) of the albumin in the nanoparticles is in the form of albumin polymers (or albumin trimers); (4) the nanoparticles have a volume weighted mean particle size and / or Z-average particle size of about 200 nm or less (or as otherwise provided herein, such as between about 50 nm and about 200 nm); (5) the nanoparticles have a polydispersity index of less than about 0.2 (or as otherwise provided herein, such as between about 0.03 and about 0.2); (6) the span of the particle size distribution ((Dv95- Dv5) / Dv50) is about 0.8 to about 1.2 (or as otherwise provided herein); (7) the nanoparticles are about 25% to about 45% albumin by weight (or as otherwise provided herein); (8) the nanoparticles are about 55% to about 75% rapamycin by weight (or as otherwise provided herein); (9) the weight ratio of albumin to rapamycin in the nanoparticles is about 1:1 to about 1:4 (or as otherwise provided herein); (10) the zeta potential of the nanoparticles in the composition is about -25 mV to about -50 mV (or as otherwise provided herein); (11) the nanoparticles have an amorphous morphology; (12) the rapamycin in the nanoparticles has an amorphous morphology; (13) the vinyl chain of the rapamycin in the nanoparticles interacts with the albumin in the nanoparticles; (14) at least a portion (such as at least 20%, or as otherwise provided herein) of the nanoparticles in the composition are non-spherical; (15) the nanoparticles comprise less than about 2.5% seco-rapamycin (or as otherwise provided herein, such as between about 0.2% and about 2.5%) compared to the sum of seco-rapamycin and rapamycin by weight; and / or (16) the composition comprises less than 3% seco-rapamycin (or assf-6235590Attorney Reference: 638772023240 otherwise provided herein, such as between about 0.2% and about 2.5%) compared to the sum of seco-rapamycin and rapamycin by weight. In some embodiments, the nanoparticle composition may be a nanoparticle suspension, and in some embodiments the concentration of the albumin in the nanoparticle suspension that is in the nanoparticles is about 1.8 mg / mL to about 3 mg / mL (or as otherwise provided herein).

[0176] In some embodiments, the nanoparticles of the composition have one or more of the following distinct characteristics: (1) about 25% to about 50% (or as otherwise provided herein) of the albumin in the nanoparticles is in the form of albumin monomers; (2) about 5% to about 16% (or as otherwise provided herein) of the albumin in the nanoparticles is in the form of albumin dimers; (3) about 1% to about 4.5% (or as otherwise provided herein) of the albumin in the nanoparticles is in the form of albumin oligomers; (4) about 42% to about 60% (or as otherwise provided herein) of the albumin in the nanoparticles is in the form of albumin polymers (other than oligomers); (5) the nanoparticles have a volume weighted mean particle size and / or Z-average particle size of about 200 nm or less (or as otherwise provided herein, such as between about 50 nm and about 200 nm); (6) the nanoparticles have a polydispersity index of less than about 0.2 (or as otherwise provided herein, such as between about 0.03 and about 0.2); (7) the span of the particle size distribution ((Dv95-Dv5) / Dv50) is about 0.8 to about 1.2 (or as otherwise provided herein); (8) the nanoparticles are about 25% to about 45% albumin by weight (or as otherwise provided herein); (9) the nanoparticles are about 55% to about 75% rapamycin by weight (or as otherwise provided herein); (10) the weight ratio of albumin to rapamycin in the nanoparticles is about 1:1 to about 1:4 (or as otherwise provided herein); (11) the zeta potential of the nanoparticles in the composition is about -25 mV to about -50 mV (or as otherwise provided herein); (12) the nanoparticles have an amorphous morphology; (13) the rapamycin in the nanoparticles has an amorphous morphology; (14) the vinyl chain of the rapamycin in the nanoparticles interacts with the albumin in the nanoparticles; (15) at least a portion (such as at least 20%, or as otherwise provided herein) of the nanoparticles in the composition are non-spherical; (16) the nanoparticles comprise less than about 2.5% seco-rapamycin (or as otherwise provided herein, such as between about 0.2% and about 2.5%) compared to the sum of seco-rapamycin and rapamycin by weight; and / or (17) the composition comprises less than about 3% seco-rapamycin (or as otherwise provided herein, such as between about 0.2% and about 3%) compared to the sum of seco-rapamycin and rapamycin, by weight.sf-6235590Attorney Reference: 638772023240 In some embodiments, the nanoparticle composition may be a nanoparticle suspension, and in some embodiments the concentration of the albumin in the nanoparticle suspension that is in the nanoparticles is about 1.8 mg / mL to about 3 mg / mL (or as otherwise provided herein).

[0177] In some embodiments, the non-nanoparticle portion of the composition has one or more of the following distinct characteristics: (1) about 80% to about 95% (or as otherwise provided herein) of the albumin in the non-nanoparticle portion of the composition is in the form of albumin monomers; (2) about 5% to about 14% (or as otherwise provided herein) of the albumin in the non-nanoparticle portion of the composition is in the form of albumin dimers; and / or (3) about 1% to about 5% (or as otherwise provided herein) of the albumin in the non- nanoparticle portion of the composition is in the form of albumin polymers (or albumin trimers). In some embodiments, the nanoparticle composition may be a nanoparticle suspension, and the non-nanoparticle portion of the nanoparticle suspension may have one or more of the following distinct characteristics (in addition to or in alternative to any one of the previously described district characteristics): (1) the concentration of albumin in the non-nanoparticle portion of the composition is between about 30 mg / mL and about 100 mg / mL (or as otherwise provided herein); and / or (2) the concentration of rapamycin in the non-nanoparticle portion is about 20 µg / mL to about 55 µg / mL (or as otherwise provided herein).

[0178] In some embodiments, the non-nanoparticle portion of the composition has one or more of the following distinct characteristics: (1) about 80% to about 95% (or as otherwise provided herein) of the albumin in the non-nanoparticle portion of the composition is in the form of albumin monomers; (2) about 5% to about 16% (or as otherwise provided herein) of the albumin in the non-nanoparticle portion of the composition is in the form of albumin dimers; about 0.5% to about 4% (or as otherwise provided herein) of the albumin in the non-nanoparticle portion of the composition is in the form of albumin oligomers; and / or (4) about 0.5% to about 3% (or as otherwise provided herein) of the albumin in the non-nanoparticle portion of the composition is in the form of albumin polymers (other than oligomers). In some embodiments, the nanoparticle composition may be a nanoparticle suspension, and the non-nanoparticle portion of the nanoparticle suspension may have one or more of the following distinct characteristics (in addition to or in alternative to any one of the previously described district characteristics): (1) the concentration of albumin in the non-nanoparticle portion of thesf-6235590Attorney Reference: 638772023240 composition is between about 30 mg / mL and about 100 mg / mL (or as otherwise provided herein); and / or (2) the concentration of rapamycin in the non-nanoparticle portion is about 20 µg / mL to about 55 µg / mL (or as otherwise provided herein).

[0179] The compositions (such as pharmaceutical compositions) described herein can be in liquid (e.g., as a nanoparticle suspension) or powder forms. For example, in some embodiments, the composition is a liquid nanoparticle suspension (for example prior to lyophilization). In some embodiments, the composition is a reconstituted suspension (e.g., in an aqueous solution such as a saline solution). In some embodiments, the composition is dried, such as lyophilized. In some embodiments, the composition is sterile. In some embodiments, the composition is contained in a sealed container, such as a sealed vial (e.g., a glass vial) or sealed bag.

[0180] In some embodiments, the nanoparticle composition comprises (a) nanoparticles comprising rapamycin and albumin (such as human albumin), and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin. In some embodiments, about 0.5% to about 5% of the albumin in the non-nanoparticle portion or the total albumin in the nanoparticle composition is in the form of polymeric albumin (or trimeric albumin). In some embodiments, about 4% to about 14% of the albumin in the non-nanoparticle portion or the total albumin in the nanoparticle composition is in the form of dimeric albumin. In some embodiments, about 80% to about 95% of the albumin in the non-nanoparticle portion or the total albumin in the nanoparticle composition is in the form of monomeric albumin. In some embodiments, the weight ratio of the albumin to the rapamycin in the composition is about 1:1 to about 10:1. In some embodiments, about 90% or more of the albumin in the composition is in the non-nanoparticle portion. In some embodiments, about 90% or more of the rapamycin in the composition is in the nanoparticles. In some embodiments, the concentration of albumin in the nanoparticle composition that is in the non-nanoparticle portion or the concentration of total albumin in the nanoparticle composition is about 30 mg / mL to about 100 mg / mL. In some embodiments, the osmolality of the composition is about 300 mOsm / kg to about 350 mOsm / kg. In some embodiments, the viscosity of the composition is about 1.2 cP to about 1.5 cP. In some embodiments, the pH of the composition is about 6.0 to about 7.5. In some embodiments, the composition is stable at 4 °C and / or 25 °C for at least 24 hours. In some embodiments, the rapamycin in the nanoparticles has an amorphous morphology. In some embodiment, thesf-6235590Attorney Reference: 638772023240 nanoparticle composition is a nanoparticle suspension. In some embodiments, the nanoparticle composition is a dried composition. In some embodiments, the nanoparticle composition is sterile, for example by filtration. In some embodiments, the nanoparticle composition is contained within a sealed container, such as a sealed vial or a sealed bag. In some embodiments, the nanoparticle composition comprises less than 10 µg / mL tert-butanol and / or comprises less than 5 µg / mL chloroform.

[0181] In some embodiments, the nanoparticle composition comprises (a) nanoparticles comprising rapamycin and albumin (such as human albumin), wherein about 70% to about 85% of the albumin in the nanoparticles is in the form of monomeric albumin; and (b) a non- nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0182] In some embodiments, the nanoparticle composition comprises (a) nanoparticles comprising rapamycin and albumin (such as human albumin), wherein about 25% to about 50% of the albumin in the nanoparticles is in the form of monomeric albumin; and (b) a non- nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0183] In some embodiments, the nanoparticle composition comprises (a) nanoparticles comprising rapamycin and albumin (such as human albumin), wherein about 5% to about 15% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0184] In some embodiments, the nanoparticle composition comprises (a) nanoparticles comprising rapamycin and albumin (such as human albumin), wherein about 25% to about 50% of the albumin in the nanoparticles is in the form of polymeric albumin (other than oligomeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0185] In some embodiments, the nanoparticle composition comprises (a) nanoparticles comprising rapamycin and albumin (such as human albumin), wherein about 9% to about 20% of the albumin in the nanoparticles is in the form of dimeric albumin; and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0186] In some embodiments, the nanoparticle composition comprises (a) nanoparticles comprising rapamycin and albumin (such as human albumin), wherein about 5% to about 16%sf-6235590Attorney Reference: 638772023240 of the albumin in the nanoparticles is in the form of dimeric albumin; and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0187] In some embodiments, the nanoparticle composition comprises (a) nanoparticles comprising rapamycin and albumin (such as human albumin), wherein about 70% to about 85% of the albumin in the nanoparticles is in the form of monomeric albumin, about 9% to about 20% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 5% to about 15% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0188] In some embodiments, the nanoparticle composition comprises (a) nanoparticles comprising rapamycin and albumin (such as human albumin), wherein about 25% to about 50% of the albumin in the nanoparticles is in the form of monomeric albumin, about 1% to about 4.5% of the albumin in the nanoparticles is in the form of oligomeric albumin, about 5% to about 16% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 25% to about 50% of the albumin in the nanoparticles is in the form of polymeric albumin (other than oligomeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0189] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 200 nm or less (such as about 50 nm to about 200 nm), comprising rapamycin and albumin (such as human albumin), wherein about 70% to about 85% of the albumin in the nanoparticles is in the form of monomeric albumin, about 9% to about 20% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 5% to about 15% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0190] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 200 nm or less (such as about 50 nm to about 200 nm), comprising a coating comprising albumin (such as human albumin) and a core comprising rapamycin, wherein about 70% to about 85% of the albumin in the nanoparticles is in the form of monomeric albumin, about 9% to about 20% of the albumin in the nanoparticles is in the formsf-6235590Attorney Reference: 638772023240 of dimeric albumin, and about 5% to about 15% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0191] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 200 nm or less (such as about 50 nm to about 200 nm), comprising about 55% to about 65% (by weight) rapamycin and about 25% to about 45% (by weight) albumin (such as human albumin), wherein about 70% to about 85% of the albumin in the nanoparticles is in the form of monomeric albumin, about 9% to about 20% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 5% to about 15% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0192] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 200 nm or less (such as about 50 nm to about 200 nm), comprising a coating comprising albumin (such as human albumin) and a core comprising rapamycin, wherein the albumin comprises about 25% to about 45% of the nanoparticles by weight and the rapamycin comprises about 55% to about 75% of the nanoparticles by weight, wherein about 70% to about 85% of the albumin in the nanoparticles is in the form of monomeric albumin, about 9% to about 20% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 5% to about 15% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0193] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 200 nm or less (such as about 50 nm to about 200 nm), comprising about 55% to about 75% (by weight) rapamycin and about 25% to about 45% (by weight) albumin (such as human albumin), wherein about 70% to about 85% of the albumin in the nanoparticles is in the form of monomeric albumin, about 9% to about 20% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 5% to about 15% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin; whereinsf-6235590Attorney Reference: 638772023240 the concentration of the rapamycin in the nanoparticle composition is about 1 mg / mL to about 100 mg / mL (such as about 1 mg / mL to about 15 mg / mL).

[0194] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 200 nm or less (such as about 50 nm to about 200 nm), comprising a coating comprising albumin (such as human albumin) and a core comprising rapamycin, wherein the albumin comprises about 25% to about 45% of the nanoparticles by weight and the rapamycin comprises about 55% to about 75% of the nanoparticles by weight, wherein about 70% to about 85% of the albumin in the nanoparticles is in the form of monomeric albumin, about 9% to about 20% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 5% to about 15% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin; wherein the concentration of the rapamycin in the nanoparticle composition is about 1 mg / mL to about 100 mg / mL (such as about 1 mg / mL to about 15 mg / mL).

[0195] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 200 nm or less (such as about 50 nm to about 200 nm) and a zeta potential of about -25 mV to about -50 mV, comprising about 55% to about 75% (by weight) rapamycin and about 25% to about 45% (by weight) albumin (such as human albumin), wherein about 70% to about 85% of the albumin in the nanoparticles is in the form of monomeric albumin, about 9% to about 20% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 5% to about 15% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin; wherein the concentration of the rapamycin in the nanoparticle composition is about 1 mg / mL to about 100 mg / mL (such as about 1 mg / mL to about 15 mg / mL).

[0196] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 200 nm or less (such as about 50 nm to about 200 nm) and a zeta potential of about -25 mV to about -50 mV, comprising a coating comprising albumin (such as human albumin) and a core comprising rapamycin, wherein the albumin comprises about 25% to about 45% of the nanoparticles by weight and the rapamycin comprises about 55%sf-6235590Attorney Reference: 638772023240 to about 75% of the nanoparticles by weight, wherein about 70% to about 85% of the albumin in the nanoparticles is in the form of monomeric albumin, about 9% to about 20% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 5% to about 15% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin; wherein the concentration of the rapamycin in the nanoparticle composition is about 1 mg / mL to about 100 mg / mL (such as about 1 mg / mL to about 15 mg / mL).

[0197] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 200 nm or less (such as about 50 nm to about 200 nm) and a zeta potential of about -25 mV to about -50 mV, comprising about 55% to about 75% (by weight) rapamycin and about 25% to about 45% (by weight) albumin (such as human albumin), wherein about 70% to about 85% of the albumin in the nanoparticles is in the form of monomeric albumin, about 9% to about 20% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 5% to about 15% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin; wherein the concentration of the rapamycin in the nanoparticle composition is about 1 mg / mL to about 100 mg / mL (such as about 1 mg / mL to about 15 mg / mL); and wherein about 3% or less of the rapamycin in the nanoparticle composition is free rapamycin.

[0198] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 200 nm or less (such as about 50 nm to about 200 nm) and a zeta potential of about -25 mV to about -50 mV, comprising a coating comprising albumin (such as human albumin) and a core comprising rapamycin, wherein the albumin comprises about 25% to about 45% of the nanoparticles by weight and the rapamycin comprises about 55% to about 75% of the nanoparticles by weight, wherein about 70% to about 85% of the albumin in the nanoparticles is in the form of monomeric albumin, about 9% to about 20% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 5% to about 15% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin; wherein the concentration of the rapamycin in the nanoparticle composition is about 1 mg / mL to aboutsf-6235590Attorney Reference: 638772023240 100 mg / mL (such as about 1 mg / mL to about 15 mg / mL); and wherein about 3% or less of the rapamycin in the nanoparticle composition is free rapamycin.

[0199] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 200 nm or less (such as about 50 nm to about 200 nm) and a zeta potential of about -25 mV to about -50 mV, comprising about 55% to about 75% (by weight) rapamycin and about 25% to about 45% (by weight) albumin (such as human albumin), wherein about 70% to about 85% of the albumin in the nanoparticles is in the form of monomeric albumin, about 9% to about 20% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 5% to about 15% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin; wherein the concentration of the rapamycin in the nanoparticle composition is about 1 mg / mL to about 100 mg / mL (such as about 1 mg / mL to about 15 mg / mL); and wherein the sum of seco-rapamycin and rapamycin in the nanoparticles is less than 3% (such as about 0.2% to about 3%) seco-rapamycin, by weight. In some embodiments, the sum of seco-rapamycin and rapamycin in the composition is less than 3% (such as about 0.2% to about 3%) seco-rapamycin, by weight.

[0200] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 200 nm or less (such as about 50 nm to about 200 nm) and a zeta potential of about -25 mV to about -50 mV, comprising a coating comprising albumin (such as human albumin) and a core comprising rapamycin, wherein the albumin comprises about 25% to about 45% of the nanoparticles by weight and the rapamycin comprises about 55% to about 75% of the nanoparticles by weight, wherein about 70% to about 85% of the albumin in the nanoparticles is in the form of monomeric albumin, about 9% to about 20% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 5% to about 15% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non- nanoparticle portion comprising albumin (such as human albumin) and rapamycin; wherein the concentration of the rapamycin in the nanoparticle composition is about 1 mg / mL to about 100 mg / mL (such as about 1 mg / mL to about 15 mg / mL); and wherein the sum of seco-rapamycin and rapamycin in the nanoparticles is less than 3% (such as about 0.2% to about 3%) seco-sf-6235590Attorney Reference: 638772023240 rapamycin, by weight. In some embodiments, the seco-rapamycin is less than 3% (such as about 0.2% to about 3%) of the sum of seco-rapamycin and rapamycin in the composition.

[0201] In some embodiments, the nanoparticle composition comprises (a) nanoparticles comprising rapamycin and albumin (such as human albumin), wherein about 74% to about 80% of the albumin in the nanoparticles is in the form of monomeric albumin; and (b) a non- nanoparticle portion comprising albumin (such as human albumin) and rapamycin. In some embodiments, about 1.5% to about 3% of the albumin in the non-nanoparticle portion or the total albumin in the nanoparticle composition is in the form of polymeric albumin (or trimeric albumin). In some embodiments, about 7% to about 11% of the albumin in the non-nanoparticle portion in the nanoparticle composition is in the form of dimeric albumin. In some embodiments, about 7% to about 11% of the total albumin in the nanoparticle composition is in the form of dimeric albumin. In some embodiments, about 83% to about 92% of the albumin in the non-nanoparticle portion or the total albumin in the nanoparticle composition is in the form of monomeric albumin. In some embodiments, the weight ratio of the albumin to the rapamycin in the composition is about 7:1 to about 9:1. In some embodiments, about 95% or more of the albumin in the composition is in the non-nanoparticle portion. In some embodiments, about 98% to about 99.5% of the rapamycin in the composition is in the nanoparticles. In some embodiments, the concentration of albumin in the nanoparticle composition that is in the non-nanoparticle portion or the concentration of total albumin in the nanoparticle composition is about 35 mg / mL to about 45 mg / mL.

[0202] In some embodiments, the nanoparticle composition comprises (a) nanoparticles comprising rapamycin and albumin (such as human albumin), wherein about 7% to about 11% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0203] In some embodiments, the nanoparticle composition comprises (a) nanoparticles comprising rapamycin and albumin (such as human albumin), wherein about 12% to about 17% of the albumin in the nanoparticles is in the form of dimeric albumin; and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0204] In some embodiments, the nanoparticle composition comprises (a) nanoparticles comprising rapamycin and albumin (such as human albumin), wherein about 74% to about 80%sf-6235590Attorney Reference: 638772023240 of the albumin in the nanoparticles is in the form of monomeric albumin, about 12% to about 17% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 7% to about 11% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0205] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 85 nm to about 95 nm, comprising rapamycin and albumin (such as human albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0206] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 85 nm to about 95 nm, comprising rapamycin and albumin (such as human albumin), wherein about 74% to about 80% of the albumin in the nanoparticles is in the form of monomeric albumin, about 12% to about 17% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 7% to about 11% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non- nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0207] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 85 nm to about 95 nm, comprising a coating comprising albumin (such as human albumin) and a core comprising rapamycin, wherein about 74% to about 80% of the albumin in the nanoparticles is in the form of monomeric albumin, about 12% to about 17% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 7% to about 11% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0208] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a zeta potential of about -33 mV to about -39 mV, comprising rapamycin and albumin (such as human albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0209] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a zeta potential of about -33 mV to about -39 mV, comprising a coating comprisingsf-6235590Attorney Reference: 638772023240 albumin (such as human albumin) and a core comprising rapamycin; and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin

[0210] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a zeta potential of about -33 mV to about -39 mV, comprising rapamycin and albumin (such as human albumin), wherein about 74% to about 80% of the albumin in the nanoparticles is in the form of monomeric albumin, about 12% to about 17% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 7% to about 11% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non- nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0211] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a zeta potential of about -33 mV to about -39 mV, comprising a coating comprising albumin (such as human albumin) and a core comprising rapamycin, wherein about 74% to about 80% of the albumin in the nanoparticles is in the form of monomeric albumin, about 12% to about 17% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 7% to about 11% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0212] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 85 nm to about 95 nm and a zeta potential of about -33 mV to about -39 mV, comprising rapamycin and albumin (such as human albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0213] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 85 nm to about 95 nm and a zeta potential of about -33 mV to about -39 mV, comprising a coating comprising albumin (such as human albumin) and a core comprising rapamycin; and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0214] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 85 nm to about 95 nm and a zeta potential of about -33 mV to about -39 mV, comprising rapamycin and albumin (such as human albumin), wherein about 74% to about 80% of the albumin in the nanoparticles is in the form of monomericsf-6235590Attorney Reference: 638772023240 albumin, about 12% to about 17% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 7% to about 11% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0215] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 85 nm to about 95 nm and a zeta potential of about -33 mV to about -39 mV, comprising a coating comprising albumin (such as human albumin) and a core comprising rapamycin, wherein about 74% to about 80% of the albumin in the nanoparticles is in the form of monomeric albumin, about 12% to about 17% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 7% to about 11% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non- nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0216] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 85 nm to about 95 nm, comprising about 62% to about 68% (by weight) rapamycin and about 32% to about 38% (by weight) albumin (such as human albumin), wherein about 74% to about 80% of the albumin in the nanoparticles is in the form of monomeric albumin, about 12% to about 17% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 7% to about 11% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin.

[0217] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 85 nm to about 95 nm, comprising about 62% to about 68% (by weight) rapamycin and about 32% to about 38% (by weight) albumin (such as human albumin), wherein about 74% to about 80% of the albumin in the nanoparticles is in the form of monomeric albumin, about 12% to about 17% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 7% to about 11% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin; wherein the concentration of the rapamycin in the nanoparticle composition is about 1 mg / mL to about 100 mg / mL (such as about 1 mg / mL to about 15 mg / mL).sf-6235590Attorney Reference: 638772023240

[0218] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 85 nm to about 95 nm and a zeta potential of about -33 mV to about -39 mV, comprising about 62% to about 68% (by weight) rapamycin and about 32% to about 38% (by weight) albumin (such as human albumin), wherein about 74% to about 80% of the albumin in the nanoparticles is in the form of monomeric albumin, about 12% to about 17% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 7% to about 11% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin; wherein the concentration of the rapamycin in the nanoparticle composition is about 1 mg / mL to about 100 mg / mL (such as about 1 mg / mL to about 15 mg / mL).

[0219] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 85 nm to about 95 nm and a zeta potential of about -33 mV to about -39 mV, comprising about 62% to about 68% (by weight) rapamycin and about 32% to about 38% (by weight) albumin (such as human albumin), wherein about 74% to about 80% of the albumin in the nanoparticles is in the form of monomeric albumin, about 12% to about 17% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 7% to about 11% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin; wherein the concentration of the rapamycin in the nanoparticle composition is about 1 mg / mL to about 100 mg / mL (such as about 1 mg / mL to about 15 mg / mL); and wherein about 1% or less of the rapamycin in the nanoparticle composition is free rapamycin.

[0220] In some embodiments, the nanoparticle composition comprises (a) nanoparticles having a Z-average particle size of about 85 nm to about 95 nm and a zeta potential of about of about -33 mV to about -39 mV, comprising about 62% to about 68% (by weight) rapamycin and about 32% to about 38% (by weight) albumin (such as human albumin), wherein about 74% to about 80% of the albumin in the nanoparticles is in the form of monomeric albumin, about 12% to about 17% of the albumin in the nanoparticles is in the form of dimeric albumin, and about 7% to about 11% of the albumin in the nanoparticles is in the form of polymeric albumin (or trimeric albumin); and (b) a non-nanoparticle portion comprising albumin (such as human albumin) and rapamycin; wherein the concentration of the rapamycin in the nanoparticlesf-6235590Attorney Reference: 638772023240 composition is about 1 mg / mL to about 100 mg / mL (such as about 1 mg / mL to about 15 mg / mL); and wherein the sum of seco-rapamycin and rapamycin in the nanoparticles is less than 1% (such as about 0.5% to about 1%) seco-rapamycin, by weight. In some embodiments, seco- rapamycin is greater than about 0.2% (such as about 0.2% to about 3%) of the sum of seco- rapamycin and rapamycin in the composition.

[0221] Also provided herein are commercial batches of the nanoparticle compositions (such as the pharmaceutical compositions) for use of any one of the treatment methods described here. “Commercial batch” as used herein refers to a batch size that is at least about 20 grams (by mass of rapamycin). Commercial batches are produced at a larger scale than experimental or bench- scale batches. The increased scale is associated with longer production times, including longer steps (such as evaporation steps) or longer hold times between steps.

[0222] In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered subcutaneously. In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered intravenously. In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered at a dose between about 1 mg / m2and about 150 mg / m2, between about 5 mg / m2and about 75 mg / m2, e.g., via intravenous infusion. In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered at a dose of about any one of 5, 7.5, 10, 15, 30, 56, 75 or 100 mg / m2, e.g., via intravenous infusion. In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered to the individual having cancer in one or more 21-day cycles (e.g., three-week cycles). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered to the individual once during each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered during Week 1, Week 2, or Week 3 during each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticlesf-6235590Attorney Reference: 638772023240 composition, such as FYARRO™) is administered on Day 1, Day 8, or Day 15 of each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered to the individual twice during each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered during Week 1 and Week 2 during each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered during Week 2 and Week 3 during each 21-day cycle (e.g., three- week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered during Week 1 and Week 3 during each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered on Day 1 and Day 8 of each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered on Day 1 and Day 15 of each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered on Day 8 and Day 15 of each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered to the individual three times during each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered during Week 1, Week 2, and Week 3 during each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is administered on Day 1, Day 8, and Day 15 of each 21-day cycle (e.g., three- week cycle). In some embodiments, the dosage of the mTOR inhibitor nanoparticle composition (e.g., a sirolimus / albumin nanoparticle composition, such as FYARRO™) is modified (e.g., if the individual experiences one or more adverse effects). Details regarding dosage modificationsf-6235590Attorney Reference: 638772023240 for FYARRO™ and circumstances under which dosage modifications are made are detailed at www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2021 / 213312lbl.pdf. V. Articles of Manufacture and Kits

[0223] In some embodiments, there is provided an article of manufacture containing materials useful for a method described herein, the article of manufacture, such as a medicament or medicament combination, comprising an mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition, e.g., nab-sirolimus). The article of manufacture can comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. Generally, the container holds a composition which is effective for treating a disease or disorder described herein, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used for treating the particular condition in an individual, such as described herein. The label or package insert will further comprise instructions for administering the composition to the individual according to the methods described herein. Articles of manufacture and kits comprising combination therapies described herein are also contemplated.

[0224] Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products. In some embodiments, the package insert indicates that the composition is used for the methods provided herein.

[0225] Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0226] Kits are also provided that are useful for various purposes, e.g., for a treatment described herein. Kits of the invention include one or more containers comprising an mTORsf-6235590Attorney Reference: 638772023240 inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) (or unit dosage form and / or article of manufacture) and / or instructions for use in accordance with any of the methods described herein. The kit may further comprise a description of selection of individuals suitable for treatment, such as an individual having an inactivating mutation in TSC1 or TSC2. Also encompassed herein are methods and kits useful for determining if an individual has an inactivating mutation in TSC1 or TSC2. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0227] The kits of the invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretative information. The present application thus also provides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.

[0228] The instructions relating to the use of the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. For example, kits may be provided that contain sufficient dosages of an mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) as disclosed herein to provide effective treatment of an individual for an extended period, such as any of a week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or more. Kits may also include multiple unit doses of the mTOR inhibitor nanoparticle composition (such as sirolimus / albumin nanoparticle composition) and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.

[0229] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this invention. The invention will now be described in greater detail by reference to the following non-limiting examples. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.sf-6235590Attorney Reference: 638772023240 EXAMPLES Example 1. Phase 2 Basket Trial of Nab-sirolimus in Patients With Malignant Solid Tumors With Pathogenic Alterations in TSC1 / TSC2 Genes (PRECISION 1)

[0230] This study was a phase 2 multi-center open-label basket trial of nab-sirolimus for adult and adolescent patients with malignant solid tumors harboring pathogenic inactivating alterations in TSC1 or TSC2 genes, to determine the efficacy and safety profile of nab-sirolimus intravenously administered to patients with malignant solid tumors harboring pathogenic inactivating alterations in TSC1 or TSC2 genes. Patients were treated with single agent IV nab- sirolimus until disease progression, or unacceptable toxicity, or until in the opinion of the investigator the patient is no longer benefiting from therapy, or at patient discretion. Patient inclusion criteria:

[0231] Patients must have a malignant solid tumor with a pathogenic inactivating TSC1 or TSC2 alteration. Genetic alterations should be identified using NGS in tumor tissue or liquid biopsy). Patients were enrolled after the central evaluation of NGS report confirms eligibility.

[0232] Patients must have solid tumors that are metastatic or locally advanced where surgical resection is not an option or likely to result in severe morbidity.

[0233] Patients must have received all standard therapies appropriate for their tumor type and stage of disease or, in the opinion of the Investigator, the patient would be unlikely to tolerate or derive clinically meaningful benefit from appropriate standard of care therapy, or the patient has no satisfactory alternative treatments.

[0234] Patients must have 1 or more measurable target lesions by computed tomography (CT) scan or magnetic resonance imaging (MRI) (RECIST v1.1).

[0235] Patients must be 12 years or older.

[0236] Patients must have an Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1 or Karnofsky Performance Status (KPS) ≥80 or Lansky play-performance scale for pediatric patients ≥80.sf-6235590Attorney Reference: 638772023240

[0237] Patients must have: a) adequate liver function: total bilirubin ≤1.5 × upper limit of normal (ULN) (unless due to Gilbert's syndrome, then ≤3 × ULN), b) aspartate aminotransferase (AST) ≤2.5 × ULN (≤5 × ULN if attributable to liver metastases), c) adequate renal function: creatinine clearance ≥30 mL / min, Cockcroft-Gault CCr = ((140-age) × weight[kg]) / (72 × SCr[mL / min]) × 0.85, if female, d) adequate hematologic parameters: absolute neutrophil count (ANC) ≥1.0 × 109 / L (growth factor support allowed), e) platelet count ≥100,000 / mm3 (100 × 109 / L) (transfusion and / or growth factor support allowed), f) hemoglobin ≥8.0 g / dL (transfusion and / or growth factor support allowed), g) fasting serum triglyceride must be ≤300 mg / dL; fasting serum cholesterol must be ≤350 mg / dL.

[0238] There must be a minimum of 4 weeks since any major surgery, completion of radiation, or completion of prior systemic anticancer therapy, or at least 5 half-lives if the prior therapy is a single agent small-molecule therapeutic, and adequately recovered from the acute toxicities of any prior therapy, including neuropathy, to Grade ≤1.

[0239] Male or non-pregnant and non-breastfeeding female.

[0240] Females of childbearing potential must agree to use effective contraception or abstinence without interruption from 28 days prior to starting investigational product (IP) throughout 3 months after last dose of IP and have a negative serum pregnancy test (beta human chorionic gonadotropin, β-hCG) result at screening and agree to ongoing pregnancy testing during the course of the study, and after the end of study treatment. A second form of birth control is required even if she has had a tubal ligation.

[0241] Male patients must agree not to donate sperm and must practice abstinence or agree to use a condom during sexual contact with a pregnant female or a female of childbearing potential while participating in the study and throughout 3 months after last dose of IP. A second form of birth control is required even if he has undergone a successful vasectomy.

[0242] The patient or the patient's parent(s) or legal guardian(s) understand(s) and sign(s) the informed consent.

[0243] Willingness and ability to comply with scheduled visits, laboratory tests, and other study procedures.sf-6235590Attorney Reference: 638772023240 Patient exclusion Criteria:

[0244] Prior treatment with an mTOR inhibitor, including nab-sirolimus.

[0245] Severe (Grade ≥3) ongoing infection requiring parenteral or oral anti-infective treatment, either ongoing or completed ≤7 days prior to enrollment.

[0246] Patients with primary brain tumors or PEComa.

[0247] Patients who have any severe and / or uncontrolled medical or psychiatric conditions or other conditions that could affect their participation including:

[0248] Patients with meningeal carcinomatosis, leptomeningeal carcinomatosis, spinal cord compression, untreated brain metastases or symptomatic or unstable brain metastases. Note: Patients with stable brain metastases (defined as asymptomatic or no requirement for high-dose [defined as dexamethasone 10 mg daily or higher] or increasing dose of systemic corticosteroids) and without imminent need of radiation therapy are eligible. If applicable, patients must have completed brain radiation therapy and recovered adequately from any associated toxicity and / or complications prior to eligibility assessment. For patients who have received prior radiation therapy, post-treatment MRI scan should show no increase in brain lesion size / volume.

[0249] Unstable angina pectoris, symptomatic congestive heart failure (New York Heart Association, NYHA class III or IV), myocardial infarction ≤6 months prior to first study treatment, serious uncontrolled cardiac arrhythmia or any other clinically significant cardiac disease.

[0250] Pre-existing severely impaired lung function. If a patient has a pre-existing pulmonary condition, eligible patients should have a spirometry and diffusing capacity for carbon monoxide (DLCO) that is >50% of the normal predicted value and / or O2 saturation that is >88% at rest on room air (Note: spirometry and pulmonary function tests [PFTs] not required to be performed unless clinically indicated).

[0251] Nonmalignant medical illnesses that are uncontrolled or whose control may be jeopardized by the treatment with the study therapy.sf-6235590Attorney Reference: 638772023240

[0252] A history of malignancies other than the one under treatment unless the patient is disease-free for more than 5 years from diagnosis. Note, controlled non-melanoma skin cancers, carcinoma in situ of the cervix, resected incidental prostate cancer, certain low grade hematologic malignancies (eg CLL, follicular lymphoma, etc), or other adequately treated carcinoma-in-situ may be eligible, after discussion with the medical monitor.

[0253] Uncontrolled hypertension (systolic blood pressure ≥160 mm-Hg and / or diastolic blood pressure ≥100 mm Hg).

[0254] Patients with history of interstitial lung disease and / or pneumonitis, or pulmonary hypertension.

[0255] Individuals with known human immunodeficiency virus (HIV) infection are excluded from this study as combination antiretroviral therapy could potentially result in significant pharmacokinetic interactions. In addition, these individuals are at increased risk of serious infections due to the immunosuppressive effects of mTOR inhibition.

[0256] Active Hepatitis B or Hepatitis C, with detectable viral load.

[0257] Regarding concomitant medications with significant CYP3A4 and P-gp interactions, discontinuation of strong inhibitors (eg, ketoconazole, itraconazole, voriconazole, erythromycin, clarithromycin, telithromycin, and others), strong inducers (eg, rifampin, rifabutin), and known CYP3A4 substrates with a narrow therapeutic window (eg, fentanyl, alfentanil, astemizole, cisapride, dihydroergotamine, pimozide, quinidine, or terfenadine) is required at least 5 half lives prior to receiving the first dose of nab-sirolimus, whichever is longer. Study Design

[0258] See the design of the study in FIG. 1. Arm A includes patients with pathogenic inactivating TSC1 alterations. Arm B includes patients with pathogenic inactivating TSC2 alterations. Central confirmation of TSC1 and TSC2 pathogenic inactivating alterations was via evaluation of NGS reports. Patients were enrolled only after central confirmation of eligibility. TSC1 and TSC2 alterations was determined by analytically validated NGS tests performed in CLIA-certified laboratories. 2b Patients enrolled in South Korea must be ≥18 years of age. 4c Patients with a history of hypersensitivity to sirolimus, rapamycin analogues, or albumin are excluded from enrollment in South Korea. 4d Follow-up is for survival and initiation ofsf-6235590Attorney Reference: 638772023240 anticancer therapy. Follow-up is initiated after the end-of-treatment visit. 2 CLIA, Clinical Laboratory Improvement Amendments; CT, computerized tomography; DCR, disease control rate; DOR, duration of response; ECOG PS, Eastern Cooperative Oncology Group performance status; IRR, independent radiographic review; KPS, Karnofsky Performance Scale; LPPS, Lansky Play-Performance Scale; MRI, magnetic resonance imaging; mTORi, mechanistic target of rapamycin inhibitor; nab, nanoparticle albumin-bound; NGS, next-generation sequencing; ORR, overall response rate; OS, overall survival; PFS, progression-free survival; QoL, quality of life; RECIST, Response Evaluation Criteria for Solid Tumors; TSC1 / TSC2, tuberous sclerosis complex 1 / 2; TTR, time to response. Outcome measures

[0259] 1. Primary outcome measure: overall response rate (ORR). ORR based on the proportion of patients with best overall response (BOR) of confirmed partial response (PR) or complete response (CR) from the time of study treatment initiation until disease progression as determined by IRR using Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. Time frame for this measure: 9 months.

[0260] 2. Secondary outcome measures:

[0261] 2.1. Duration of response (DOR). Determined for patients with BOR of confirmed CR or PR (by IRR). Time frame for this measure: 9 months.

[0262] 2.2. Disease control rate. BOR of confirmed CR or PR (either of any duration) or stable disease (SD) following study treatment initiation (by IRR). Time frame for this measure: 9 months.

[0263] 2.3. Time to response. Time from first dose of study drug to initial measurement of CR or PR, where CR or PR is subsequently confirmed. Time frame for this measure: 9 months.

[0264] 2.4. Progression-free survival. Number of months from study treatment initiation to the date of disease progression (by IRR) or death due to any cause. Time frame for this measure: 9 months.

[0265] 2.5. Overall survival. Number of months from study treatment initiation to the date of death due to any cause. Time frame for this measure: 24 months.sf-6235590Attorney Reference: 638772023240

[0266] 2.6. Patient-reported outcome. Changes from baseline in the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire v3.0 (EORTC-QOQ-C30) scores. Time frame for this measure: 9 months.

[0267] 2.7. Incidence and severity of treatment-emergent and treatment-related adverse events (AEs). Incidence and severity of treatment-emergent and treatment-related AEs as assessed by the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0. Time frame for this measure: 9 months. Interim analysis

[0268] An interim analysis of a total of 37 enrolled patients revealed a wide spectrum of cancer / tumor types that bear a TSC1 or TSC2 pathogenic inactivating alteration. Specifically, the cancer / tumor types included ovarian cancer, colon cancer, leiomyosarcoma, breast cancer, bladder cancer, endometrial cancer, cervical cancer, hepatocellular carcinoma, soft tissue sarcomas, adrenocortical cancer, gastrointestinal stromal tumor (GIST), pancreatic neuroendocrine tumors (PNET), vaginal cancer, osteosarcoma, mesothelioma, and head and neck cancer.

[0269] FIG. 2 shows the demographics and efficacy evaluable populations. As shown, these patients represent a heavily treated population that are particularly difficult to treat. Specifically, for the TSC1 arm, the median number of prior treatments was 3, and for the TSC2 arm, the median number of prior treatments was 3.5. About 80% of the patients in each arm had 2 or more lines of prior treatments prior to nab-rapamycin. Moreover, 26% of patients in the TSC1 arm and 50% of patients in the TSC2 arm had 4 or more lines of prior treatments prior to being treated with nab-rapamycin. FIG. 3 shows the tumor types of the patients enrolled in each of the TSC1 arm and TSC2 arm.

[0270] Nonetheless, as shown in FIG. 4, of the efficacy evaluable patients in the TSC1 arm, 26% achieved a partial response, 47% achieved stable disease, and 16% demonstrated stable disease of more than six months. Responses were observed in four different epithelial carcinoma types: gastrointestinal (hepatocellular carcinoma), gynecologic (ovarian cancer), breast, and genitourinary cancer (bladder cancer). The total clinical benefit rate was 42%. Strikingly, 78.9% of the patients showed a tumor shrinkage post nab-sirolimus administration (FIG. 6), demonstrating the high effectiveness of nab-sirolimus monotherapy for this heavilysf-6235590Attorney Reference: 638772023240 treated TSC1 patient population. More strikingly, the responses appeared to be early (e.g., only 1.3 months of median time to response), deep (e.g., more than half of the responding patients demonstrated a tumor size reduction of more than 50%) and durable (e.g., more than 50% of the responding patients exhibited durable response of more than 9 months with all responses still ongoing). See FIGS. 4-7. Such results are highly encouraging especially considering that the majority of patients in the TSC1 arm had failed on or progressed following at least three lines of different prior treatments.

[0271] The TSC2 arm patients were even more heavily treated as discussed above with half of the patients having failed on or progressed following at least 4 lines of different prior treatments. Nonetheless, 11% of the patients showed a partial response, 67% of the patients showed stable disease, and 17% of the patients showed stable disease of more than 6 months (FIGS. 8-9) The total clinical benefit rate was 28%. 61.1% of the patients showed tumor shrinkage (FIG. 10), consistently demonstrating the high effectiveness of nab-sirolimus monotherapy for this heavily treated TSC2 patient population. Responses were observed in epithelial carcinoma (e.g., breast cancer) and sarcoma. See FIGS. 8-10.

[0272] There were no new safety signals observed as of the interim analysis. No grade 4 therapy-related adverse events or deaths due to study drug was observed. See FIG. 12.sf-6235590

Claims

Attorney Reference: 638772023240 CLAIMS What is claimed is:

1. A method of treating a cancer in an individual in need thereof, wherein the individual has been subjected to two or more prior treatments for the cancer, the method comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin.

2. The method of claim 1, wherein the individual has failed the two or more prior treatments for the cancer.

3. The method of claim 1, wherein the individual has been subjected to three or more prior treatments for the cancer.

4. The method of claim 3, wherein the individual has failed the three or more prior treatments for the cancer.

5. The method of claim 1, wherein the individual has been subjected to five or more prior treatments for the cancer.

6. The method of claim 5, wherein the individual has failed the five or more prior treatments for the cancer.

7. The method of any one of claims 1-6, wherein the cancer is refractory, relapsed, or recurrent to the prior treatments.

8. The method of any one of claims 1-7, wherein the prior treatments do not comprise treatment with an mTOR inhibitor.

9. The method of any one of claims 1-8, wherein the prior treatments do not comprise treatment with a composition comprising nanoparticles comprising an mTOR inhibitor and an albumin.sf-6235590Attorney Reference: 638772023240 10. A method of reducing the size of a tumor in an individual having a cancer, the method comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin.

11. The method of claim 10, wherein the cancer is unresectable prior to the administering to the individual the composition comprising nanoparticles comprising sirolimus and the albumin.

12. The method of claim 10 or 11, wherein the reducing the size of the tumor is a reduction of at least about 30% of the tumor as compared to tumor size prior to the administering to the individual the composition comprising nanoparticles comprising sirolimus and the albumin.

13. The method of any one of claims 10-12, wherein the reducing the size of the tumor results in the tumor being suitable for surgical resection.

14. The method of claim 13, further comprising surgically resecting the tumor following the administering to the individual the composition comprising nanoparticles comprising sirolimus and the albumin.

15. The method of any one of claims 1-14, wherein the cancer is selected from the group consisting of a bladder cancer, ovarian cancer, endometrial cancer, colon cancer, leiomyosarcoma, breast cancer, cervical cancer, adrenocortical carcinoma, hepatocellular carcinoma, sarcoma, soft tissue sarcoma, gastrointestinal stromal tumor (GIST), primitive neuroectodermal tumor (PNET), vaginal cancer, osteosarcoma, mesothelioma, head and neck cancer, hepatobiliary cancer, esophagogastric cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, gynecological cancer, and genito-urinary cancer.

16. A method of treating a cancer of the adrenal cortex in an individual in need thereof, the method comprising administering to the individual a composition comprising nanoparticles comprising sirolimus and an albumin.sf-6235590Attorney Reference: 638772023240 17. The method of any one of claims 1-16, wherein the cancer is locally advanced, advanced, malignant, advanced malignant, or metastatic.

18. The method of any one of claims 1-17, wherein the individual is selected for treatment on the basis of having a TSC1 or TSC2 inactivating mutation.

19. The method of claim 18, wherein the individual is selected for treatment on the basis of having the TSC1 inactivating mutation.

20. The method of claim 18, wherein the individual is selected for treatment on the basis of having the TSC2 inactivating mutation.

21. The method of any one of claims 18-20, wherein the inactivating mutation in TSC1 or TSC2 comprises a homozygous deletion, bi-allelic mutations, splice site mutation, frameshift mutation, somatic mutation, truncation, deletion, nonsense mutation in coding region, missense mutation with confirmed impact, or a loss or deletion of TSC1 or TSC2.

22. The method of claim 21, wherein the inactivating mutation in TSC1 or TSC2 is a somatic mutation.

23. The method of claim 21, wherein the inactivating mutation in TSC1 or TSC2 is a truncation, deletion, or frameshift mutation.

24. The method of claim 21, wherein the inactivating mutation in TSC1 or TSC2 comprises bi-allelic mutations.

25. The method of any one of claims 1-24, wherein the method further comprises assessing for the inactivating mutation in TSC1 or TSC2.

26. The method of any one of claims 1-25, wherein the method further comprises assessing if the inactivating mutation in TSC1 or TSC2 is pathogenic.

27. The method of any one of claims 1-26, wherein sirolimus in the sirolimus nanoparticle composition is administered at an amount of about 10 mg / m2to about 150 mg / m2.sf-6235590Attorney Reference: 638772023240 28 The method of any one of claims 1-27, wherein sirolimus in the sirolimus nanoparticle composition is administered at an amount of about 30 mg / m2to about 100 mg / m2.

29. The method of claim 27 or 28, wherein sirolimus in the sirolimus nanoparticle composition is administered at an amount of about 100 mg / m2.

30. The method of claim 27 or 28, wherein sirolimus in the sirolimus nanoparticle composition is administered at an amount of about 75 mg / m2.

31. The method of claim 27 or 28, wherein sirolimus in the sirolimus nanoparticle composition is administered at an amount of about 56 mg / m2.

32. The method of claim 27 or 28, wherein sirolimus in the sirolimus nanoparticle composition is administered at an amount of about 45 mg / m2.

33. The method of claim 27 or 28, wherein sirolimus in the sirolimus nanoparticle composition is administered at an amount of about 35 mg / m2.

34. The method of any one of claims 1-33, wherein the sirolimus nanoparticle composition is administered twice out of every 3 weeks.

35. The method of any one of claims 1-34, wherein the sirolimus nanoparticle composition is administered on days 1 and 8 of a 21-day cycle.

36. The method of any one of claims 1-35, wherein the average diameter of the nanoparticles in the composition is no greater than about 150 nm.

37. The method of claim 36, wherein the average diameter of the nanoparticles in the composition is no greater than about 120 nm.

38. The method of any one of claims 1-7, wherein the sirolimus nanoparticle composition is administered intravenously.

39. The method of any one of claims 1-38, wherein the individual is human.sf-6235590

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