WO2020072090A1 - Use of antineoplastic agents to stimulate the immune system for production of tertiary lymphoid structures (tls) - Google Patents

Abstract

Disclosed is a method of producing tertiary lymphoid structures in a subject with a malignant solid tumor, the method comprising locally administering a composition comprising antineoplastic particles to the tumor of the subject, wherein the antineoplastic particles reside at the tumor site after administration of the composition exposing the tumor to the antineoplastic particles for a sustained amount of time sufficient to stimulate the endogenous immune system of the subject resulting in the production of tertiary lymphoid structures, and infiltration of the tertiary lymphoid structures in and/or around the tumor site. The methods include local administration methods such as topical application, pulmonary administration, intratumoral injection, intravesical instillation, direct injection into tissues surrounding a tumor, and intraperitoneal injection. The presence of tertiary lymphoid structures in and around the tumor site induces tumor destruction.

Description

Use of Antineoplastic Agents to Stimulate the Immune System for Production of Tertiary Lymphoid Structures (TLS)

[0001] This application claims priority to U.S. Provisional Patent Application Serial Nos. 62/740501 filed October 3, 2018 and 62/779320 filed December 13, 2018, each incorporated by reference herein in their entirety.

FIELD

[0002] The present disclosure generally relates to the field of cancer treatment and to the stimulation of the immune system.

[0003] Millions of patients are diagnosed each year world-wide as having cancer, and millions more die from cancer or cancer-related complications each year. The risk of cancer increases significantly with age, many cancers occur more commonly in developed countries, and cancer rates are increasing as life expectancy increases in the developed world. Current therapies include systemic treatments such as intravenous (IV) infusion injection of antineoplastic agents. These therapies, however, generally have significant undesired side effects to the patient due to systemic toxicity, and the antineoplastic agents generally do not reside at the tumor site for very long because of their short half-life in the body.

SUMMARY

[0004] In one aspect, disclosed is a method of producing tertiary lymphoid structures (TLSs) in a subject with a malignant solid tumor, the method comprising locally administering a composition comprising antineoplastic particles to the tumor of the subject, wherein the antineoplastic particles reside at the tumor site after administration of the composition exposing tire tumor to the antineoplastic particles for a sustained amount of time sufficient to stimulate the endogenous immune system of the subject resulting in the production of tertiary lymphoid structures, and infiltration of the tertiary lymphoid structures in and/or around the tumor site. In some embodiments the sustained amount of exposure time is at least 4 weeks. In some embodiments, the sustained amount of exposure time is at least 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, 324, or 336 hours. In various further embodiments, the sustained amount of exposure time is at least 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, tire administering comprises two or more separate administrations. In some embodiments, the administering comprises two or more separate administrations once a week for at least two weeks. In other embodiments, the administering comprises two or more separate administrations twice a week for at least one week, wherein the two or more separate administrations are separated by at least one day. In some embodiments, the two or more separate administrations are administered at or at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 14 days apart. In some embodiments, the two or more separate administrations are administered 2 to 12 weeks apart. In some embodiments, the composition is administered in two to five separate administrations. In some embodiments, the composition is administered in 1, 2, 3, 4, 5, or 6 separate administrations. In other embodiments, the composition is administered in 7 or more separate administrations. In some embodiments, the administering is by pulmonar ' administration, by intratumoral injection administration, by intraperitoneal injection administration, by topical administration, by intravesical instillation or by direct in_jection into tissues surrounding the tumor such as prostate tissue, bladder tissue, and kidney tissue. In some embodiments, the administering is by pulmonary administration. In some embodiments, the pulmonary administration comprises nebulization, wherein the composition further comprises a liquid carrier, wherein the antineoplastic particles are dispersed in the carrier, and wherein the nebulizing results in pulmonary_' delivery_' of aerosol droplets of the composition. In some embodiments, the aerosol droplets have a mass median aerodynamic diameter (MMAD) of between about 0.5 pm to about 6 pm diameter, or between about 1 pm to about 3 pm diameter, or about 2. pm to about 3 pm diameter in some embodiments, the antineoplastic particles have a mean particle size (number) of from 0.1 microns to 5 microns, or from 0.1 microns to 1.5 microns, or from 0.4 microns to 1.2 microns. In some embodiments, the antineoplastic particles are taxane particles. In some embodiments, the taxane particles comprise paclitaxei particles, docetaxel particles, cabazitaxel particles, or combinations thereof. In some embodiments, the taxane particles comprise at least 95% of the taxane. In some embodiments, the taxane particles are paclitaxei particles. In some embodiments the taxane particles are docetaxel particles. In some embodiments, the taxane particles comprise paclitaxei particles and have a specific surface area (SSA) of at least 18 m²/g, 20 m7g, 25 m²/g, 30 m²/g, 32 nf/g, 34 nf/g, or 35 m²/g; or from about 18 m²/g to about 50 m²/g. In other embodiments, the taxane particles comprise docetaxel particles and have an SSA of at least 18 m /g, 20 mVg, 25 ni7g, 30 m g, 35 m /g, 40 m7g, or 42 m7g; or from about 18 m7g and about 60 nf/g. In some embodiments, the taxane particles and/or the docetaxel particles have a bulk density (not-tapped) of 0.05 g/cnr to 0.15 g/cm³. In some embodiments, the

7 taxane particles are not bound to, encapsulated in, or coated with one or more of a monomer, a polymer (or biocompatible polymer), a protein, a surfactant, or albumin. In some embodiments, the taxane particles are in crystalline form

[0005] In some embodiments, the stimulation of the endogenous immune systems produces a cellular (cell-mediated) immune response. In other embodiments, the stimulation of the endogenous immune system produces a humoral immune response. In some embodiments, the tumor is treated as a result of the production and tumor infiltration of the tertiary lymphoid structures. In some embodiments, metastases are reduced or eliminated. [QQ06] Disclosed herein are the following embodiments 1 to 23:

Embodiment 1 is a method of producing tertiary lymphoid structures in a subject with a malignant solid tumor, the method comprising locally administering a composition comprising antineoplastic particles to the tumor of the subject, wherein the antineoplastic particles reside at the tumor site after administration of the composition exposing the tumor to the antineoplastic particles for a sustained amount of time sufficient to stimulate the endogenous immune system of the subject resulting in the production of tertiary' lymphoid structures, and infiltration of the tertiary' lymphoid structures in and/or around the tumor site. Embodiment 2 is the method of embodiment 1, wherein the sustained amount of time is at least 4 weeks.

Embodiment 3 is the method of any one of embodiments 1 or 2, wherein the administering comprises two or more separate administrations.

Embodiment 4 is the method of any one of embodiments 1 -3, wherein the administering comprises two or more separate administrations once a week for at least two weeks.

Embodiment 5 is the method of any one of embodiments 1-3, wherein the administering comprises two or more separate administrations twice a week for at least one week, wherein the two or more separate administrations are separated by at least one day.

Embodiment 6 is the method of any' one of embodiments 1-5, wherein the administering is by pulmonary' administration, by intratumoral injection administration, by intrapentoneal injection administration, or by topical administration.

Embodiment 7 is the method of any one of embodiments 1 -6, wherein the antineoplastic particles have a mean particle size (number) of from 0.1 microns to 5 microns, or from 0.1 microns to 1.5 microns, or from 0.4 microns to 1.2 microns.

Embodiment 8 is the method of any one of embodiments 1-7, wherein the antineoplastic particles are taxane particles. Embodiment 9 is the method of embodiment 8, wherein the taxane particles comprise paclitaxel particles, docetaxel particles, cabazitaxel particles, or combinations thereof.

Embodiment 10 is the method of any one of embodiments 8 or 9, wherein the taxane particles comprise at least 95% of the taxane.

Embodiment 1 1 is the method of any one of embodiments 8-10, wherein the taxane particles are paclitaxel particles.

Embodiment 12 is the method of embodiment 11, wherein the paclitaxel particles have a specific surface area (SSA) of at least 18 m2/g, 20 m2/g, 25 m2/g, 30 m2/g, 32 m2/g, 34 m2/g, or 35 m2/g: or from about 18 m2/g to about 50 m2/g.

Embodiment 13 is the method of any one of embodiments 11 or 12, wherein the paclitaxel particles have a bulk density (not-tapped) of 0.05 g/cm3 to 0.15 g/cm3.

Embodiment 14 is the method of any one of embodiments 8-10, wherein the taxane particles are docetaxel particles.

Embodiment 15 is the method of embodiment 14, wherein the docetaxel particles have a specific surface area (SSA) of at least 18 m2/g, 20 m2/g, 25 m2/g, 30 m2/g, 35 m2/g, 40 m2/g, or 42 m2/g: or from about 18 m2/g and about 60 m2/g.

Embodiment 16 is the method of any one of embodiments 14 or 15, wherein the docetaxel particles have a bulk density (not-tapped) of 0.05 g/cm3 to 0.15 g/cm3.

Embodiment 17 is the method of any one of embodiments 8-16, wherein, the taxane particles are not bound to, encapsulated in, or coated with one or more of a monomer, a polymer (or biocompatible polymer), a protein, a surfactant, or albumin.

Embodiment 18 is the method of any one of embodiments 8-17, wherein tire taxane particles are in crystalline form.

Embodiment 19 is the method of any one of embodiments 1-18, wherein the stimulation of the endogenous immune system produces a cellular immune response.

Embodiment 20 is the method of any one of embodiments 1-18, wherein the stimulation of the endogenous immune system produces a humoral immune response.

Embodiment 21 is the method of any one of embodiments 6-20, wherein the administering is by pulmonar ^ administration .

Embodiment 22 is the method of embodiment 21, wherein the pulmonary administration comprises nebulization, wherein the composition further comprises a liquid carrier, wherein the antineoplastic particles are dispersed in the carrier, and wherein the nebulizing results in pulmonary delivery of aerosol droplets of the composition. Embodiment 23 is the method of embodiment 22, wherein the aerosol droplets have a mass median aerodynamic diameter (MMAD) of between about 0.5 mhi to about 6 pm diameter, or between about 1 pm to about 3 pm diameter, or about 2 pm to about 3 pm diameter.

Embodiment 24 is the method of any one of embodiments 21 -23, wherein the tumor is a lung tumor.

Embodiment 25 is the method of embodiment 24, wherein the lung tumor is non-small-cell lung carcinoma (NSCLC).

Embodiment 26 is the method of embodiment 24, wherein the lung tumor is small-cell lung carcinoma.

Embodiment 27 is the method of any one of embodiments 6-20, wherein the administering is by intratumoral injection.

Embodiment 28 is the method of embodiment 27, wherein the tumor is a bladder tumor.

[0007] The term“antineoplastic agents” as used herein are drugs used to treat neoplasms including cancer, and include “chemotherapeutic agents”, which are drags used to treat cancer. In some embodiments, the antineoplastic agent is a taxane.

[QQ08] The terms“antineoplastic agent particles”,“antineoplastic particles” or“particles of an antineoplastic agent(s)”, as used herein are particles of an antineoplastic agent and have a mean particle size (number) of from about 0.1 microns to about 5 microns (about 100 nm to about 5000 nm) in diameter. In some embodiments, the antineoplastic particles are taxane particles.

[0009] The term“solid tumor” as used herein means one or more abnormal masses of tissue that usually does not contain cysts or liquid areas and that results when cells divide more than they should or do not die when they should. Tumors may be benign (not cancer), or malignant (cancer).

[0010] The term“hydrophobic,” as used herein, describes compounds, compositions, or carriers that have a solubility in water of less than or equal to 10 mg/mL at room temperature.

[0011] The term “volatile,” as used herein, describes compounds, compositions, or carriers that have a vapor pressure greater than or equal to 10 Pa at room temperature.

[0012] Tire term“non-volatile,” as used herein, describes compounds, compositions, or carriers that have a vapor pressure less than 10 Pa at room temperature.

[QQ13] The term“anhydrous,” as used herein with regard to the compositions or carriers of the disclosure, means that less than 3% w/w, less than 2% w/w, less than 1 % w/w, or 0% w/w of water is present in the compositions or carriers. This can account for small amounts of water being present (e.g., water inherently contained in any of the ingredients of the compositions or earners, water contracted from the atmosphere, etc.).

[0014] The terms“skin” or“cutaneous” as used herein mean the epidermis and/or the dennis.

[0015] The term“skin tumor” as used herein means a solid tumor that includes benign skin tumors and malignant skin tumors.

[0016] The terms“skin malignancy” or“malignant skin tumor” as used herein means a solid tumor that includes cancerous skin tumors which includes skin cancers and cutaneous metastases.

[0017] The“affected area” of a skin tumor or skin malignancy as used herein means at least a portion of the skin where the skin tumor or skin malignancy is visibly present on the outermost surface of the skin or directly underneath the surface of the skin (epithelial/dermal covering), and includes areas of the skin in the proximity of the skin tumor or skin malignancy likely to contain visibly undetectable preclinical lesions.

[0018] The terms“cutaneous (skin) metastasis” or“cutaneous (skin) metastases” (plural) as used herein means the manifestation of a malignancy in the skin as a secondary growth (malignant tumor) arising from the primary growth of a cancer tumor at another location of the body. Spread from the primar tumor can be through the lymphatic or blood circulation systems, or by other means.

[0019] The terms “treat”, “treating”, or“treatment” as used herein with respect to treatment of cancer and/or treatment of a tumor means accomplishing one or more of the following: (a) reducing tumor size; (b) reducing tumor growth; (c) eliminating a tumor; (d) reducing or limiting development and/or spreading of metastases, or eliminating metastases; (e) obtaining partial or complete remission of cancer.

[002Q] The terms“subject” or“patient” as used herein mean a vertebrate animal. In some embodiments, the vertebrate animal can be a mammal. In some embodiments, the mammal can be a primate, including a human.

[0021] The term“room temperature” (RT) as used herein, means 15-30°C or 20-25°C

[0022] Tire term“penetration enhancer” or“skin penetration enhancer” as used herein, means a compound or a material or a substance that facilitates drug absorption into the skin (epidermis and dermis).

[0023] The term “surfactant” or “surface active agent” as used herein, means a compound or a material or a substance that exhibits the ability to lower the surface tension of water or to reduce the interfacial tension between two immiscible substances. [0024] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.“And” as used herein is interchangeably used with“or” unless expressly stated otherwise.

[0025] The terms“about” or“approximately” as used herein mean +/- five percent (5%) of the recited unit of measure.

[0026] For this application, a number value with one or more decimal places can be rounded to the nearest whole number using standard rounding guidelines, i.e. round up if the number being rounded is 5, 6, 7, 8, or 9; and round down if the number being rounded is 0, 1 , 2, 3, or 4. For example, 3.7 can be rounded to 4.

[0027] Unless the context clearly requires otherwise, throughout the description and the claims, the words“comprise”,“comprising”, and the like are to he construed in an inclusive or open-ended sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and“below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application. The compositions and methods for their use can“comprise,”“consist essentially of,” or“consist of’ any of the ingredients or steps disclosed throughout the specification. With respect to the phrase“consisting essentially of,” a basic and novel property of the methods of the present disclosure is their ability to stimulate the endogenous immune system of a subject who has a solid tumor and induce the production of tertiary lymphoid structures.

[0028] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.

[0029] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While the specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize.

[0030] FIG. 1 is a graph of animal body weight over time from Orthotopic Lung Cancer study. [0031] FIG. 2 is a graph of animal body weight change over time from Orthotopic Lung Cancer study.

[0032] FIG. 3 is a plot of animal lung weights from Orthotopic Lung Cancer study.

[0033] FIG. 4 is a plot of animal lung to body weight ratios from Orthotopic Lung Cancer study.

[0034] FIG. 5 is a plot of animal lung to brain weight ratios from Orthotopic Lung Cancer study.

[0035] FIG. 6 is a graph of average tumor areas from Orthotopic Lung Cancer study.

[0036] FIG. 7 is a plot of average tumor areas from Orthotopic Lung Cancer study.

[0037] FIG. 8 is a plot of tumor regression from Orthotopic Lung Cancer study.

[0038] FIG. 9 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 1006 (Control) Adenocarcinoma-3, Primitive-1, Regression-0. Primary characteristics of the lung tumor masses. (2x).

[0039] FIG. 10 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide --- 1006 Control, Adenocarcinoma-3, Primitive-1, Regression-0. Primary characteristics of undifferentiated cells within the lung tumor masses.

[0040] FIG. 11 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 1006 (Control) Adenocarcinoma-3, Primitive-! , Regression-0. Primary characteristics of undifferentiated cells within the lung tumor masses.

[0041] FIG. 12 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 1006 (Control) Adenocarcinoma-3, Primitive-! , Regression-0. Primary characteristics of undifferentiated cells within the lung tumor masses showing masses within alveolar spaces. a(20x).

[0042] FIG. 13 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 1006 (Control) Adenocarcinoma-3, Primitive-! , Regression-0. Primary characteristics of primitive ceils within the lung tumor masses. b(10x).

[0043] FIG. 14 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 1006 (Control) Adenocarcinoma-3, Primitive-! , Regression-0. Primary characteristics of primitive cells within the lung tumor masses. b20x.

[0044] FIG. 15 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 1006 (Control) Adenocarcinoma-3, Primitive- 1, Regression-0. Primary characteristics of primitive cells within the lung tumor masses. b(40x). [0045] FIG. 16 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide --- 1006 (Control) Adenocarcinoma-3, Primitive- 1, Regression-0. Primary characteristics of primitive ceils within the lung tumor masses. b(40x).

[0046] FIG. 17 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 1006 (Control) Adenocarcinoma-3, Primitive-1, Regression-0 bronchiole. Primary characteristics of undifferentiated cells showing within bronchiole. c(20x).

[0047] FIG. 18 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 1006 (Control) Adenocarcinoma-3, Primitive-1, Regression-0 glands. Primary characteristics of acinar gland differentiation within the lung tumor masses. d(10x).

[0048] FIG. 19 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 1006 (Control) Adenocarcinoma-3, Primitive-] , Regression-0 glands. Primary characteristics of acinar gland differentiation within the lung tumor masses. d(20x).

[0049] FIG. 20 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 2001 (IV Abraxane®) Adenocarcinoma-2, Primitive- 1, Regression-0. Primary characteristics of the lung tumor mass pushing underneath a bronchiole and showing no evidence of intravascular invasion. (2x).

[0050] FIG. 21 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 2001 (IV Abraxane®) Adenocarcinoma-2, Primitive- 1, Regression-0. Primary characteristics of the lung tumor mass pushing underneath a bronchiole and showing no e vidence of intravascular invasion. (4x).

[0051] FIG. 22 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide -- 2001 (IV Abraxane®¹) Adenocarcinoma-2, Primitive- 1, Regression-0. Primary characteristics of the lung tumor mass pushing underneath a bronchiole. (lOx).

[0052] FIG. 23 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 2003 (IV Abraxane®) Adenocarcinoma- 1, Primitive- 1, Regression- 1 . Characteristics of the lung tumor masses undergoing regression. (4x).

[0053] FIG. 24 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 2003 (IV Abraxane®)) Adenocarcinoma- 1, Primitive- 1, Regression- 1. Characteristics of the lung tumor masses undergoing regression. (l Ox).

[0054] FIG. 25 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide -- 2003 (IV Abraxane®) Adenocarcinoma- 1, Primitive- 1, Regression- 1. Characteristics of the lung tumor masses undergoing regression. (20x).

[0055] FIG. 26 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 2003 (IV Abraxane®¹) Adenocarcinoma- 1, Primitive- 1, Regression- 1. Characteristics of the lung tumor masses undergoing regression. Note lymphocytes and macrophages along the edge. l(40x).

[0056] FIG. 27 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 2003 (IV Abraxane®) Adenocarcinoma- 1 , Primitive- 1, Regression- 1 . Characteristics of the lung tumor masses undergoing regression. Note lymphocytes and macrophages along the edge. 2(40x).

[0057] FIG. 28 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide --- 2003 (IV Abraxane®¹) Adenocarcinoma- 1, Primitive- 1, Regression- 1. Characteristics of the lung tumor masses undergoing regression. Note larger foamy and pigmented macrophages. 2, 2 x(40x).

[0058] FIG. 29 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 2010 (IV Abraxane®)) Adenocarcinoma-3, Primitive- 1, Regression-0. Primary characteristics of the lung tumor masses. (2x).

[0059] FIG. 30 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide --- 2010 (IV Abraxane®¹) Adenocarcinoma-3, Primitive- 1, Regression-0. Primary characteristics of the lung tumor masses. (20x).

[0060] FIG. 31 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 2010 (IV Abraxane®)) Adenocarcinoma-3, Primitive- 1, Regression-0. Primary characteristics of the lung tumor masses. Note subtle evidence of macrophages along the edge. (40x).

[0061] FIG. 32 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide -- 4009 (IH nPac lx High) Adenocarcinoma-0, Primitive-0, Regression-4.

Characteristics of the lung tumor masses that have undergone complete regression. (2x).

[0062] FIG. 33 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 4009 (IH nPac lx High) Adenocarcinoma-0, Primitive-0, Regression-4

Characteristics of a lung tumor mass that has undergone complete regression. Note stromal fibrosis. (lOx).

[0063] FIG. 34 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 4009 (IH nPac lx High) Adenocarcinoma-0, Primitive-0, Regression-4.

Characteristics of a lung tumor mass that has undergone complete regression. Note stromal fibrosis, and lymphocytes and macrophages along the edge. (40x).

[0064] FIG. 35 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 5010 (IH nPac 2x Low) Adenocarcinoma- 1, Primitive-0, Regression-3.

Characteristics of the lung tumor masses undergoing regression. (2x). [0065] FIG. 36 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide -- 5010 (1H nPac 2x Low) Adenocarcinoma- 1, Primitive-0, Regression-3.

Characteristics a lung tumor mass that is undergoing regression. (lOx).

[0066] FIG. 37 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 5010 (IH nPac 2x Low) Adenocarcinoma- 1, Primitive-0, Regression-3.

Characteristics a lung tumor mass that is undergoing regression. (20x).

[0067] FIG. 38 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide -- 5010 (IH nPac 2x Low) Adenocarcinoma- 1, Primitive-0, Regression-3.

Characteristics a lung tumor mass that is undergoing regression. (40x).

[0068] FIG. 39 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 6005 (IH nPac 2x High) Adenocarcinoma- 1 , Primitive-0, Regression-4.

Characteristics a lung tumor mass drat is undergoing regression. (2x).

[0069] FIG. 40 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 6005 (IH nPac 2x High) Adenocarcinoma- 1, Primitive-0, Regression-4.

Characteristics a lung tumor mass that is undergoing regression. Note stromal fibrosis, and lymphocytes and macrophages along the edge. ( 1 Ox) .

[0070] FIG. 41 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 6005 (IH nPac 2x High) Adenocarcinoma- 1 , Primitive-0, Regression-4.

Characteristics a lung tumor mass that is undergoing regression. Note lymphocytes and macrophages along the edge. (20x).

[0071] FIG. 42 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide -- 6005 (IH nPac 2x High) Adenocarcinoma- 1, Primitive-0, Regression-4. Note lymphocytes and macrophages along the edge. (40x).

[0072] FIG. 43 is a photomicrograph of H&E Stained Orthotopic Lung Cancer tissue slide - 6005 (IH nPac 2x High) Adenocarcinoma- 1 , Primitive-0, Regression-4. Note the presence of a focal area of residual tumor cells within an alveolus. 2(40x).

[0073] FIG. 44 are various photomicrographs of tire Orthotopic Lung Cancer tissue slides - (Control). Top row: H/E stained sections. Bottom row: Immunohistochemical staining with Keratin or CD 1 lb.

[0074] FIG. 45 are various photomicrographs of the Orthotopic Lung Cancer tissue slides - (IV Abraxane®). Top row: H/E stained sections. Bottom row: Immunohistochemical staining with Keratin or CD 1 lb.

[0075] FIG. 46 are various photomicrographs of the Orthotopic Lung Cancer tissue slides - (Inhaled nPac). Various staining with H/E stain, Trichrome, Keratin and CD l ib. [0076] FIG. 47 are various photomicrographs of the Orthotopic Lung Cancer tissue slides showing presence of TLSs.

[0077] FIG. 48 is a graph of mean tumor volumes over time from the bladder cancer xenograft study. The arrows on the x-ax!S represent the administration points.

[0078] FIG. 49 is a graph of individual tumor volumes over time for Vehicle 3 cycles from the bladder cancer xenograft study. The triangles on the x-axis represent an administration point.

[0079] FIG. 50 is a graph of individual tumor volumes over time for the Docetaxel IV 3 cycles from the bladder cancer xenograft study. The triangles on the x-axis represent the administration points .

[0080] FIG. 51 is a graph of individual tumor volumes over time for the nanoparticulate docetaxel (nDoce) IT 1 cycle from the bladder cancer xenograft study. The triangle on the x- axis represent the single administration point.

[0081] FIG. 52 is a graph of individual tumor volumes over time for the nDoce IT 2 cycles from the bladder cancer xenograft study. The triangles on the x-axis represent the administration points.

[0082] FIG. 53 is a graph of individual tumor volumes over time for the nDoce 3 cycles from the bladder cancer xenograft study. The triangles on the x-axis represent the administration points.

[0083] FIG. 54 is a scatter plot of tumor volumes at end of study over tumor volumes at Day 1 treatment from the bladder cancer xenograft study.

[0084] FIG. 55 is a graph of mean body weights over time from the bladder cancer xenograft study. The arrows on the x-axis represent the administration points.

[0085] FIG. 56 is a graph of mean tumor volumes at Day 61 for each administration group from the bladder cancer xenograft study.

[0086] FIG. 57 are photos of animals from each administration group at Day 27, Day 40 and Day 61 post tumor implant from tire bladder cancer xenograft study.

[0087] FIG. 58 a graph of concentrations of docetaxel in tumor tissue for nDoce 1 cycle, 2 cycles, and 3 cycles from the bladder cancer xenograft study.

[0088] FIG. 59 is a photomicrograph of bladder cancer xenograft tissue slide - IT Vehicle Control. H&E. Magnification 2.52 x.

[0089] FIG. 60 is a photomicrograph of bladder cancer xenograft tissue slide - IT Vehicle Control. H&E. Magnification 6.3 x. [0090] FIG. 61 is a photomicrograph of bladder cancer xenograft tissue slide - IT Vehicle Control. H&E. Magnification 25.2 x.

[0091] FIG. 62 is a photomicrograph of bladder cancer xenograft tissue slide - IV Docetaxel 3 cycles. H&E. Magnification 2.52 x.

[0092] FIG. 63 is a photomicrograph of bladder cancer xenograft tissue slide - IV Docetaxel 3 cycles. H&E. Magnification 6.3 x.

[0093] FIG. 64 is a photomicrograph of bladder cancer xenograft tissue slide - IV Docetaxel 3 cycles. H&E. Magnification 25.2 x.

[0094] FIG. 65 is a photomicrograph of bladder cancer xenograft tissue slide - IT nDoce 2 cycles. H&E. Magnification 2.52 x.

[0095] FIG. 66 is a photomicrograph of bladder cancer xenograft tissue slide - IT nDoce

2 cycles. H&E. Magnification 6.3 x.

[0096] FIG. 67 is a photomicrograph of bladder cancer xenograft tissue slide --- IT nDoce

3 cycles. H&E. Magnification 2.52. x.

[0097] FIG. 68 is a photomicrograph of bladder cancer xenograft tissue slide - IT nDoce 3 cycles. H&E. Magnification 2.52 x.

[0098] FIG. 69 is a photomicrograph of bladder cancer xenograft tissue slide - IT nDoce 3 cycles. H&E. Magnification 25.2 x.

[0099] FIG. 70 is a photomicrograph of bladder cancer xenograft tissue slide --- IT Vehicle Control 3 cycles F4/80 stain. Magnification 2.52 x.

[00100] FIG. 71 is a photomicrograph of bladder cancer xenograft tissue slide - IV Docetaxel 3 cycles F4/80 stain. Magnification 2.52 x.

[00101] FIG. 72 is a photomicrograph of bladder cancer xenograft tissue slide - IT nDoce 3 cycles F4/80 stain. Magnification 2.52 x.

[00102] FIG. 73 are various photomicrographs of Control Cases of bladder cancer xenograft tissue slides. H&E stain and CD68 stain.

[00103] FIG. 74 are various photomicrographs of IT nDoce cases of bladder cancer xenograft tissue slides. Top row: One cycle nDoce ( lx). Second row: Two cycles of nDoce treatment (2x). Third row: Two cycles of nDoce treatment (2x). Fourth row: Three cycles of nDoce treatment (3x).

DETAILED DESCRIPTION

[00104] Disclosed are methods for stimulating the endogenous immune system of a subject who has a malignant solid tumor to produce tertiary lymphoid structures (TLSs). Disclosed is a method of producing tertiary lymphoid structures in a subject with a malignant solid tumor, the method comprising locally administering a composition comprising antineoplastic particles to the tumor of the subject, wherein the antineoplastic particles reside at the tumor site after administration of the composition exposing the tumor to the antineoplastic particles for a sustained amount of time sufficient to stimulate the endogenous immune system of the subject resulting in the production of tertiary lymphoid structures, and infiltration of the tertiary lymphoid structures in and/or around the tumor site. The stimulation of the endogenous immune systems can produce a cellular (cell -mediated) immune response or a humoral immune response. In some embodiments, metastases are reduced or eliminated. The sustained amount of exposure time can be at least 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, 324, or 336 hours, or can be at least 3, 4, 5, 6, 7, or 8 weeks.

[00105] The inventors have surprisingly discovered that the methods disclosed herein stimulate the endogenous immune system resulting in the production of tertiary lymphoid structures that have infiltrated in and around the tumor site inducing tumor destruction. Secondary lymphoid organs develop as part of a genetically preprogrammed process during embryogenesis and primarily serve to initiate adaptive immune response providing a location for interactions between rare antigen-specific naive lymphocytes and antigen-presenting cells draining from local tissue. Organogenesis of secondary_' lymphoid tissues can also be recapitulated in adulthood during de novo lymphoid neogenesis of tertiary lymphoid structures (TLSs) and form m the inflamed tissue afflicted by various pathological conditions, including cancer. Organogenesis of mucosal-associated lymphoid tissue such as bronchial- associated lymphoid tissue is one such example. The term TLS can refer to structures of varying organization, from simple clusters of lymphocytes, to sophisticated, segregated structures highly reminiscent of secondary_' lymphoid organs. A notable difference between lymph nodes and TLSs is the that where lymph nodes are encapsulated, TLSs represent a congregation of immune and stromal cells confined within an organ or tissue.

[00106] Without being limited to any specific mechanism, such effect may comprise, for example, providing sufficient time for lymphocytes to activate both their innate as well as adaptive immunological response to the tumor, all without the added associated toxicities of IV chemotherapy. For example, and without being limited to any specific mechanism, local tumor cell killing by the administration of antineoplastic particles such as taxane particles releases tumor cell antigens which are identified by antigen presenting cells. The activated antigen-presenting cells may then present tumor-specific antigen to T-cells, B-cells and other tumoricidal cells that circulate throughout the patient’s vascular system as woll as enter tissues tliat contain tumor allowing for destruction of cancer throughout the patient. Thus, methods disclosed herein allow for direct local therapy, as well as indirect immune system- mediated local and systemic cancer cell killing. For example, the methods disclosed herein provide the local anti neoplastic agent particles, such as taxane particles, to act as an adjuvant to stimulate die immune response. Local concentration of taxane remains elevated in the tumor for greater than 4 days, or at least 14 days, or at least 4 weeks, which provides sufficient time for the tumor to be exposed to the taxane for killing of local tumor cells as well as stimulation of the immune response appropriate for killing of cancer that may be widely disseminated through the body. This stimulation of die immune system by local administration of taxane particles occurs without producing concomitant high levels of taxane m the patient’s circulating blood. Thus, local administration of particle taxane does not reduce hematopoiesis in the bone marrow involving reduction in white blood cell numbers such as lymphocytes. Bone marrow suppression is a common side effect of taxanes when given IV due to the high concentrations of circulating taxane. Thus, locally administering the taxane particles directly to a tumor is in effect, a tumor vaccine given its effect in stimulating the endogenous immune system.

[00107] Without being limited to any specific mechanism, the methods disclosed herein may produce sufficient concentrations of taxanes for a prolonged period to stimulate local immunological response through activation of dendritic cells, one type of antigen presenting ceil. Activation of dendritic cells can occur most notably in tire skin or lung where they are found in abundance. Topical administration of taxane particles to skin tumors causes entry of taxane into tumor cells which kills them during their division cycle rendering them more accessible to immune recognition. Dendritic cells in the area would become activated by the increased access to tumor antigen and would subsequently present antigen to lymphocytes. The lymphocytes would then circulate throughout the patient’s body producing humoral mediators that are specific to the cell surface antigens of the tumor cells. The lymphocytes destroy tumor located in the skin as well as distant metastasis. Lymphocyte tumor killing could also occur via the cellular route of immune surveillance. For example, topical administration of taxane particles to a cutaneous metastasis would result in eradication of the patient’s cancer throughout their body, not just the cutaneous metastasis. The same elimination of cancer in the body would happen to metastatic lung cancer in response to inhaled taxane particles. Likewise, the same elimination of cancer in the body would happen to malignant solid tumors in response to intratumoral injection of taxane particles or to intraperitonea! injection of taxane particles. I. Antineoplastic Agents and Antineoplastic Agent Particles

[QQ108] Antineoplastic agents are drags used to treat neoplasms including cancer, and include "‘chemotherapeutic agents”, which are drags used to treat cancer. Suitable antineoplastic agents include those that stimulate an immunological response when administered to a subject. The antineoplastic agent can be a taxane such as paclitaxel or docetaxel. Non-limiting examples of antineoplastic agents include taxane s (paclitaxel, derivatives of paclitaxel, docetaxel, cabazitaxel, etc.); epithilones; Vinca alkaloids, such as vinblastine, vincristine, vindesine, vinorelbine; camptothecin analogs; epipodophyllotoxins, such as cisplatin, carboplatin, oxaliplatin, etoposide and teniposide; doxorubicin, anthrcyclines, 5-fluorouracil, topotecan, gemcitabine, peroxisome prohferator-activated receptor (PPAR) ligands, and antiangiogenics. Other non-limiting examples of antineoplastic agents can be found listed in the“Ashgate Handbook of Antineoplastic Agents”, published by- Gower Publishing Limited, 2000, herein incorporated by reference. Antineoplastic agents can be in the form of particles (antineoplastic particles). Antineoplastic agent particles have a mean particle size (number) of from about 0.1 microns to about 5 microns (about 100 nm to about 5000 nm) in diameter. In some embodiments, the antineoplastic agent particles have a mean particle size (number) of from about 0.1 microns to about 1.5 microns (about 100 nm to about 1500 nm) in diameter. In some embodiments, the antineoplastic agent particles have a mean particle size (number) of from about 0.1 microns to less than 1 micron (about 100 nm to less than 1000 nm) m diameter. In some embodiments, the antineoplastic agent particles have a mean particle size (number) of from about 0.4 microns to about 1.2 microns (about 400 nm to about 1200 nm). The antineoplastic agent particles are in a size range where they are unlikely to be carried out of the tumor by systemic circulation and yet benefit from the high specific surface area to provide enhanced solubilization and release of the drag.

[00109] In some embodiments, the antineoplastic particles are solid, uncoated (“neat” or “naked”) individual particles. In some embodiments, the antineoplastic particles are not bound to any substance. In some embodiments, no substances are absorbed or adsorbed onto the surface of the antineoplastic particles. In some embodiments, the antineoplastic agents or antineoplastic particles are not encapsulated, contained, enclosed or embedded within any substance. In some embodiments, the antineoplastic particles are not coated with any substance. In some embodiments, the antineoplastic particles are not microemulsions, nanoemulsions, microspheres, or liposomes containing an antineoplastic agent. In some embodiments, the antineoplastic particles are not bound to, encapsulated in, or coated with one or more of a monomer, a polymer (or biocompatible polymer), a protein, a surfactant, or albumin. In some embodiments, a monomer, a polymer (or biocompatible polymer), a protein, a surfactant, or albumin is not absorbed or adsorbed onto the surface of the antineoplastic particles. In some embodiments, the antineoplastic particles are in crystalline form. In other embodiments, the antineoplastic particles are in amorphous form, or a combination of both crystalline and amorphous form. In some embodiments, the antineoplastic particles of the disclosure contain traces of impurities and byproducts typically found during preparation of the antineoplastic agent. In some embodiments, the antineoplastic particles comprise at least 90 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the antineoplastic agent, meaning tire antineoplastic particles consist of or consist essentially of substantially pure antineoplastic agent.

[00110] In some embodiments, the antineoplastic particles are coated with or bound to a substance such as a protein (e.g., albumin), a monomer, a polymer, a biocompatible polymer, or a surfactant. In some embodiments, a substance such as a protein (e.g., albumin), a monomer, a polymer, a biocompatible polymer, or a surfactant is adsorbed or absorbed onto the surface of the antineoplastic particles. In some embodiments, the antineoplastic particles are encapsulated, contained, enclosed, or embedded within a substance such as a protein (e.g., albumin), a monomer, a polymer, a biocompatible polymer, and/or a surfactant. In some embodiments, the antineoplastic particles are microemulsions, nanoemulsions, microspheres, or liposomes containing an antineoplastic agent. In some embodiments, the antineoplastic particles are non-agglomerated individual particles and are not clusters of multiple antineoplastic particles that are bound together by interactive forces such as non-covaient interactions, van der Waal forces, hydrophilic or hydrophobic interactions, electrostatic interactions, Coulombic forces, interactions with a dispersion material, or interactions via functional groups. In some embodiments, the taxane particles are individual antineoplastic particles that are fonned by the agglomeration of smaller particles which fuse together forming the larger individual antineoplastic particles, all of which occurs during the processing of the antineoplastic particles. In other embodiments, the antineoplastic particles are clusters or agglomerates of antineoplastic particles that are bound together by interactive forces such as non-covalent interactions, van der Waal forces, hydrophilic or hydrophobic interactions, electrostatic interactions, Coulombic forces, interactions with a dispersion material, or interactions via functional groups.

[00111] In some embodiments, the antineoplastic particles are taxane particles. Taxane s are poorly water-soluble compounds generally having a solubility' of less than or equal to 10 mg/niL m water at room temperature. Taxanes are widely used as antineoplastic agents and chemotherapy agents. The term“taxanes” as used herein include paditaxel (I), docetaxel (II), cabazitaxel (III), and any other taxane or taxane derivatives, non-limiting examples of which are taxoi B (cephalomannine), taxol C, taxol D, taxol E, taxol F, taxol G, taxadiene, baccatin III, 10-deacetylbaccatm, taxchinin A, hrevifoliol, and taxuspme D, and also include pharmaceutically acceptable salts of taxanes.

(I) paditaxel

[00112] Paditaxel and docetaxel active pharmaceutical ingredients (APIs) are commercially available from Phyton Biotech LLC, Vancouver, Canada The docetaxe! API contains not less than 90%, or not less than 95%, or not less than 97.5% docetaxel calculated on the anhydrous, solvent-free basis. The paditaxel API contains not less than 90%, or not less than 95%, or not less than 97% paditaxel calculated on the anhydrous, solvent-free basis. In some embodiments, the paditaxel API and docetaxel API are USP and/or EP grade Paditaxel API can be prepared from a semisynthetic chemical process or from a natural source such as plant cell fermentation or extraction. Paditaxel is also sometimes referred to by the trade name TAXOL®, although this is a misnomer because TAXOL® is the trade name of a solution of paclitaxel in polyoxy ethylated castor oil and ethanol intended for dilution with a suitable parenteral fluid prior to intravenous infusion Taxane APIs can be used to make taxane particles. The taxane particles can be paclitaxel particles, docetaxel particles, or cabazitaxel particles, or particles of other taxane derivatives, including particles of pharmaceutically acceptable salts oftaxanes.

[00113] Taxane particles have a mean particle size (number) of from about 0.1 microns to about 5 microns (about 100 nrn to about 5000 nm) in diameter. In some embodiments, the taxane particles are solid, uncoated (neat) individual particles. The taxane particles are in a size range where they are unlikely to be carried out of the tumor by systemic circulation and yet benefit from the high specific surface area to provide enhanced solubilization and release of the drug. In some embodiments, the taxane particles are not bound to any substance. In some embodiments, no substances are absorbed or adsorbed onto the surface of the taxane particles. In some embodiments, the taxane or taxane particles are not encapsulated, contained, enclosed or embedded within any substance. In some embodiments, the taxane particles are not coated with any substance. In some embodiments, the taxane particles are not microemulsions, nanoemulsions, microspheres, or liposomes containing a taxane. In some embodiments, the taxane particles are not bound to, encapsulated in, or coated with one or more of a monomer, a polymer (or biocompatible polymer), a protein, a surfactant, or albumin. In some embodiments, a monomer, a polymer (or biocompatible polymer), a protein, a surfactant, or albumin is not absorbed or adsorbed onto the surface of the taxane particles. In some embodiments, the composition and tire taxane particles exclude albumin. In some embodiments, the taxane particles are in crystalline form. In other embodiments, the taxane particles are in amorphous form, or a combination of both crystalline and amorphous form. In some embodiments, the taxane particles of the disclosure contain traces of impurities and byproducts typically found during preparation of the taxane. In some embodiments, the taxane particles comprise at least 90 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the taxane, meaning the taxane particles consist of or consist essentially of substantially pure taxane.

[00114] In some embodiments, the taxane particles are coated with or bound to a substance such as a protein (e.g., albumin), a monomer, a polymer, a biocompatible polymer, and/or a surfactant. In some embodiments, a substance such as a protein (e.g , albumin), a monomer, a polymer, a biocompatible polymer, or a surfactant is adsorbed or absorbed onto the surface of the taxane particles. In some embodiments, the taxane particles are encapsulated, contained, enclosed, or embedded within a substance such as a protein (e.g., albumin), a monomer, a polymer, a biocompatible polymer, or a surfactant. In some embodiments, the taxane particles are microemulsions, nanoemulsions, microspheres, or liposomes containing a taxane. In some embodiments, the taxane particles are non agglomerated individual particles and are not clusters of multiple taxane particles that are bound together by interactive forces such as non-covalent interactions, van der Waal forces, hydrophilic or hydrophobic interactions, electrostatic interactions, Coulombic forces, interactions with a dispersion material, or interactions via functional groups. In some embodiments, the taxane particles are individual taxane particles that are formed by the agglomeration of smaller particles which fuse together forming the larger individual taxane particles, all of which occurs during the processing of the taxane particles. In some embodiments, the taxane particles are clusters or agglomerates of taxane particles that are bound together by interactive forces such as non-covalent interactions, van der Waal forces, hydrophilic or hydrophobic interactions, electrostatic interactions, Coulombic forces, interactions with a dispersion material, or interactions via functional groups.

[00115] The antineoplastic particles or taxane particles (including but not limited to paclitaxel particles, docetaxel particles, or cabazitaxel particles) can have a mean particle size (number) of from 0.1 microns to 5 microns, or from 0.1 microns to 2 microns, or from 0.1 microns to 1.5 microns, or from 0.1 microns to 1.2 microns, or from 0.1 microns to 1 micron, or from 0.1 microns to less than 1 micron, or from 0.1 microns to 0.9 microns, or from 0.1 microns to 0.8 microns, or from 0.1 microns to 0.7 microns, or from 0.2 microns to 5 microns, or from 0.2 microns to 2 microns, or from 0.2 microns to 1.5 microns, or from 0.2 microns to 1.2 microns, or from 0.2 microns to 1 micron, or from 0.2 microns to less than 1 micron, or from 0.2 microns to 0.9 microns, or from 0.2 microns to 0.8 microns, or from 0.2. microns to 0.7 microns, or from 0.3 microns to 5 microns, or from 0.3 microns to 2 microns, or from 0.3 microns to 1.5 microns, or from 0.3 microns to 1.2 microns, or from 0.3 microns to 1 micron, or from 0.3 microns to less than 1 micron, or from 0.3 microns to 0.9 microns, or from 0.3 microns to 0.8 microns, or from 0.3 microns to 0.7 microns, or from 0.4 microns to 5 microns, or from 0.4 microns to 2 microns, or from 0.4 microns to 1.5 microns, or from 0.4 microns to 1.2 microns, or from 0.4 microns to 1 micron, or from 0.4 microns to less than 1 micron, or from 0.4 microns to 0.9 microns, or from 0.4 microns to 0.8 microns, or from 0.4 microns to 0.7 microns, or from 0.5 microns to 5 microns, or from 0.5 microns to 2 microns, or from 0.5 microns to 1.5 microns, or from 0.5 microns to 1.2 microns, or from 0.5 microns to 1 micron, or from 0.5 microns to less than 1 micron, or from 0.5 microns to 0.9 microns, or from 0.5 microns to 0.8 microns, or from 0.5 microns to 0.7 microns, or from 0.6 microns to 5 microns, or from 0.6 microns to 2 microns, or from 0.6 microns to 1.5 microns, or from 0.6 microns to 1.2 microns, or from 0.6 microns to 1 micron, or from 0.6 microns to less than 1 micron, or from 0.6 microns to 0.9 microns, or from 0.6 microns to 0.8 microns, or from 0.6 microns to 0.7 microns. The antineoplastic particles or taxane particles are in a size range where they are unlikely to be carried out of the tumor by systemic circulation and yet benefit from the high specific surface area to provide enhanced solubilization and release of the drag.

[00116] The particle size of the antineoplastic particles including taxane particles can be determined by a particle size analyzer instrument and the measurement is expressed as the mean diameter based on a number distribution (number). A suitable particle size analyzer instrument is one which employs the analytical technique of light obscuration, also referred to as photozone or single particle optical sensing (SPOS). A suitable light obscuration particle size analyzer instrument is the ACCUSIZER, such as the ACCUSIZER 780 SIS, available from Particle Sizing Systems, Port Richey, Florida. Another suitable particle size analyzer instrument is one which employs laser diffraction, such as the Shimadzu SALD-7101.

[QQ117] Antineoplastic agent particles including taxane particles can be manufactured using various particle size-reduction methods and equipment known in the art. Such methods include, but are not limited to conventional particle size-reduction methods such as wet or dry milling, micronizing, disintegrating, and pulverizing. Other methods include“precipitation with compressed anti-solvents” (PCA) such as with supercritical carbon dioxide. In various embodiments, the antineoplastic and/or taxane particles are made by PCA methods as disclosed in US patents US 5874029, US 5833891, US 6113795, US 7744923, US 8778181, US 9233348; US publications US 2015/0375153, US 2016/0354336, US 2016/0374953: and international patent application publications WO 2016/197091, WO 2016/197100, and WO 2016/197101 ; all of which are herein incorporated by reference.

[00118] In PCA particle size reduction methods using supercritical carbon dioxide, supercritical carbon dioxide (anti-solvent) and solvent, e.g. acetone or ethanol, are employed to generate uncoated antineoplastic or taxane particles as small as 0.1 to 5 microns within a well-characterized particle-size distribution Tire carbon dioxide and solvent are removed during processing (up to 0.5% residual solvent may remain), leaving antineoplastic or taxane particles as a powder. Stability' studies show' that the pachtaxel particle pow'der is stable in a vial dose form when stored at room temperature for up to 59 months and under accelerated conditions (40°C/75% relative humidity) for up to six months. [00119] Taxane particles produced by various supercritical carbon dioxide particle size reduction methods can have unique physical characteristics as compared to taxane particles produced by conventional particle size reduction methods using physical impacting or grinding, e.g., wet or dry milling, micronizing, disintegrating, comminuting, microfluidizing, or pulverizing. As disclosed in US publication 2016/0374953, herein incorporated by- reference, such unique characteristics include a bulk density (not tapped) between 0.05 g/cm³ and 0.15 g/cm³ and a specific surface area (SSA) of at least 18 m²/g of taxane (e.g., paclitaxel and docetaxel) particles, which are produced by the supercritical carbon dioxide particle size reduction methods described in US publication 2016/0374953 and as described below'. This bulk density range is generally lower than the bulk density of taxane particles produced by conventional means, and the SSA is generally higher than the SSA of taxane particles produced by conventional means. These unique characteristics result in significant increases in dissolution rates m water / methanol media as compared to taxanes produced by conventional means. As used herein, the“specific surface area” (SSA) is the total surface area of the taxane particle per unit of taxane mass as measured by the Brunauer-Emmett- Teller (“BET”) isotherm by the following method: a known mass between 200 and 300 mg of the analyte is added to a 30 mL sample tube. The loaded tube is then mounted to a Porous Materials Inc. SORPTOMETER^®, model BET-202A. The automated test is then canted out using the BETWIN^® software package and the surface area of each sample is subsequently- calculated. As will be understood by those of skill in the art, the “taxane particles” can include both agglomerated taxane particles and non-aggiomerated taxane particles; since the SSA is determined on a per gram basis it takes into account both agglomerated and non agglomerated taxane particles in the composition. The agglomerated taxane particles are defined herein as individual taxane particles that are fonned by the agglomeration of smaller particles which fuse together forming the larger individual taxane particles, all of which occurs during the processing of the taxane particles. The BET specific surface area test procedure is a compendial method included in both the United States Pharmaceopeia and the European Pharmaceopeia. The bulk density measurement can be conducted by pouring the taxane particles into a graduated cylinder without tapping at room temperature, measuring the mass and volume, and calculating the bulk density.

[00120] As disclosed in US publication 2016/0374953, studies showed a SSA of 15.0 m²/g and a bulk density of 0.31 g/cm³ for paclitaxel particles produced by milling paclitaxel in a Deco-PBM-V-0.41 ball mill suing a 5 mm ball size, at 600 RPM for 60 minutes at room temperature. Also disclosed in US publication 2016/0374953, one lot of paclitaxel particles had a SSA of 37.7 m²/g and a bulk density of 0.085 g/euri when produced by a supercritical carbon dioxide method using the following method: a solution of 65 mg/ml of paclitaxei was prepared in acetone. A BETE MicroWhirl'⁸' fog nozzle (BETE Fog Nozzle, Inc.) and a sonic probe (Qsonica, model number Q700) were positioned in the crystallization chamber approximately 8 mm apart. A stainless steel mesh filter with approximately 100 nm holes was attached to the crystallization chamber to collect the precipitated paclitaxei particles. The supercritical carbon dioxide was placed in the crystallization chamber of the manufacturing equipment and brought to approximately 1200 psi at about 38 °C and a flow' rate of 24 kghour. The sonic probe was adjusted to 60% of total output power at a frequency of 20 kHz. The acetone solution containing the paclitaxei was pumped through the nozzle at a flow rate of 4.5 mL/mmute for approximately 36 hours. Additional lots of paclitaxei particles produced by the supercritical carbon dioxide method described above had SSA values of: 22.27 m²/g, 23.90 m²/g, 26.19 m²/g, 30.02 m²/g, 31.16 m²/g, 31.70 m²/g, 32.59 m²/g, 33.82 m²/g, 35.90 m²/g, 38.22 m²/g, and 38.52 m²/g

[00121] As disclosed in US publication 2016/0374953, studies showed a SSA of 15.2 m²/g and a bulk density of 0.44 g/cm^J for docetaxel particles produced by milling docetaxel in a Deco-PBM-V-0.41 ball mill suing a 5 mm ball size, at 600 RPM for 60 minutes at room temperature. Also disclosed in US publication 2016/0374953, docetaxel particles had a SSA of 44.2 m²/g and a bulk density of 0.079 g/cm³ when produced by a supercritical carbon dioxide method using the following method: A solution of 79.32 mg/mi of docetaxel was prepared in ethanol. The nozzle and a sonic probe were positioned in the pressurizable chamber approximately 9 mm apart. A stainless steel mesh filter with approximately 100 nm holes was attached to die pressurizable chamber to collect the precipitated docetaxel particles. The supercritical carbon dioxide was placed in the pressurizable chamber of the manufacturing equipment and brought to approximately 1200 psi at about 38 °C and a flow rate of 68 slpm. Hie sonic probe was adjusted to 60% of total output power at a frequency of 20 kHz. The ethanol solution containing the docetaxel was pumped through the nozzle at a flow rate of 2 mL/minute for approximately 95 minutes). The precipitated docetaxel agglomerated particles and smaller docetaxel particles were then collected from the supercritical carbon dioxide as the mixture is pumped through the stainless steel mesh filter. The filter containing the particles of docetaxel was opened and the resulting product was collected from the filter.

[00122] As disclosed in US publication 2016/0374953, dissolution studies showed an increased dissolution rate in methanol/water media of paclitaxei and docetaxel particles made by the supercritical carbon dioxide methods described in US publication 2016/0374953 as compared to paclitaxel and docetaxel particles made by milling paclitaxel and docetaxel using a Deco-PBM-V-0 41 ball mill suing a 5 mm ball size, at 600 RPM for 60 minutes at room temperature. The procedures used to determine the dissolution rates are as follows. For paclitaxel, approximately 50 rng of material were coated on approximately 1.5 grams of 1 mm glass beads by tumbling the material and beads in a vial for approximately 1 hour. Beads were transferred to a stainless steel mesh container and placed in the dissolution bath containing methanol/water 50/50 (v/v) media at 37°C, pH 7, and a USP Apparatus II (Paddle), operating at 75 rpm. At 10, 20, 30, 60, and 90 minutes, a 5 mL aliquot was removed, filtered through a 0.22 pm filter and analyzed on a UV/VIS spectrophotometer at 227 nm. Absorbance values of the samples were compared to those of standard solutions prepared in dissolution media to determine the amount of material dissolved. For docetaxel, approximately 50 mg of material was placed directly in the dissolution bath containing methanol/water 15/85 (v/v) media at 37°C, pH 7, and a USP Apparatus II (Paddle), operating at 75 rpm. At 5, 15, 30, 60, 120 and 225 minutes, a 5 mL aliquot was removed, filtered through a 0.22 pm filter, and analyzed on a UV/VIS spectrophotometer at 232 mu. Absorbance values of the samples were compared to those of standard solutions prepared in dissolution media to determine the amount of material dissolved. For paclitaxel, the dissolution rate was 47% dissolved in 30 minutes for the particles made by the supercritical carbon dioxide method versus 32% dissolved in 30 minutes for the particles made by milling. For docetaxel, the dissolution rate was 27% dissolved in 30 minutes for the particles made by the supercritical carbon dioxide method versus 9% dissolved in 30 minutes for the particles made by milling.

[00123] In some embodiments, the antineoplastic particles have a SSA of at least 10, at least 12, at least 14, at least 16, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, or at least 35 m²/g. In one embodiment, the antineoplastic particles have an SSA of between about 10 m²/g and about 50 in /g. In some embodiments, the antineoplastic particles have a bulk density between about 0.050 g/cm³ and about 0.20 g/cm^J.

[00124] In further embodiments, the antineoplastic particles have a SSA of:

(a) between 16 m²/g and 31 m²/g or between 32 m g and 40 m7g;

(b) betweenl 6 m²/g and 30 m²/g or between 32 m7g and 40 m7g;

(c) between 16 m²/g and 29 m²/g or between 32 m²/g and 40 m²/g; (d) between 17 m²/g and 31 m²/g or between

(e) between!7 m²/g and 30 m²/g or between

(f) between! 7 m²/g and 29 m²/g, or between

(g) between! 6 m²/g and 3 ! m²/g or between

(h) betweenlb m7g and 30 m²/g or between

(i) between 16 m²/g and 29 m²/g or between

(j) between!? m²/g and 31 m²/g or between

(k) between 17 m²/g and 30 m²/g or between

(!) between!7 m²/g and 29 m²/g, or between

(m) between ! 6 m²/g and 31 m²/g, or >32 m7g;

(h) between! 7 m²/g and 3 ! m²/g, or > 32 m7g;

(i) betweenlb m²/g and 30 m7g, or > 32 m7g;

(j) between! 7 m²/g and 30 m²/g, or > 32 m7g;

(k) between 16 m²/g and 29 m²/g, or > 32 m7g;

(l) between! 7 m²/g and 29 m²/g, or > 32 m7g;

(m) between i 6 m²/g and 3 ! m²/g, or >33 m7g;

(n) between! 7 m²/g and 3 ! m²/g, or > 33 m7g;

(o) between 16 m²/g and 30 m²/g, or > 33 m7g;

(p) between! 7 m²/g and 30 m²/g, or > 33 m7g;

(q) between 16 m²/g and 29 m²/g, or > 33 m7g; or

(r) between! 7 m²/g and 29 m²/g, or > 33 m7g.

[00125] In some embodiments, the antmeopiastic particles are taxane particles. In some embodiments, the antineopiastic particles or taxane particles are agglomerated particles that are formed by the agglomeration of smaller particles which fuse together forming the larger individual antineopiastic or taxane particles, all of which occurs during the processing of the particles. In some embodiments, the antineopiastic particles or taxane particles are formed by the agglomeration of smaller particles which fuse together forming the larger individual antineopiastic or taxane particles, all of which occurs during the processing of the particles. In some embodiments, the antineopiastic particles or taxane particles are non-agglomerated individual particles and are not clusters of multiple antineopiastic or taxane particles that are bound together by interactive forces such as non-cova!ent interactions, van der Waal forces, hydrophilic or hydrophobic interactions, electrostatic interactions, Coulombic forces, interactions with a dispersion material, or interactions via functional groups. In some embodiments, the antineoplastic particles or taxane particles comprise both agglomerated and non-agglomerated particles.

[00126] In some embodiments, the taxane particles are paclitaxel particles and have an SSA of at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, or at least 35 m²/g. In other embodiments, the pachtaxei particles have

[00127] In some embodiments, the paditaxei particles have a bulk density (not-tapped) of 0.05 g/em’ to 0.15 g/em^J, or 0.05 g/cm’ to 0.20 g/em’.

[00128] In some embodiments, the paditaxei particles have a dissolution rate of at least 40% w/w dissolved in 30 minutes or less in a solution of 50% methanol/50% water (v/v) in a USP II paddle apparatus operating at 75 RPM, at 37°C, and at a pH of 7.

[00129] In some embodiments, the taxane particles are docetaxel particles and have an SSA of at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41 , or at least 42 m²/g in other embodiments, the docetaxel particles have an SSA of 18

[00130] In some embodiments, the docetaxel particles have a bulk density (not-tapped) of 0.05 g/cm’ to 0.15 g/cm³.

[00131] In some embodiments, the docetaxel particles have a dissolution rate of at least 20% w/w dissolved in 30 minutes or less in a solution of 15% methanol/85% water (v/v) hr a USP II paddle apparatus operating at 75 RPM, at 37°C, and at a pH of 7.

11. Compositions for Local Administration

[00132] The compositions useful for local administration are compositions that comprise antineoplastic agents, and/or antineoplastic particles, including taxane particles, described herein and throughout tins disclosure, and are compositions suitable for the various types of local administration, i.e. topical application, pulmonary administration, intratumoral (IT) injection, and mtraperitoneal (IP) injection. The composition can be a suspension. For example, the composition can comprise a carrier wherein the antineoplastic particles are dispersed within the carrier such that the carrier is a continuous phase and the antineoplastic particles are a dispersed (suspended) phase. In a suspension, the antineoplastic particles can be completely dispersed, or partially dispersed and partially dissolved in the composition and/or carrier, but the antineoplastic particles cannot be completely dissolved in the composition and/or carrier.

[QQ133] The composition can be administered in two or more separate administrations. In some embodiments, the two or more separate administrations are administered at or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 14 days apart. In some embodiments, the two or more separate administrations are administered 2 to 12, 2-11, 2-10, 2-9, 2-8 2-7, 2-6, 2-5, 2-4, 2-3, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-12, 5- 11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-12, 7-1 1, 7-10, 7-9, 7-8, 8-12, 8- 11, 8-10, 8-9, 9-12, 9-11, 9-10, 10-12, 10-11, 11-12, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks apart. In some embodiments, the composition is administered in 2-5, 2-4, 2-3, 3-5, 3-4, 2, 3, 4, 5, or more separate administrations. In some embodiments, the two or more separate administrations are administered once a week for at least two weeks. In other embodiments, the two or more separate administrations are administered twice a week for at least one week, wherein the two or more separate administrations are separated by at least one day. In some embodiments the method results in elimination (eradication) of the tumor. In some embodiments, the composition is administered in 1, 2, 3, 4, 5, 6 or more separate administrations. In other embodiments, the composition is administered in 7 or more separate administrations.

A, Compositions for Topical Application

[00134] The compositions for topical application (topical compositions) comprise antineoplastic particles, such as taxane particles. The antineoplastic particles can be dispersed (suspended) in the topical composition. The topical composition can be any composition suitable for topical delivery. The topical composition can be a hydrophobic composition. The topical composition can be an anhydrous composition, which can include an anhydrous, hydrophilic composition or an anhydrous, hydrophobic composition. Non- limiting examples of anhydrous, hydrophilic compositions include compositions based on polyols, glycols (e.g. propylene glycol, PEG), and/or poloxamers. The topical composition can be non-anhydrous, such as an aqueous-based composition. The topical compositions can be stenle, can be self-preserved, or can include preservatives.

[00135] The topical compositions can be formulated in various forms suitable for topical delivery'. Non-limiting examples include semi-solid compositions, lotions, liquid suspensions, emulsions, creams, gels, ointments, pastes, aerosol sprays, aerosol foams, non aerosol sprays, non-aerosol foams, films, and sheets. Semi-solid compositions include ointments, pastes, and creams. The topical compositions can be impregnated in gauzes, bandages, or other skin dressing materials. In some embodiments, the topical compositions are semi-solid compositions. In some embodiments, the topical compositions are ointments. In other embodiments, the topical compositions are gels in still other embodim nts, the topical compositions are liquid suspensions. In some embodiments, the topical compositions are not sprays and are not sprayable.

[00136] In some embodiments, the topical compositions are free of / do not include or contain a polymer/copolymer or biocompatible polymer/copolymer. In some embodiments, the compositions are free of / do not include or contain a protein. In some aspects of the disclosure, the compositions are free of / do not include or contain albumin. In some aspects of the disclosure, the compositions are free of / do not include or contain hyaluronic acid. In some aspects of the disclosure, the compositions are free of / do not include or contain a conjugate of hyaluronic acid and a taxane. In some aspects of the disclosure, the compositions are free of / do not include or contain a conjugate of hyaluronic acid and paclitaxel. In some aspects of the disclosure, the compositions are free of / do not include or contain poloxamers, polyanions, polycations, modified polyanions, modified polycations, chitosan, chitosan derivatives, metal ions, nanovectors, poly-gamma-glutamic acid (PGA), polyacrylic acid (PAA), alginic acid (ALG), Vitamin E-TPGS, dimethyl isosorbide (DMT), methoxy PEG 350, citric acid, anti-VEGF antibody, ethylceiluiose, polystyrene, polyanhydrides, polyhydroxy acids, polyphosphazenes, polyorthoesters, polyesters, polyamides, polysaccharides, polyproteins, styrene-isobutylene-styrene (SIBS), a polyanhydride copolymer, polycaprolactone, polyethylene glycol (PEG), Poly (bis(P- carboxyphenoxy)propane-sebacic acid, poly(d,l-iactic acid) (PLA), poly (d,l -lactic acid-co- glycolic acid) (PLAGA), and/or poly(D, L lactic-co-glycolic acid (PLGA).

[QQ137] The topical compositions can be packaged in any package configuration suitable for topical products. Non-limiting examples include bottles, bottles with pumps, tottles, tubes (aluminum, plastic or laminated), jars, non-aerosol pump sprayers, aerosol containers, pouches, and packets. The packages can be configured for single-dose or multiple-dose administration.

[00138] Non-limiting examples of suitable topical compositions are disclosed in Internationa] patent publication WO 2017/049083, herein incorporated by reference.

1. Hydrophobic Topical Compositions

[00139] In some embodiments, the topical composition is a hydrophobic composition. For purposes of this disclosure, a hydrophobic composition is a composition in which the total amount of the hydrophobic constituents in the composition is greater than the total amount of the non-hydrophobic constituents in the composition. In some embodiments, the hydrophobic composition is anhydrous. In some embodiments, the hydrophobic composition comprises a hydrophobic carrier.

[00140] The hydrophobic carrier can comprise substances from plant, animal, paraffinic, and/or synthetically derived sources. Hydrophobic substances are generally known as substances that lack an affinity for and repel water. The hydrophobic carrier can be the continuous phase of the topical composition and the antineoplastic particles can be the dispersed phase. In various embodiments, tire hydrophobic carriers are non-polar and/or non volatile. Non-limiting examples of hydrophobic carriers include fats, butters, greases, waxes, solvents, and oils; mineral oils; vegetable oils; petrolatums; water insoluble organic esters and triglycerides; and fluorinated compounds. The hydrophobic carriers can also comprise silicone materials. Silicone materials are defined as compounds based on polydialkylsiloxanes and include polymers, elastomers (crosslinked silicones), and adhesives (branched silicones). Non-limiting examples of silicone materials include dimethicone (polydimethylsiloxane), dimethicone copolyol, cyclomethicone, simethicone, silicone elastomers such as ST-elastomer 10 (DOW CORNING), silicone oils, silicone polymers, volatile silicone fluids, and silicone waxes. In some embodiments, the hydrophobic carrier does not comprise silicone materials. Plant derived materials include, but are not limited to, arachis (peanut) oil, balsam Peru oil, carnauba wax, candelli!a wax, castor oil, hydrogenated castor oil, cocoa butter, coconut oil, com oil, cotton seed oil, jojoba oil, macadamia seed oil, olive oil, orange oil, orange wax, palm kernel oil, rapeseed oil, safflower oil, sesame seed oil, shea butter, soybean oil, sunflower seed oil, tea tree oil, vegetable oil, and hydrogenated vegetable oil. Non-limiting examples of animal derived materials include beeswax (yellow' wax and white wax), cod liver oil, emu oil, lard, mink oil, shark liver oil, squalane, squalene, and tallow. Non-limiting examples of paraffinic materials include isoparaffin, microcrystalline wax, heavy mineral oil, light mineral oil, ozokerite, petrolatum, white petrolatum, and paraffin wax. Non-limiting examples of organic esters and triglycerides include C12-15 alkyl benzoate, isopropyl myristate, isopropyl paimitate, medium chain triglycerides, mono- and di~ glycerides, trilaurin, and trihydroxy stearin. A non-limiting example of a f!uorinated compound is perfluoropolyether (PFPE), such as FOMBLTN®HC04 commercially available from Solvay Specialty Polymers. The hydrophobic carrier can comprise pharmaceutical grade hydrophobic substances.

[00141] In various embodiments, the hydrophobic carrier comprises petrolatum, mineral oil, or paraffin, or mixtures thereof. Petrolatum is a purified mixture of semi-solid saturated hydrocarbons obtained from petroleum, and varies from dark amber to light yellow in color. White petrolatum is wholly or nearly decolorized and varies from cream to snow white in color. Petrolatums are available with different melting point, viscosity, and consistency characteristics. Petrolatums may also contain a stabilizer such as an antioxidant. Pharmaceutical grades of petrolatum include Petrolatum USP and White Petrolatum USP. Mineral oil is a mixture of liquid hydrocarbons obtained from petroleum. Mineral oil is available in various viscosity grades, such as light mineral oil, heavy mineral oil, and extra heavy mineral oil. Light mineral oil has a kinematic viscosity' of not more than 33.5 centistokes at 40°C. Heavy mineral oil has a kinematic viscosity of not less than 34.5 centistokes at 40°C. Pharmaceutical grades of mineral oil include Mineral Oil USP, which is heavy mineral oil, and Light Mineral Oil NF, which is light mineral oil. In some embodiments, tire mineral oil is heavy mineral oil. Paraffin wax is a purified mixture of solid hydrocarbons obtained from petroleum. It may also be synthetically derived by the Fischer-Tropsch process from carbon monoxide and hydrogen wliich are catalytically converted to a mixture of paraffin hydrocarbons. Paraffin wax may contain an antioxidant. Pharmaceutical grades of paraffin wax include Paraffin NF and Synthetic Paraffin NF.

[00142] In some embodiments, the concentration of the hydrophobic carrier in the hydrophobic composition is greater than 10% w/w of the total composition w'eight. In other embodiments, the concentration of the hydrophobic carrier in the hydrophobic composition is greater than 15%, or greater than 20%, or greater than 25%, or greater than 30%, or greater than 35%, or greater than 40%, or greater than 45%, or greater than 50%, or greater than 55%, or greater than 60%, or greater than 65%, or greater than 70%, or greater than 75%, or greater than 80%, or greater than 82%, or greater than 85%, or greater than 87%, or greater than 90% w/w of the total composition weight. In other embodiments, the concentration of the hydrophobic carrier in the hydrophobic composition is from greater than 10% w7w to 95% w/w of the total composition weight. In other embodiments, the concentration of the hydrophobic carrier in the hydrophobic composition is from 1 1% w/w to 95% w/w, or from 12% w/w to 95% w/w, or from 13% w/w to 95% w/w, or from 14% w/w to 95% w/w, or from 15% w7w to 95% w/w, or from 16% w/w to 95% w/w, or from 17% w/w to 95% w/w, or from 18% wVw to 95% w/w, or from 19 % w/w to 95% w/w, or from 20% w/w to 95% w/w of the total composition weight. In a some embodiment, the hydrophobic carrier in the hydrophobic composition is greater than 50% of the hydrophobic composition.

[00143] The hydrophobic composition can comprise a hydrophobic carrier and further comprise one or more volatile silicone fluids. Volatile silicone fluids, also known as volatile sdicone oils, are volatile liquid polysiloxanes which can by cyclic or linear. They are liquid at room temperature. Volatile silicone fluids are hydrophobic materials. Linear volatile silicone fluids include po!ydimethylsiloxane, hexamethyldisiioxane and octamethyltrisiloxane and are commercially available from Dow' Coming under the trade names DOW CORNING Q7-9180 Silicone Fluid 0.65 cSt and DOW CORNING Q7-9180 Silicone Fluid 1.0 cSt, respectively. Cyclic volatile silicone fluids are generally known as cyclomethicones. Cyclomethicone is a fully methylated cyclic siloxane containing repeating units of formula (IV):

(IV) [-(CH₃)₂SiO-]„

in which n is 3, 4, 5, 6, or 7; or mixtures thereof Cyclomethicone is a clear, colorless volatile liquid silicone fluid. Cyclomethicone has emollient properties and helps to improve the tactile feel of an oil based product by making it feel less greasy on the skin. Pharmaceutical grade cyclomethicone includes Cyclomethicone NF. Cyclomethicone NF is represented by formula (IV) in which n is 4 (cyclotetrasi!oxane), 5 (cyclopentasiloxane), or 6 (cyclohexasiloxane); or mixtures thereof. Cyclopentasiloxane, also known as deearnethy!ey!eopemtasiioxane, cyclomethicone D5, or cyclomethicone 5, is the cyclomethicone represented by formula (IV) in which n is 5 (pentamer), but it can contain small amounts (generally less than 1%) of one or more of the other cyclic chain length cyclomethicones. Cyclopentasiloxane is available in a pharmaceutical grade as Cyclomethicone NF. Cyclomethicones are commercially available from Dow Coming under the trade names DOW CORNING ST-Cyclomethicone 5-NF, DOW CORNING ST~ Cyclomethicone 56-NF, and XIAMETER PMX-0245. It is also commercially available from the Spectrum Chemical Mfg. Corp. Cyclopentasiloxane has a vapor pressure of about 20 to about 27 Pa at 25°C.

[00144] In one embodiment, the concentration of cyclomethicone in the hydrophobic composition is less than 25% w/w. In another embodiment, the cyclomethicone in the hydrophobic composition is at a concentration from 5 to 24% w/w. In another embodiment, the concentration of cyclomethicone is from 5 to 20% w/w. In another embodiment, the cyclomethicone is at a concentration of from 5 to 18% w/w. In another embodiment, the concentration of cyclomethicone is 13% w/w. In various embodiments, the concentration of cyclomethicone can be 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13,

13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23,

23.5, or 24% w/w or any percentage derivable therein of the total composition weight. In some embodiments, the volatile silicone fluid is a cyclomethicone. In some embodiments, the cyclomethicone is cyclopentasiloxane.

[00145] The hydrophobic composition can be a suspension of the antineoplastic particles, such as taxane particles, within a mixture of the hydrophobic carrier and the volatile silicone fluid. The antineoplastic particles can be completely dispersed, or partially dispersed and partially dissolved in the hydrophobic composition, but the antineopiastic particles cannot be completely dissolved in the hydrophobic composition. The hydrophobic carrier can be the continuous phase of the hydrophobic composition and the antineopiastic particles can be the dispersed phase. Therefore, the hydrophobic compositions can include at least two phases, a continuous hydrophobic carrier phase and a dispersed (suspended) antineopiastic particle phase. The volatile silicone fluid can be solubilized and/or dispersed within the continuous phase.

[00146] In some embodiments, the hydrophobic compositions are free of / do not include or contain additional penetration enhancers. In some embodiments, the hydrophobic compositions are free of / do not include or contain laurocapram. In some embodiments, the hydrophobic compositions are free of / do not include diethylene glycol monoethyl ether (DOME) In some embodiments, the hydrophobic compositions are free of / do not include isopropyl myri state. In other embodiments, the hydrophobic compositions are free of / do not include alpha tocopherol. In other embodiments, the hydrophobic compositions are free of do not include or contain additional volatile solvents or compounds. In some embodiments, the hydrophobic compositions are free of / do not include or contain any alcohols or Ci - C₄ aliphatic alcohols. In some embodiments, the hydrophobic compositions are free of / do not include or contain alcohol or C - C aliphatic alcohols. In other embodiments, the hydrophobic compositions are free of / do not include or contain surfactants. In other embodiments, the hydrophobic compositions are free of / do not include polymers/copolymers (or biodegradable polymers/copolymers). In other embodiments, the hydrophobic compositions are free of / do not include poloxamers, styrene-isobutylene - styrene (SIBS), a polyanhydride copolymer, polycaprolactone, polyethylene glycol. Poly (bis(P-carboxyphenoxy)propane-sebacic acid, and/or poly(D, L lactic-co-glycolic acid (PLGA).

[00147] In some embodiments, the hydrophobic compositions are semi -solid compositions. In some embodiments, the hydrophobic compositions are ointments. In some embodiments, the hydrophobic compositions are semi-solid compositions, including ointments, and have a viscosity of from 12,500 cps to 247,500 cps, or from 25,000 cps to 150,000 cps as measured at room temperature by a Brookfield RV viscometer using a small sample adapter with a SC4-14 spindle and a 6R chamber at 5 rpm with an equilibration time of 2 minutes. An alternative method for performing viscosity measurements of the hydrophobic, semi-solid compositions is using a Brookfield RV viscometer on a helipath stand with the helipath on, with a T-E spindle at 10 RPM at room temperature for 45 seconds. In some embodiments, the hydrophobic compositions are semi-solid compositions, including ointments, and have a viscosity of from 25,000 cps to 500,000 cps, or from 25,000 cps to 400,000 cps, or from 25,000 cps to 350,000 cps, or from 25,000 cps to 300,000 cps, or from 50,000 cps to 500,000 cps, or from 50,000 cps to 400,000 cps, or from 50,000 cps to 350,000 cps, or from 50,000 cps to 300,000 cps, or from 75,000 cps to 500,000 cps, or from 75,000 cps to 400,000 cps, or from 75,000 cps to 350,000 cps, or from 75,000 cps to 300,000 cps, or from 100,000 cps to 500,000 cps, or from 100,000 cps to 400,000 cps, or from 100,000 cps to 350,000 cps, or from 100,000 cps to 300,000 cps using a Brookfield RV viscometer on a helipath stand with the helipath on, with a T-E spindle at 10 RPM at room temperature for 45 seconds.

2. Aqueous-Based Topical Compositions

[00148] Topical aqueous-based compositions comprise antineoplastic particles, such as taxane particles, and an aqueous earner. Tire aqueous compositions are dispersions (suspensions) of the antineoplastic particles in an aqueous earner. The antineoplastic particles can be completely dispersed, partially dispersed and partially dissolved, but not completely dissolved in the aqueous carrier. An aqueous-based composition is a composition in which water is the major constituent (greater than 50%). Aqueous earners can include single phase aqueous solutions, and multi-phase aqueous-based emulsions such as oil-in water and water-in-oil emulsions. Non-limiting examples of aqueous carriers include water and buffer solutions.

[00149] A non-limiting example of a topical aqueous-based composition comprises an aqueous carrier (e.g. water) comprising poloxamer 407, a quaternary ammonium compound, and/or or a cross-linked acrylic acid polymer, as disclosed in international patent publication WO 2017/049083. Non-limiting examples of a quaternary ammonium compound include benzalkonium chloride and benzethonium chloride. Non-limiting examples of cross-linked acrylic acid polymers include Carbomer (INCI name), Acrylates Copolymer (INCI name), Acrylates/C 10-30 Alkyl Acrylate Crosspolymer (INCI name), Acrylates Crosspolymer-4 (INCI name), and Polyacrylate- 1 Crosspolymer (INCI name).

3. Additional Ingredients and Excipients for Topical Compositions

[00150] The topical compositions can further comprise functional ingredients suitable for use in topical compositions. Non-limiting examples include absorbents, acidifying agents, antimicrobial agents, antioxidants, binders, biocides, buffering agents, bulking agents, crystal growth inhibitors, chelating agents, colorants, deodorant agents, emulsion stabilizers, film formers, fragrances, humectants, lytic agents, enzymatic agents, opacifying agents, oxidizing agents, pH adjusters, plasticizers, preservatives, reducing agents, emollient skin conditioning agents, humectant skin conditioning agents, moisturizers, surfactants, emulsifying agents, cleansing agents, foaming agents, hydrotopes, solvents, suspending agents, viscosity control agents (rheology modifiers), viscosity increasing agents (thickeners), and propellants. Listings and monographs of the examples of the functional ingredients described herein are disclosed in The International Cosmetic Ingredient Dictionary' and Handbook (INCI), 12^th Edition, 2008, herein incorporated by reference.

[00151] In some embodiments, the topical compositions comprise penetration enhancers. In other embodiments, the topical compositions are free of / do not include additional penetration enhancers. Tire term “penetration enhancer’ has been used to describe compounds or materials or substances that facilitate drug absorption through the skin. These compounds or materials or substances can have a direct effect on the permeability of the sk , or they can augment percu taneous absorption by increasing the thermodynamic activity' of the penetrant, thereby increasing the effective escaping tendency and concentration gradient of tire diffusing species. The predominant effect of these enhancers is to either increase the stratum comeum’s degree of hydration or disrupt its l ipoprotein matrix, the net result in either case being a decrease in resistance to drug (penetrant) diffusion (Remington, The Science and Practice of Pharmacy, 22“^d ed., 2013). Non-limiting examples of skin penetration enhancers include oieyi alcohol, isopropyl myristate, dimethyl isosorbide (DM!) available under the tradename ARLASOLVE DMT, and Diethylene Glycol Monoethyl Ether (DGME) which is available under the trade name TRANSCUTOL P. Other examples of skin penetration enhancers can be found in“Skin Penetration Enhancers Cited in the Technical Literature”, Osborne, David W., and Henke, Jill J., Pharmaceutical Technology, pages 58-66, November 1997, herein incorporated by reference. Such examples include: Fatty alcohols such as aliphatic alcohols, Decanol, Lauryl alcohol (dodecanol), Linolenyl alcohol, Nerolidol, 1 - Nonanol, n-Octanol, Oleyl alcohol, Fatty acid esters, Butylacetate, Cetyl lactate, Decyl N,N- dimethylamino acetate, Decyl A Y-dimethylamino isopropionate, Diethyleneglycol oleate, Diethyl sebacate, Diethyl succinate, Diisopropyl sebacate, Dodecyl AUV-dimethylamuno acetate, Dodecyl (AUA-dimethyiamino)-butyrate, Dodecyl AGV-dimethylainino isopropionate, Dodecyl 2-(dimethylamino) propionate, EO-5-oieyi ester, Ethyl acetate, Ethylaceto acetate, Ethyl propionate, Glycerol monoethers, Glycerol monolaurate. Glycerol monooleate, Glycerol monolinoleate, Isopropyl isostearate, Isopropyl linoleate. Isopropyl myristate. Isopropyl myri state/fatty acid monoglyceride combination, Isopropyl myristate/ethanol/L- lactic acid (87: 10:3) combination, Isopropyl palmitate, Methyl acetate, Methyl caprate, Methyl laurate, Methyl propionate, Methyl valerate, 1-Monocaproyl glycerol, Monoglycerides (medium chain length), Nicotinic esters (benzyl). Octyl acetate, Octyl N.N- dimethylamino acetate, Oleyl oleate, «-Pentyl A-acetylprolinate, Propylene glycol monolaurate, Sorbitan dilaurate, Sorbitan dioleate, Sorbitan monolaurate, Sorbitan monooleates, Sorbitan trilaurate, Sorbitan trioleate, Sucrose coconut fatty ester mixtures, Sucrose monolaurate, Sucrose monooleate, and Tetradecyl ty/V-dimethylamino acetate; Fatty- acids such as Alkanoic acids, Capric acid, Diacid, Ethyloctadecanoic acid, Hexanoic acid, Lactic acid, Laurie acid, Linoelaidic acid, Linoleic acid, Linolenic acid, Neodecanoic acid, Oleic acid, Palmitic acid, Pelargonic acid, Propionic acid, and Vaccenic acid; Faty alcohol ethers such as a-Monoglyceryl ether, EO-2-oleyl ether, EO-5 -oleyl ether, EO-lO-oleyl ether, and Ether derivatives of polyglycerols and alcohols (l-0-dodecyl-3-0-methyl-2-0-(2', 3' - dihydroxypropyl) glycerol); Biologies such as L-a-amino-acids, Lecithin, Phospholipids, Saponin/phospholipids, Sodium deoxycholate. Sodium taurocholate, and Sodium tauroglycocholate; Enzymes such as Acid phosphatase, Calonase, Orgelase, Papain, Phospholipase A-2, Phospholipase C, and Tnacylglycerol hydrolase; Amines and Amides such as Acetamide derivatives, Acyclic amides, iV-Adamantyl «-alkanamides, Clofibric acid amides, AyA-Didodecyl acetamide, Di-2-ethylhexylamine, Diethyl methyl benzamide, N,N~ Diethyl-m-toluarnide, AOV-Dimethyl-m-toluamide, Ethomeen S 12 fbis-(2-hydroxyethyl) oleylamine], Hexamethylene lauramide, Lauryl-amine (dodecylamine). Octyl amide, Oleylamine, Unsaturated cyclic ureas, and Urea; Complexing Agents such as, b - and g- cyclodextrin complexes, Hydroxypropyl methylcellulose. Liposomes, Naphthalene diamide diimide, and Naphthalene diester diimide; Macrocyclics such as Macrocyclic lactones, ketones, and anhydrides (optimum ring- 16), and Unsaturated cyclic ureas; Classical surfactants such as Brij 30, Brij 35, Bri_j 36T, Bri_j 52, Brij 56, Brij 58, Bri_j 72, Brij 76, Brij 78, Brij 92, Brij 96, Brij 98, Cetyl trimethyl ammonium bromide, Empicol ML26/F, HCO-60 surfactant, Hydroxypolyethoxydodecane, Ionic surfactants (ROONa, ROSO Na, RNEECl, R ^::: 8-16), Lauroyl sarcosine, Nonionic surface active agents, Nonoxynol, Octoxynol, Phenylsulfonate CA, Pluronic F68, Pluronic F 127, Pluronic L62, Polyoleates (nonionic surfactants), Rewopal HV 10, Sodium laurate, Sodium lauryl sulfate (sodium dodecyl sulfate), Sodium oleate, Sorbitan dilaurate, Sorbitan dioleate, Sorbitan monolaurate, Sorbitan monooleates, Sorbitan trilaurate, Sorbitan trioleate, Span 20, Span 40, Span 85, Synperonic NP, Triton X-100, Tween 20, Tween 40, Tween 60, Tween 80, and Tween 85; /V-methyl pyrrolidone and related compounds such as N-Cyclohexyl-2-pyrrolidone, l-Butyl-3-dodecyl- 2-pyrrolidone, l,3-Dirneihyi-2-irnidazolikinone, 1,5 Dimethyl-2-pyrrolidone, 4,4-Dimethyl- 2-undecyl-2-oxazoline, l-Etliyi-2-pyrrolidone, 1 -Hexyl -4-methyloxy carbonyl -2 -pyrrolidone, 1 -Hexyl-2-pyrro 1 idone, 1 -(2-Hydroxyethyl) pyrrolidinone, 3-Hydroxy-N -methyl -2- pyrrolidinone, I-Tsopropyl-2-undecyl-2 -imidazoline, l-Lauryl-4-methyloxycarbonyl-2- pyrrolidone, TV-Methyl-2-pyrrolidone, Poly(N-vinylpyrrolidone), Pyroglutamic acid esters, and 2-Pyrrolidone (2-pyrrolidinone); ionic compounds such as Ascorbate, Amphoteric cations and anions, Calcium thioglycolate, Cetyl trimethyl ammonium bromide, 3,5- Diiodosalicylate sodium, Lauroylcholine iodide, 5 -Metlioxy salicylate sodium, Monoalkyl phosphates, 2-PAM chloride, 4-PAM chloride (derivatives of N-methyl picolinium chloride), Sodium carboxylate, and Sodium hyaluronate; Dimethyl sulfoxide and related compounds such as Cyclic sulfoxides, Decylrnethyl sulfoxide, Dimethyl sulfoxide (DMSO), and 2- Hydroxyundecyl methyl sulfoxide; Solvents and related compounds such as Acetone, n- Alkanes (chain length between 7 and 16), Cyclohexyl -1,1 -dimethyl ethanol, Dimethylacetamide, Dimethyl formamide. Ethanol, Ethanol /d-limonene combination, 2- Ethyl-l,3-hexanediol, Ethoxydiglycol (TRANSCUTOL), Glycerol, Glycols, Lauryl chloride, Limonene, N-Methylformamide, 2-Phenylethanol, 3 -Phenyl- 1 -propanol, 3 -Phenyl-2 -propen- l~ol, Polyethylene glycol, Polyoxyethylene sorbitan monoesters, Polypropylene glycol, Primary alcohols (tridecanol), Propylene glycol, Squalene, Triacetin, Trichloroethanol, Trifluoroethanol, Trimethylene glycol, and Xylene; Azone and related compounds such as N- Acyl-hexahydro-2-oxo- lH-azepines, N-Alkyl-dihydro- 1 ,4-oxazepine-5,7-diones, N -

Alkylmorpholine-2,3-diones, N-Alkylmorpholine-3,5-diones, Azacycloalkane derivatives (- ketone, -tliione), Azacycloalkenone derivatives, l-[2-(Decylthio)ethyl]azacyclopentan-2-one (HPE-101), 2V-(2,2-Dihydroxyethyl)dodecylamine, 1-Dodecanoylhexaliydro-l-H-azepine, 1 - Dodeeyl azacycloheptan-2-one (AZONE or laurocaprarn), N-Dodecyl diethanolamine, N- Dodecyl-hexahydro-2-thio- IH-azepme, N-Dodecyl- -(2-methoxyethyl)acetamide, N - Dodecyl-N-(2-methoxyethyl) isobutyramide, N-Dodecyl-piperidine-2-thione, N-Dodecyl-2- piperidinone, N-Dodecyl pyrrolidine-3, 5-dione, N-Dodecyl pyrrolidine-2-thione, N- Dodecyl-2-pyrrolidone, 1 -Famesy lazacycloheptan-2-one, 1 -Famesylazacyclopentan-2-one, 1- Geranylazacycioheptan-2 -one, l-Geranylazacyclopentan-2 -one, Hexahy dro-2-oxo-azepine- 1 - acetic acid esters, N-(2-Hydroxyethyl)-2-pyrrolidone, 1-Laurylazacycloheptane, 2-(l-Nonyl)- 1,3-dioxoiane, l-N-Octylazacyclopentan-2-one, N-(l -Oxododecyl)-hexahydro-lH-azepine, N -( 1 -Oxododecyl)-morpholines, 1 -Oxohydrocarbyl-substituted azacydohexanes, N-( 1- Gxotetradeeyl)-hexahydro~2-oxo~l H-azepine, and N-(l-Thiododecyl)-morpholines; and others such as Aliphatic thiols. Alkyl A^/V-dialkyl-substituted amino acetates. Anise oil, Anticholinergic agent pretreatment, Ascaridole, Biphasic group derivatives, Bisahoiol, Cardamom oil, 1-Carvone, Chenopodium (70% ascaridole), Chenopodium oil, 1,8 Cineole (eucalyptol), Cod liver oil (fatly_' acid extract), 4~Decyloxazoiidin-2-one, Dicyclohexylmethylamine oxide, Diethyl hexadecylphosphonate. Diethyl hexadecylphosphoramidate, A/A-Dimethyl dodecylamine-iV-oxide, 4, 4-Dimethyl-2-undecyl- 2-oxazoline, N-Dodecanoyl-L-amino acid methyl esters, 1 ,3-Dioxacycloalkanes (SEPAs), Dithiothreitol, Eucalyptol (cineole), Eucalyptus oil, Eugenol, Herbal extracts, Lactam N- acetic acid esters, N-Hydroxyethalaceamide, N-Hydroxyethylacetamide, 2 -Hydroxy-3 - oleoyloxy-l-pyroglutamyloxypropane. Menthol, Menthone, Morpholine derivatives, N- Oxide, Nerolidol, Octyl-P-D-(thio)glucopyranosides, Oxazolidinones, Piperazine derivatives, Polar lipids, Poiydimethylsiloxanes, Poly | 2-(methylsulfinyi)ethyl acrylate], Polyrotaxanes, Polyvinylbenzyldimethylalkylammonium chloride, Poly(A_'-vinyl~A'-methyl acetamide). Sodium pyroglutaminate, Terpenes and azacyclo ring compounds, Vitamin Pi (a-tocopherol). Vitamin E TPGS and Ylang-ylang oil . Additional examples of penetration enhancers not listed above can be found in “Handbook of Pharmaceutical Excipients”, Fifth edition, Pharmaceutical Press, 2006, and include glycofurol, lanolin, light mineral oil, myristic acid, polyoxyethylene alky ethers, and thymol. Other examples of penetration enhancers include ethanolamine, diethanolamine, triethanolamine, diethylene glycol, monoethyl ether, citric acid, succinic acid, borage oil, tetrahydropiperine (THP), methanol, ethanol, propanol, octanol, benzyl alcohol, myristyl alcohol, cetyl alcohol, stearyl alcohol, and polyethylene glycol monolaurate.

[00152] In some embodiments, the topical compositions comprise alcohols, Ci -C₄ aliphatic alcohols, and/or Ci -C5 aliphatic alcohols. In other embodiments, the topical compositions are free of / do not include or contain C -C4 aliphatic alcohols, and/or C -C5 aliphatic alcohols. In some embodiments, the topical compositions comprise volatile solvents. In other embodiments, the topical compositions are free of / do not include volatile solvents. Volatile solvents are also known as“fugitive” solvents. Non-limiting examples of volatile solvents include volatile alcohols, such as Ci to C₄ aliphatic alcohols; C to C5 alcohols: and volatile Ci to C4 aliphatic ketones, such as acetone.

[00153] In some embodiments, the topical compositions comprise surfactants. In other embodiments, the topical compositions are free of / do not include surfactants. The term “surfactant” or“surface active agent” means a compound or material or substance that exhibits the ability to lower the surface tension of water or to reduce the interfacial tension between two immiscible substances and includes anionic, cationic, nonionic, amphoteric, and/or phospholipid surfactants. Non-limiting examples of surfactants can be found in McCutcheon s Emulsifiers & Detergents, 2001 North American Edition, The Manufacturing Confectioner Publishing Co. herein incorporated by reference and also in the International Cosmetic Ingredient Dictionary and Handbook (INCI), 12th Edition, 2008, herein incorporated by reference. Such examples include, but are not limited to, the following: block polymers, e.g., Poloxamer 124; ethoxy lated alcohols e.g., Ceteth-2, Ceteareth-20, Laureth-3; ethoxylated fatty esters and oils, e.g., PEG-40 Hydrogenated Castor Oil, PEG-36 Castor Oil, PEG-150 Distearate; glycerol esters, e.g., Polyglyceryl-3 Diisostearate, Glyceryl Stearate; glycol esters, PEG-12 Dioleate, LEXEMUL P; phosphate esters, e.g., Cetyl Phosphate; polymeric surfactants, e.g., PVM/MA Copolymer, Acrylates/C 10-30 Alkyl Acrylate Crosspolymer; quaternary' surfactants, e.g., Cetrimonium Chloride; Silicone Based Surfactants, e.g., PEG/PPG-20/6 Dimethicone; Sorbitan Derivatives, e.g., Sorbitan Stearate, Polysorbate 80; sucrose and glucose esters and derivatives, e.g., PEG-20 Methyl Glucose Sesquistearate; and sulfates of alcohols, e.g , Sodium Lauryl Sulfate. More generally, surfactants can be classified by their ionic type such as anionic, cationic, nonionic, or amphoteric. They can also be classified by their chemical structures, such as block polymers, ethoxylated alcohols, ethoxylated faty esters and oils, glycerol esters, glycol esters, phosphate esters, polymeric surfactants, quaternary surfactants, silicone-based surfactants, sorbitan derivatives, sucrose and glucose esters and derivatives, and sulfates of alcohols. [QQ154] In some embodiments, the topical compositions comprise proteins, such as albumin. In other embodiments, the topical compositions are free of / do not include proteins, such as albumin. [00155] In one embodiment, the topical composition is a hydrophobic composition comprising a hydrophobic carrier, one or more volatile silicone fluids, and taxane particles, wherein the mean particle size (number) of the taxane particles is from 0.1 microns to 1.5 microns. In further embodiments, the hydrophobic carrier comprises petrolatum, mineral oil, or paraffin wax, or mixtures thereof. In further embodiments, the one or more volatile silicone fluid is cyclomethicone at a concentration of from 5 to 25% w/w of the composition. In further embodiments, the taxane particles are paclitaxel particles.

4. Concentration of Antineoplastic Particles in Topical Compositions

[QQ156] The concentration or amount of the antineoplastic particles in the topical composition is at an“effective amount” to (1) stimulate an immunological response to the immunotherapeutic agent in the subject, and (2) treat the tumor(s) of the subject, i.e., to provide a therapeutic effect on the tumor by accomplishing one or more of the following: (a) reducing tumor size; (b) reducing tumor growth rate; (c) eliminating the tumor. Idle concentration of the antineoplastic particles, which can be taxane particles, can be from 0.05 to 10% w/w, or the concentration of the antineoplastic particles can be from 0.05 to 5% w/w, or the concentration of the antineoplastic particles can be from 0.1 to 5% w/w, or the concentration of the antineoplastic particles can be 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0, 1 1 , 1 2, 1.25, 1.3, 1.4, 1.5, 1.6, 1 7, 1.75, 1.8, 1.9, 2.0, 2.1, 2.2, 2.25, 2.3, 2.4, 2.5, 2.6, 2.7, 2.75, 2.8, 2.9, 3.0, 3.1, 3.2, 3.25, 3.3, 3.4, 3.5, 3.6, 3.7, 3.75, 3.8, 3.9, 4.0, 4.1, 4.2, 4.25, 4.3, 4.4, 4.5, 4.6, 4.7, 4.75, 4.8, 4.9, 5, 6, 7, 8, 9, or 10% w/w or any percentage derivable therein of the total composition weight. In some embodiments, the antineoplastic particles are taxane particles, such as paclitaxel nanoparticles, docetaxel nanoparticles, or cabazitaxel nanoparticles. In some embodiments, the taxane particles are paclitaxel particles. In some embodiments, the taxane particles are at a concentration of about 0.05 to less than 3% w/w, or about 0.05 to about 2% w/w, or about 0.05 to about 1 % w/w, or about 0.05 to about 0.3% w/w, or about 0.05 to about 0.2% wAv, or about 0.05 to about 0.15% w7w, or about 0.1 to about 5% w/w, or about 0.1 to about 4% w7w, or about 0.1 to about 3% w/w, or about 0.1 to about 2% w/w, or about 0.1 to about 1% w/w, or about 0.1 to about 0.3% w/w, or about 0.1 to about 0.2% w/w, or about 0.15 to about 5% w/w, or about 0.15 to about 4% w/w, or about 0.15 to about 3% w/w, or about 0.15 to about 2% w/w, or about 0.15 to about 1% w/w, or about 0.15 to about 0.3% w/w, or about 0.3 to about 5% w/w, or about 0.3 to about 4% w/w, or about 0.3 to about 3% w/w, or about 0.3 to about 2% w/w, or about 0.3 to about 1% w/w, or about 1 to about 5% w7w, or about 1 to about 4% w/w, or about 1 to about 3% w/w or about 1 to about 2% w/w, or about 0.2 to about 0.4% w/w, or about 0.5 to about 1.5% w/w, or about 1.5 to about 2.5% w/w, or about 2 to about 5% w/w, or about 2 to about 4% w/w, or about 2 to about 3% w/w, or about 0.2 to about 0.4% w/w, or about 0.5 to about 1.5% w/w, or about 1.5 to about 2.5% w/w in the compositions. In other embodiments, the concentration of the taxane particles is 80 to 120% of 1% w/w (i.e., 0.8 to 1.2% w/w), or 80 to 120% of 0.05% w/w, or 80 to 120% of 0.1% w/w, or 80 to 120% of 0.15% w/w, or 80 to 120% of 0.2% w/w, or 80 to 120% of 0.25% w/w, or 80 to 120% of 0.3% w/w, or 80 to 120% of 0.35% w/w, or 80 to 120% of 0.4% w/w, or 80 to 120% of 0.45% w/w, or 80 to 120% of 0.5% w/w, or 80 to 120% of 0.55% w/w, or 80 to 120% of 0.6% w/w, or 80 to 120% of 0.65% w/w, or 80 to 120% of 0.7% w/w, or 80 to 120% of 0.75% w/w, or 80 to 120% of 0.8% w/w, or 80 to 120% of 0.85% w/w, or 80 to 120% of 0.9% w/w, or 80 to 120% of 0.95% w/w, or 80 to 120% of 1.5% w/w, or 80 to 120% of 2% w/w, or 80 to 120% of 2.5% w/w, or 80 to 120% of 3% w/w, or 80 to 120% of 4% w/w, or 80 to 120% of 5% w/w.

B. Compositions for Pulmonary Administration, Intratumoral (IT) Injection, Intraperitoneal (IP) Injection, Intravesical Instillation, and/or Direct Injection into Tissues

[00157] The compositions suitable for pulmonary administration, intratumoral (IT) injection, intraperitoneal (IP) injection, intravesical instillation, and/or direct injection into tissues surrounding the tumor such as prostate tissue, bladder tissue, and kidney tissue comprise an antineoplastic agent and/or antineoplastic particles, such as taxane particles and are described below . The compositions can further comprise a carrier. The compositions can be anhydrous and include an anhydrous carrier. The carrier can be a liquid (fluid) carrier, such as an aqueous earner. Non-limiting examples of suitable aqueous carriers include water, such as Sterile Water for In_jection USP; 0.9% saline solution (normal saline), such as 0.9% Sodium Chloride for Injection USP; dextrose solution, such as 5% Dextrose for Injection USP; and Lactated Ringer’s Solution for Injection USP. Non-aqueous based liquid carriers and other aqueous-based liquid earners can be used. The carrier can be a pharmaceutically acceptable carrier, i.e., suitable for administration to a subject by injection, pulmonary route, or other routes of administration. The carrier can be any other type of liquid such as emulsions or flowable semi-solids. Non-limiting examples of flowable semisolids include gels and thermosetting gels. The composition can be a suspension, i.e., a suspension dosage form composition where the antineoplastic particles, such as taxane particles, are dispersed (suspended) within a continuous carrier/and or diluent. In a suspension the antineoplastic particles can be completely dispersed, partially dispersed and partially dissolved, but not completely dissolved in the carrier in some embodiments, the composition is a suspension of taxane particles dispersed within a continuous carrier. In one embodiment, the carrier is a pharmaceutically acceptable carrier. In other embodiments, the composition is sterile. In various embodiments, the composition comprises, consists essentially of, or consists of antineoplastic particles and a liquid carrier, wherein the composition is a suspension of the antineoplastic particles dispersed within the liquid carrier. In some embodiments, the composition consists essentially of or consists of antineoplastic particles and a carrier, wherein the carrier is an aqueous carrier and wherein the composition is a suspension.

[00158] The composition of antineoplastic particles and a carrier can be administered as- is. Optionally, the composition of antineoplastic particles and a carrier can further comprise a suitable diluent to dilute the composition in order to achieve a desired concentration (dose) of antineoplastic particles. In some embodiments, the carrier can serve as the diluent; stated another way, the amount of carrier in the composition provides the desired concentration of antineoplastic particles in the composition and no further dilution is needed. A suitable diluent can be a fluid, such as an aqueous fluid. Non-limiting examples of suitable aqueous diluents include water, such as Sterile Water for Injection USP; 0.9% saline solution (normal saline), such as 0.9% Sodium Chloride for Injection USP; dextrose solution, such as 5% Dextrose for Injection USP; and Lactated Ringer’s Solution for Injection USP. Other liquid and aqueous-based diluents suitable for administration by injection can be used and can optionally include salts, buffering agents, and/or other excipients. In some embodiments, the diluent is sterile. The composition can be diluted with the diluent at a ratio to provide a desired concentration dosage of the antineoplastic particles. For example, the volume ratio of composition to diluent might be in the range of 1: 1 - 1: 100 v/v or other suitable ratios. In some embodiments, the composition comprises antineoplastic particles, a carrier, and a diluent, wherein the carrier and diluent form a mixture, and wherein the composition is a suspension of antineopiastic particles dispersed in the carrier/diluent mixture. In some embodiments, the carrier/diluent mixture is a continuous phase and the antineopiastic particles are a dispersed phase

[QQ159] The composition, carrier, and/or diluent can further comprise functional ingredients such as buffers, salts, osmotic agents, surfactants, viscosity modifiers, rheology modifiers, suspending agents, pH adjusting agents such as alkalinizmg agents or acidifying agents, tonicity adjusting agents, preservatives, antimicrobial agents including quaternary ammonium compounds such as benzalkonium chloride and benzethonium chloride, demulcents, antioxidants, antifoaming agents, chelating agents, and/or colorants. For example, the composition can comprise taxane particles and a carrier comprising water, a salt, a surfactant, and optionally a buffer in one embodiment, the carrier is an aqueous carrier and comprises a surfactant, wherein the concentration of the surfactant is 1% or less on a w/w or w/v basis; in other embodiments, the surfactant is less than 0.5%, less than 0.25%, less than 0.1%, or about 0.1%. In other embodiments, the aqueous carrier excludes the surfactants GELUCIRE® (polyethylene glycol glycerides composed of mono-, di- and triglycerides and mono- and diesters of polyethylene glycol) and/or CREMOPHOR® (polyethoxylated castor oil). In some embodiments, the composition or carrier excludes polymers, proteins (such as albumin), polyethoxylated castor oil, and/or polyethylene glycol glycerides composed of mono-, di- and triglycerides and mono- and diesters of polyethylene glycol.

[00160] The composition, carrier, and/or diluent can comprise one or more surfactants. Suitable surfactants include by way of example and without limitation polysorbates, lauryl sulfates, acetylated monoglycerides, diacetylated monoglycerides, and poloxamers, such as poloxamer 407. Polysorbates are polyoxyethylene sorbitan fatty acid esters which are a series of partial fatty acid esters of sorbitol and its anhydrides copolymerized with approximately 20, 5, or 4 moles of ethylene oxide for each mole of sorbitol and its anhydrides. Non-limiting examples of polysorbates are polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, and polysorbate 120. Polysorbates containing approximately 20 moles of ethylene oxide are hydrophilic nonionic surfactants. Examples of polysorbates containing approximately 20 moles of ethylene oxide include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, and polysorbate 120. Polysorbates are available commercially from Croda under the tradename TWEEN™. The number designation of the polysorbate corresponds to the number designation of the WEEN, e.g, polysorbate 20 is TWEEN 20, polysorbate 40 is TWEEN 40, polysorbate 60 is TWEEN 60, polysorbate 80 is TWEEN 80, etc. USP/NF grades of polysorbate include polysorbate 20 NF, polysorbate 40 NF, polysorbate 60 NF, and polysorbate 80 NF. Polysorbates are also available in PhEur grades (European Pharmacopoeia), BP grades, and JP grades. The term“polysorbate” is a non-proprietary name. The chemical name of polysorbate 20 is polyoxyethylene 20 sorbitan monolaurate. The chemical name of polysorbate 40 is polyoxyethylene 20 sorbitan monopalmitate. The chemical name of polysorbate 60 is polyoxyethylene 20 sorbitan monostearate. The chemical name of polysorbate 80 is polyoxyethylene 20 sorbitan monooleate. In some embodiments, the composition, carrier, and/or diluent can comprise mixtures of polysorbates. In some embodiments, the composition, carrier, and/or diluent comprises polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, and/or polysorbate 120 In some embodiments, the composition, carrier, and/or diluent comprises polysorbate 20, polysorbate 40, polysorbate 60, and/or polysorbate 80 In one embodiment, the composition, carrier, and/or diluent comprises polysorbate 80.

[00161] In some embodiments, the composition comprises antineoplastic particles, a carrier, and optionally a diluent, wherein the earner and/or diluent comprises water and a polysorbate. In one embodiment, the composition is a suspension, the antineoplastic particles are taxane particles, and the polysorbate is polysorbate 80 In other embodiments, the polysorbate or polysorbate 80 is present in the composition, carrier, and/or diluent at a concentration of between about 0.01% v/v and about 1.5% v/v. The inventors have surprisingly discovered that the recited very small amounts of polysorbate 80 reduce the surface tension at the interface of the antineoplastic particles and the aqueous carrier (such as saline solution). These embodiments are typically formulated near the time of use of the composition. In some embodiments, the particles may be coated with the polysorbate or polysorbate 80. In other embodiments, the particles are not coated with the polysorbate or polysorbate 80. In various other embodiments, the polysorbate or polysorbate 80 is present in the composition, carrier, and/or diluent at a concentration of between: about 0.01% v/v and about 1 % v/v, about 0.01% v/v and about 0.5% v/v, about 0.01% v/v and about 0 4% v/v, about 0.01% v/v and about 0.35% v/v, about 0.01% v/v and about 0.3% v/v, about 0.01% v/v and about 0.25% v/v, about 0.01% v/v and about 0.2% v/v, about 0.01% v/v and about 0.15% v/v, about 0.01% v/v and about 0 1% v/v, about 0.05% v/v and about 1% v/v, about 0.05% v/v and about 0.5% v/v, about 0.05% v/v and about 0.4% v/v, about 0.05% v/v and about 0.35% v/v, about 0.05% v/v and about 0.3% v/v, about 0.05% v/v and about 0.25% v/v, about 0.05% v/v and about 0.2% v/v, about 0.05% v/v and about 0.15% v/v, about 0.05% v/v and about 0.1% v/v, about 0.1% v/v and about 1% v/v, about 0.1% v/v and about 0.5% v/v, about 0.1% v/v and about 0.4% v/v, about 0.1% v/v and about 0.35% v/v, about 0.1 % v/v and about 0.3% v/v, about 0.1% v/v and about 0.25% v/v, about 0.1% v/v and about 0.2% v/v, about 0.1% v/v and about 0.15% v/v, about 0.2% v/v and about 1% v/v, about 0.2% v/v and about 0.5% v/v, about 0.2% v/v and about 0.4% v/v, about 0 2% v/v and about 0 35% v/v, about 0.2% v/v and about 0.3% v/v, about 0.2% v/v and about 0.25% v/v, about 0.3% v/v and about 1% v/v, about 0.3% v/v and about 0.5% v/v, about 0.3% v/v and about 0.4% v/v, or about 0.3% v/v and about 0.35% v/v; or about 0.01%, about 0.05%, about 0.1% v/v, about 0.15% v/v, about 0.16% v/v, about 0.2% v/v, about 0.25% v/v, about 0.3% v/v, about 0.35% v/v, about 0.4% v/v, about 0.45% v/v, about 0.5% v/v, or about 1 % v/v.

[00162] The composition, carrier, and/or diluent can comprise one or more tonicity adjusting agents. Suitable tonicity adjusting agents include by way of example and without limitation, one or more inorganic salts, electrolytes, sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, sodium, potassium sulfates, sodium and potassium bicarbonates and alkaline earth metal salts, such as alkaline earth metal inorganic salts, e.g., calcium salts, and magnesium salts, mannitol, dextrose, glycerin, propylene glycol, and mixtures thereof.

[00163] The composition, carrier, and/or diluent can comprise one or more buffering agents. Suitable buffering agents include by way of example and without limitation, dibasic sodium phosphate, monobasic sodium phosphate, citric acid, sodium citrate, tris(hydroxymetliyl)aminomethane, bis(2-hydroxyethyl)iminotris-{hydroxymethyl)metliane, and sodium hydrogen carbonate and others known to those of ordinary skill in the art. Buffers are commonly used to adjust the pH to a desirable range for mtraperitoneal use. Usually a pH of around 5 to 9, 5 to 8, 6 to 7.4, 6.5 to 7.5, or 6.9 to 7.4 is desired.

[00164] The composition, earner, and/or diluent can comprise one or more demulcents A demulcent is an agent that forms a soothing film over a mucous membrane, such as the membranes lining the peritoneum and organs therein. A demulcent may relieve minor pain and inflammation and is sometimes referred to as a mucoprotective agent. Suitable demulcents include cellulose derivatives ranging from about 0.2 to about 2.5 % such as carboxymethylcellulose sodium, hydroxyethyl cellulose, hydroxypropyl methylcellulose, and methylceilulose; gelatin at about 0.01%; polyols in about 0.05 to about 1%, also including about 0.05 to about 1%, such as glycerin, polyethylene glycol 300, polyethylene glycol 400, and propylene glycol; polyvinyl alcohol from about 0.1 to about 4 %; povidone from about 0.1 to about 2%; and dextran 70 from about 0.1% when used with another polymeric demulcent described herein.

[QQ165] Tire composition, carrier, and/or diluent can comprise one or more alkalinizing agents to adjust the pH. As used herein, the term“alkalizing agent” is intended to mean a compound used to provide an alkaline medium. Such compounds include, by way of example and without limitation, ammonia solution, ammonium carbonate, potassium hydroxide, sodium carbonate, sodium bicarbonate, and sodium hydroxide and others known to those of ordinary skill in the art [00166] The composition, carrier, and/or diluent can comprise one or more acidifying agents to adjust the pH. As used herein, the term“acidifying agent” is intended to mean a compound used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acid, citric acid, nitric acid, fumaric acid and other alpha hydroxy acids, hydrochloric acid, ascorbic acid, and nitric acid and others known to those of ordinary skill in the art.

[00167] The composition, carrier, and/or diluent can comprise one or more antifoaming agents. As used herein, the term“antifoaming agent” is intended to mean a compound or compounds that pre vents or reduces tire amount of foaming that forms on the surface of the fill composition. Suitable antifoaming agents include by way of example and without limitation, dimethicone, SIMETHICONE, octoxynol and others known to those of ordinary skill in the art.

[00168] The composition, earner, and/or diluent can comprise one or more viscosity modifiers that increase or decrease the viscosity of the suspension. Suitable viscosity modifiers include methylcellulose, hydroxypropyl methycellulose, mannitol, polyvinylpyrrolidone, cross-linked acrylic acid polymers such as carbomer, and others known to those of ordinary skill in the art. The composition, carrier, and/or diluent can further comprise rheology modifiers to modify the flow characteristics of the composition to allow it to adequately flow⁷ through devices such as injection needles or tubes. Non-limiting examples of viscosity and rheology modifiers can be found in “Rheology Modifiers Handbook - Practical Use and Application” Braun, William Andrew Publishing, 2000.

[00169] The concentration or amount of antineoplastic particles in a composition for pulmonary administration, intratumora! injection, intraperitoneal injection, intravesical instillation, or direct injection into tissues is at an“effective amount” to (1) stimulate an immunological response to the immunotherapeutic agent in the subject, and (2) treat the tumor(s) of the subject, i.e., to provide a therapeutic effect on the tumor by accomplishing one or more of the following: (a) reducing tumor size: (b) reducing tumor growth rate: (c) eliminating the tumor. In one embodiment, the concentration of the antineoplastic particles, which can be taxane particles, in the composition is between about 0 1 mg/mL and about 100 mg/mL. In various further embodiments, the concentration of antineoplastic particles, which can be taxane particles, in the composition is between: about 0.5 mg/mL and about 100 mg/mL, about 1 mg/mL and about 100 mg/mL, about 2 mg/mL and about 100 mg/mL, about 5 mg/mL and about 100 mg/mL, about 10 mg/mL and about 100 mg/mL, about 25 mg/mL and about 100 mg/mL, about 30 mg/mL and about 100 mg/mL, about 0.1 mg/mL and about 75 mg/mL, about 0.5 mg/mL and about 75 mg/mL, about 1 rng/mL and about 75 rng/rnL, about 2 mg/mL and about 75 mg/mL, about 5 mg/mL and about 75 mg/mL, about 10 mg/mL and about 75 mg/mL, about 25 mg/mL and about 75 mg/mL, about 30 mg/mL and about 75 mg/mL, about 0.1 mg/mL and about 50 mg/mL, about 0.5 mg/mL and about 50 mg/mL, about 1 mg/mL and about 50 mg/mL, about 2 mg/mL and about 50 rng/mL, about 5 rng/mL and about 50 mg/mL, about 10 mg/mL and about 50 mg/mL, about 25 rng/mL and about 50 mg/mL, about 30 mg/mL and about 50 mg/mL, about 0.1 mg/mL and about 40 mg/mL, about 0.5 mg/mL and about 40 mg/mL, about 1 mg/mL and about 40 mg/mL, about 2 mg/mL raid about 40 mg/mL, about 5 mg/mL and about 40 mg/mL, about 10 mg/mL and about 40 mg/mL, about 25 mg/mL and about 40 mg/mL, about 30 mg/mL and about 40 mg/mL, about 0.1 mg/mL and about 30 mg/mL, about 0.5 mg/mL and about 30 mg/mL, about 1 mg/mL and about 30 mg/mL, about 2 mg/mL and about 30 mg/mL, about 5 mg/mL and about 30 mg/mL, about 10 mg/mL and about 30 mg/mL, about 25 mg/rnL and about 30 mg/mL, about 0.1 mg/mL and about 25 mg/ L, about 0.5 mg/mL and about 25 mg/mL, about 1 mg/mL and about 25 mg/mL, about 2 mg/mL and about 25 mg/mL, about 5 mg/mL and about 25 rng/rnL, about 10 rng/mL and about 25 mg/mL, about 0.1 mg/mL and about 20 mg/mL, about 0.5 mg/mL and about 20 mg/mL, about 1 mg/rnL and about 20 mg/mL, about 2 mg/mL and about 20 mg/mL, about 5 mg/mL and about 20 mg/mL, about 10 mg/mL and about 20 mg/mL, about 0.1 rng/mL and about 15 rng/rnL, about 0.5 mg/mL and about 15 mg/rnL, about 1 mg/mL and about 15 mg/mL, about 2 mg/mL and about 15 mg/mL, about 5 mg/mL and about 15 mg/mL, about 10 mg/rnL and about 15 mg/mL, about 0.1 mg/mL and about 10 mg/rnL, about 0.5 mg/rnL and about 10 mg/mL, about 1 mg/mL and about 10 mg/mL, about

2 mg/mL and about 10 mg/mL, about 5 mg/mL and about 10 rng/mL, about 0.1 mg/mL and about 5 mg/mL, about 0.5 mg/mL and about 5 mg/mL, about 1 mg/mL and about 5 mg/mL, about 2 mg/mL and about 5 mg/mL, about 0.1 mg/mL and about 2 mg/mL, about 0.5 mg/mL and about 2 mg/mL, about 1 mg/mL and about 2 mg/mL, about 0.1 rng/rnL and about 1 mg/mL, about 0.5 mg/rnL and about 1 mg/mL, about 0.1 mg/mL and about 0.5 mg/mL, about

3 mg/rnL and about 8 mg/rnL, or about 4 mg/mL and about 6 mg/rnL; or at least about 0.1 , 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 65, 70, 75, or 100 rng/rnL; or about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 61, 65, 70, 75, or 100 mg/mL. Tire antineoplastic particles may be the sole therapeutic agent administered, or may be administered with other therapeutic agents. [00170] In various embodiments, the composition comprises taxane particles (paelitaxel particles or docetaxel particles), a earner, and a diluent, wherein the concentration of taxane particles in the composition (including the earner and diluent) is between: about 0.1 mg/mL and about 40 mg/mL, about 5 mg/mL and about 20 mg/mL, about 5 mg/mL and about 15 mg/mL, about 5 mg/mL and about 10 mg/mL, about 6 mg/mL and about 20 mg/mL, about 6 mg/mL and about 15 mg/mL, about 6 mg/mL and about 10 mg/mL, about 10 mg/mL and about 20 mg/mL, or about 10 mg/mL and about 15 mg/mL; or about 6 mg/mL, about 10 mg/mL, or about 15 mg/mL. In further embodiments, the carrier is an aqueous carrier which can be saline solution, such as about 0.9% sodium chloride solution and the diluent is an aqueous diluent which can be saline solution, such as about 0.9% sodium chloride solution. In further embodiments, the aqueous carrier comprises a polysorbate, such as polysorbate 80.

[00171] In some embodiments, the compositions are free of / do not include or contain a polymer/copolymer or biocompatible polymer/copolymer. In some embodiments, the compositions are free of / do not include or contain a protein. In some aspects of the disclosure, the compositions are free of / do not include or contain albumin. In some aspects of die disclosure, the compositions are free of / do not include or contain hyaluronic acid. In some aspects of the disclosure, the compositions are free of / do not include or contain a conjugate of hyaluronic acid and a taxane. In some aspects of the disclosure, the compositions are free of / do not include or contain a conjugate of hyaluronic acid and paelitaxel. In some aspects of the disclosure, the compositions are free of / do not include or contain poioxamers, polyanions, polycations, modified polyanions, modified polycations, chitosan, chitosan derivatives, metal ions, nanovectors, poly-gamma-glutamic acid (PGA), polyacrylic acid (PAA), alginic acid (ALG), Vitamin E-TPGS, dimethyl isosorbide (DMI), methoxy PEG 350, citric acid, anti-VEGF antibody, ethylcellulose, polystyrene, polyanhydrides, polyhydroxy acids, polyphosphazenes, polyorthoesters, polyesters, polyamides, polysaccharides, polyproteins, styrene-isobutylene-styrene (SIBS), a polyanhydride copolymer, polycaprolactone, polyethylene glycol (PEG), Poly (bis(P- carboxyphenoxy)propane-sebacic acid, poly(d,l-lactic acid) (PLA), poly (d,l -lactic acid-co- gly colic acid) (PLAGA), and/or poly(D, L lactic-co-glycolic acid (PLGA).

[00172] In one embodiment, the composition suitable for pulmonary administration, mtratumoral injection, and/or mtraperitoneal injection comprises taxane particles and a liquid carrier, wherein the taxane particles have a mean particle size (number) of from 0.1 microns to 1.5 microns. In further embodiments, the taxane particles are paelitaxel particles. In further embodiments, the liquid carrier is an aqueous carrier. III. Methods of Administration and Treatment

A. Local Administration Methods

[00173] In some embodiments, the administration of the composition to a subject is via local administration. Local administration of compositions comprising an antineoplastic agent and/or antineoplastic agent particles directly to a tumor includes but is not limited to topical application, pulmonary administration, intratumoral injection, and mtrapentoneai injection. The compositions for local administration as described herein and throughout this disclosure are compositions suitable for use in the various types of local administration, e.g., topical application, pulmonary administration, intratumoral injection, and intraperitoneal injection.

[00174] The composition can be administered in a single administration (cycle) of a single dose, or in two or more separate administrations (2 or more cycles) of single doses. In some embodiments, the two or more separate administrations are administered at or at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 14 days apart. In some embodiments, the two or more separate administrations are administered 2 to 12, 2-11, 2-10, 2-9, 2-8 2-7, 2-6, 2-5, 2-4, 2-3, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-12, 5- 11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-12, 7-11, 7-10, 7-9, 7-8, 8-12, 8- 11, 8-10, 8-9, 9-12, 9-1 1, 9-10, 10-12, 10-1 1 , 11-12, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 weeks apart. In some embodiments, the composition is administered in 2-5, 2-4, 2-3, 3-5, 3-4, 2, 3, 4, 5, or more separate administrations. In some embodiments, the two or more separate administrations are administered once a week for at least two weeks. In other embodiments, the two or more separate administrations are administered twice a week for at least one week, wherein the two or more separate administrations are separated by at least one day. In some embodiments, the composition is administered in 1, 2, 3, 4, 5, 6, or more separate administrations. In other embodiments, the composition is administered in 7 or more separate administrations. In some embodiments the method results in elimination (eradication) of the tumor.

1. Topical Application Methods

[QQ175] In some embodiments, the local administration of the composition is topical administration whereby the composition is topically applied to an affected area of the subject, and wberein the solid tumor is a skin malignancy. The skin malignancy can be a skin cancer or a cutaneous metastasis. In some embodiments, the tumor is the only cancer in the body of the subject. In other embodiments, the subject also has cancer elsewhere in the body. The “affected area” of a skin malignancy can include at least a portion of the skin where the skin malignancy is visibly present on the outermost surface of the skin or directly underneath the surface of the skin (epithelial/dermal covering), and can include areas of the skin in the proximity of the skin malignancy likely to contain visibly undetectable preclinical lesions. The skin malignancy can be a skin cancer or a cutaneous metastasis. In some embodiments, the skin malignancy is a cutaneous metastasis. In other embodiments, the skin malignancy is a skin cancer. The cutaneous metastasis can be from a variety of primary cancers, such as the following non-limiting examples of primary cancers: breast, lung, nasal, sinus, larynx, oral cavity, colon (large intestine), rectum, stomach, ovary, testis, bladder, prostate, cervical, vaginal, thyroid, endometrial, kidney, esophagus, pancreas, liver, melanoma, and Kaposi’s sarcoma (including AIDS-related Kaposi’s sarcoma). In some embodiments, the cutaneous metastasis is from lung cancer, breast cancer, colon cancer, oral cancer, ovarian cancer, kidney cancer, esophageal cancer, stomach cancer, or liver cancer. In some embodiments, tire cutaneous metastasis is from breast cancer. Non-limiting examples of skin cancers include melanoma, basal cell carcinoma, squamous cell carcinoma, and Kaposi’s sarcoma. In some embodiments, the method does not include additional skin-directed therapies, such as electrochemotherapy (ECT), photodynamic therapy (PDT), radiotherapy (RT), or intraiesional therapy (ILT).

[00176] The amount of the composition topically applied to the affected area of the skin malignancy can vary depending on the size of the affected area and the concentration of the antineoplastic particles in the composition, but generally can be applied at approximately the thickness of a dime to fully cover the affected area. Another suitable method for determining the amount of composition to apply is the“Finger-Tip Unit” (FTU) approach. One FTU is the amount of topical composition that is squeezed out from a standard tube along an adult's fingertip (This assumes the tube has a standard 5 mm nozzle). A fingertip is from the very end of the finger to the first crease in the finger. The composition can be applied with a gloved hand or spatula or other means of topical administration. In some embodiments, the composition is applied to skin malignancies which have an intact skin covering (epithelial/dermal covering). In some embodiments, the composition is applied to ulcerated areas where the skin malignancy lesion is on the surface of the skin or where the skin covering is degraded and the skin malignancy lesion is exposed. The affected area can be gently cleansed with water (and mild soap if required) and dried prior to application. Once the composition is applied, the application site can be covered with an occlusive dressing such as TEGADERM® or SOLO SITE®. The dosing of the composition can vary, but generally can include an application once, twice, or three times daily at approximately the same time each day until the condition is improved or eliminated.

2. Pulmonary Administration Methods

[00177] In some embodiments, the local administration is pulmonary administration whereby the composition is inhaled, and wherein the solid tumor is a lung tumor. In some embodiments the subject has cancer in oilier areas of the body. In some embodiments, the lung tumor is mesothelioma A malignant lung tumor is any tumor present within the lungs and may be a primary or a metastatic lung tumor. Non-limiting examples of a malignant lung tumor include small-cell lung carcinoma (SCLC) and non-small -cell lung carcinoma (NSCLC). In one embodiment, the malignant lung tumor is a SCLC. In another embodiment, the malignant lung tumor is a NSCLC. It has been shown that pulmonary administration of taxane particles according to the methods of the disclosure result in much longer residency times of the taxane in the lungs than was previously possible using any other taxane formulation. As shown in the examples that follow, the taxane remains detectable in lung tissue of the subject for at least 96 hours (4 days) or at least 336 hours ( 14 days) after the administration. In various further embodiments, the taxane remains detectable in lung tissue of the subject for at least: 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 2/40, 252, 264, 276, 288, 300, 312, 324, or 336 hours after the administration. In some embodiments, the cancerous lung disease is the only cancer in the body. In some embodiments, the subject has cancerous lung disease and cancer in other areas of tire body.

[00178] In one specific embodiment of the disclosure, pulmonary_' administration comprises inhalation of the first composition comprising the antineoplastic particles, such as by nasal, oral inhalation, or both. In this embodiment, the first composition comprising the antineoplastic particles may be formulated as an aerosol (i.e.: liquid droplets of a stable dispersion or suspension of the antineoplastic particles in a gaseous medium). Antineoplastic particles delivered as an aerosol composition may be deposited in the airways by gravitational sedimentation, inertial impaction, and/or diffusion. Any suitable device for generating the aerosol may be used, including but not limited to pressurized metered-dose inhalers (pMDI), nebulizers, and soft-mist inhalers. In some embodiments, the antineoplastic particles may be in dry powder form and used in dry powder inhalers (DPI). The drug particles are typically placed in a capsule in a DPI device. Upon actuation, the capsule is ruptured and the cloud of dry powder is expelled. The drug powder can be adjusted to the desired mass median aerodynamic diameter (MMAD) but the most common practice is to blend the small drug powders with a earner like lactose for pulmonary delivery. The drug particles adhere to the lactose particles by static adhesion. The lactose for pulmonary delivery can be sized to the desired MMAD, such as about 2.5 microns. Other sugars such as mannitol can also be used.

[00179] In one specific embodiment, the methods comprise inhalation of the first composition comprising antineoplastic particles aerosolized via nebulization. Nebulizers generally use compressed air or ultrasonic power to create mhalable aerosol droplets of the composition comprising the aerosol particles. In this embodiment, the nebulizing results in pulmonary' delivery to the subject of aerosol droplets of the composition comprising the antineoplastic particles. In embodiment, the antineoplastic particles are taxane particles. In a further embodiment, the taxane particles are paclitaxel particles. A suitable nebulizer is a Hospitak compressed air jet nebulizer.

[00180] In another embodiment, the methods comprise inhalation of the first composition comprising antineoplastic particles aerosolized via a pMDI, wherein the composition comprising the antineoplastic particles are suspended in a suitable propellant system (including but not limited to hydrofluoroalkanes (HFAs) containing at least one liquefied gas m a pressurized container sealed with a metering valve. Actuation of the valve results in delivery of a metered dose of an aerosol spray of the composition comprising antineoplastic particles. In one embodiment, the antineoplastic particles are taxane particles. In a further embodiment, the taxane particles are paclitaxel particles.

[00181] In embodiments where the compositions comprising the antineoplastic particles are aerosolized for adm inistration, the mass median aerodynamic diameter (MMAD) of the aerosol droplets of the compositions comprising the antineoplastic particles may be any suitable diameter for use in the methods of the disclosure. In one embodiment, tire aerosol droplets have a MMAD of between about 0.5 pm to about 6 p diameter. In various further embodiments, the aerosol droplets have a MMAD of between about 0.5 pm to about 5.5 pm diameter, about 0.5 pm to about 5 pm diameter, about 0.5 pm to about 4.5 pm diameter, about 0.5 pm to about 4 pm diameter, about 0.5 pm to about 3.5 pm diameter, about 0.5 pm to about 3 pm diameter, about 0.5 pm to about 2 5 pm diameter, about 0.5 pm to about 2 p diameter, about I pm to about 5.5 pm diameter, about 1 pm to about 5 pm diameter, about 1 pm to about 4.5 pm diameter, about 1 pm to about 4 pm diameter, about 1 pm to about 3.5 pm diameter, about I pm to about 3 pm diameter, about 1 pm to about 2.5 pm diameter, about 1 pm to about 2 pm diameter, about 1.5 pm to about 5.5 pm diameter, about 1 .5 pm to about 5 pm diameter, about 1.5 pm to about 4.5 pm diameter, about 1.5 pm to about 4 pm diameter, about 1.5 pm to about 3.5 pm diameter, about 1.5 pm to about 3 pm diameter, about 1.5 mhi to about 2.5 mpi diameter, about 1.5 mth to about 2 mth diameter, about 2 mth to about 5.5 pm diameter, about 2 mhi to about 5 pm diameter, about 2 mih to about 4.5 mhi diameter, about 2 mhi to about 4 pm diameter, about 2 mhi to about 3.5 mih diameter, about 2 mth to about 3 mth diameter, and about 2 mih to about 2.5 pm diameter. In some embodiments, die antineoplastic particles are taxane particles and the aerosol droplets have a mass median aerodynamic diameter (MMAD) of between about 0.5 mih to about 6 mih diameter, or between about 1 mih to about 3 mih diameter, or about 2 mih to about 3 mih diameter. A suitable instrument for measuring the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the aerosol droplets is a seven-stage aerosol sampler such as the Mercer-Style Cascade Impactor.

3. Intratumoral (IT) Injection Methods

[00182] In some embodiments, the local administration is intratumoral injection administration whereby the composition is directly injected into die solid tumor. As used herein, a“solid tumor” is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (not cancer) or malignant (cancer). Different types of solid tumors are named for the type of cells drat form them. Examples of solid malignant tumors are sarcomas, carcinomas, and lymphomas. In one particular embodiment, the solid tumor is a malignant solid tumor. In some embodiments, the malignant solid tumor is the only cancer in the body of the subject. In other embodiments, the subject has a malignant solid tumor and cancer other areas of the body.

[00183] As used herein,“directly injected into the tumor” or“intratumoral injection (IT)” means that some or all of die composition, such as a suspension, is injected into the tumor mass. As will be understood by those of skill in the art, such direct injection may include injection of some portion of the composition, such as a suspension, for example, drug on the periphery of the solid tumor (“peritumorally”), such as if the amount of composition or suspension thereof is too large to all be directly injected into the solid tumor mass. In one embodiment, the composition or suspension thereof is injected in its entirety into the solid tumor mass. As used herein the tumor includes both the tumor mass and tumor metastases, including but not limited to bone and soft tissue metastases

[00184] Intratumoral injection of compositions of the antineoplastic particles into the tumor may be accomplished by any suitable means known by one of skill in the art. In non limiting embodiments, the injection may be carried out via magnetic resonance imaging- transrectal ultrasound fusion (MR-TRUS) guidance (such as for injecting prostate tumors), or via endoscopic ultrasound-guided fine needle injection (EUS-FNI). Suitable intratumoral injection methods and compositions are disclosed in international patent application PCT/US17/25718, herein incorporated by reference.

[00185] In various embodiments, the solid tumor is selected from sarcomas, carcinomas, and lymphomas, breast tumors, prostate tumors, head and neck tumors, glioblastomas, bladder tumors, pancreatic tumors, liver tumors, ovarian tumors, colorectal tumors, pulmonary, cutaneous, lymphoid, gastrointestinal tumors, or kidney tumors. In a specific embodiment, the solid tumor is a prostate tumor and the chemotherapeutic particles are paclitaxel or docetaxel particles. In another specific embodiment, tire solid tumor is a bladder tumor and the chemotherapeutic particles are paclitaxel or docetaxel particles. In another specific embodiment, the solid tumor is a kidney tumor and the chemotherapeutic particles are paclitaxel or docetaxel particles. In another specific embodiment, the solid tumor is an ovarian tumor and the chemotherapeutic particles are paclitaxel or docetaxel particles. In another specific embodiment, tire solid tumor is a breast tumor and tire chemotherapeutic particles are docetaxel particles. In another specific embodiment, the solid tumor is a pancreatic tumor and the chemotherapeutic particles are paclitaxel or docetaxel particles. In any of these embodiments, tire tumor may be, for example, an adenocarcinoma.

4. Intraperitoneal (IP) Injection Methods

[00186] In some embodiments, the local administration is intraperitoneal injection administration whereby the composition is injected into the peritoneal cavity, and wherein the tumor is an intraperitoneal organ tumor. Intraperitoneal organs include the stomach, ileum, jejunum, transverse colon, appendix, sigmoid colon, spl een, the liver, the tail of the pancreas, the first five centimeters of the duodenum, and the upper third part of the rectum. In females, because their peritoneal cavity is open and communicates with their reproductive organs (the oviducts facilitate this communication), the uterus, ovaries, fallopian tubes, and gonadal blood vessels are all within the intraperitoneum and are included as intraperitoneal organs for purposes of this disclosure.

[00187] Intraperitoneal injection of the compositions of antineopiastic particles into the tumor may be accomplished by any suitable means known by one of skill in the art. Suitable intraperitoneal injection methods and compositions are disclosed in US patent 8221779, herein incorporated by reference. Suitable methods for intraperitoneal injection include, but are not limited to injection via a syringe, infusion through a port, and surgical administration.

[00188] In some embodiments, the malignant solid tumor is ovarian cancer, uterine cancer, stomach cancer, colon cancer, spleen cancer, liver cancer, rectal cancer, and/or pancreatic cancer. In some embodiments, the tumor is an ovarian cancer tumor. B. Systemic Administration Methods

[QQ189] In some embodiments, the administration of the composition to a subject is via systemic administration. Systemic administration methods of systemic compositions comprising an antineoplastic agent and/or antineoplastic agent particles include suitable methods as known by one of skill in the art, such as enteral administration methods and/or parenteral administration methods. Non-limiting examples of routes of systemic administration include intravenous (IV), intramuscular, intraarticular, infusion, oral, rectal, buccal, and sublingual .

EXAMPLES

[00190] The present disclosure will he described in greater detail by way of specific examples. Tire following examples are offered for illustrative purposes only and are not intended to limit the disclosure in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters, which can be changed or modified to yield essentially the same results

[QQ191] Example 1 - Evaluating Efficacy of Inhaled nPac (i.e.: paclitaxel particles as disclosed herein, approximately 98% paclitaxel with a mean particle size (number) of 0,83 microns, a SSA of 27,9 m²/g, and a bulk density (not tapped) of 0,0805 g/cm³) in the Nude Rat Orthotopic Lung Cancer Model - Study FY17-095

EXECUTIVE SUMMARY

[00192] One hundred twenty-seven (127) NIH-mu Nude Rats were x-irradiated to induce immunosuppression on Day -1. On Day 0 animals were dosed with Calu3 tumor cells by intratracheal (IT) instillation. Animals underwent a growth period of three weeks. During the third week, animals were randomized by body weight stratification into 5 study groups. Starting Week 4, animals in Group 2 received a once weekly dose of Abraxane® by intravenous (IV) dosing (5 mg/kg) on Days 22, 29 and 36. Animals in Groups 3 and 4 received once weekly (Monday) inhalation (INH) dose of nPac at low (0.5mg/kg) and high (1.0 mg/kg) target doses, respectively. Animals in Groups 5 and 6 received a twice weekly (Monday and Thursday) target inhalation dose of nPac at low (0.50 mg/kg) and high (up to 1.0 mg/kg) doses respectively. Animals in Group 1 were left untreated as a control of normal tumor cell growth. All animals were necropsied during Week 8.

[QQ193] All animals survived to their designated necropsy timepoint. Clinical observations related to the model included skin rash and labored breathing. All groups gained weight at about the same rate throughout the course of the study. [00194] The inhalation exposure average Paclitaxel aerosol concentration for once weekly Low Dose and twice weekly Low' Dose nPac groups was 270.51 pg/L and 263.56 pg/L, respectively. The inhalation exposure average Paclitaxel aerosol concentration for once weekly High Dose and twice weekly High Dose nPac groups was 244.82 pg/L and 245.76 pg/L, respectively.

[00195] Doses were based on average aerosol paclitaxel concentration, most recent average group bodyweight, the assumed deposition fraction of 10%, and an exposure duration of 33 (Low-Dose) or 65 (High-Dose) minutes. During four weeks of treatment, the average achieved rodent deposited dose for the once weekly Low Dose nPac group and twice weekly Low Dose nPac group were 0.655 mg/kg and 0.640 mg/kg (1.28 mg/kg/week), respectively. Hie average achieved rodent deposited dose for the once weekly High Dose nPac group and twice weekly High Dose nPac group were 1.166 mg/kg and 1.176 mg/kg (2.352 mg/kg/week), respectively. For the group receiving IV injections of Abraxane ®, the average dose on Day 22, 29 and 36 was 4.94, 4.64 and 4.46 mg/kg respectively.

[00196] At scheduled necropsy, the majority of animals from each group had tan nodules on the lungs and/or red or tan patchy discolorations of the lung. Other sporadic observations included an abdominal hernia in one animal and a nodule on the pericardium in another animal. No other abnormal gross observations were noted at necropsy.

[00197] In the Abraxane® treated animal’s lung weights, the lung to BW ratios and lung to brain weight ratios were significantly lower compared to Untreated Controls. The once weekly nPac High Dose group had similar weights to the Abraxane® group and significantly lower lung weights and lung to brain ratios compared to Untreated Controls.

[QQ198] Histologically, lungs of the majority of animals in all groups contained some evidence of tumor formation. Tumor formation was characterized by the presence of expansile variably sized small masses randomly scattered within the lung parenchyma and larger expanded and coalescing masses that effaced up to 75% of the lung parenchyma, smaller airways and blood vessels. The larger masses were distributed primarily in the hilar regions or juxtaposed at the axial airway and the smaller masses were generally located peripherally.

[00199] Hie primary morphologic cellular characteristics of the lung tumor masses varied from the presence of undifferentiated to a fairly well differentiated patern of adenocarcinoma of the lung. The predominant tumor cell type showed an undifferentiated adenocarcinoma morphology; the cells were pleomorphic, large, anaplastic, pale amphophilic-staining with fine intracytoplasmic vacuoles resembling mucoid vesicles, exhibited moderate to marked anisokaryosis, and were observed to be individualized or growing in sheets and lacking clear- cut features towards differentiation to adenocarcinoma. However, the cellular morphologic characteristics that were observed within other masses or growing within the previously described undifferentiated masses were more organized and consistent with well differentiated lung adenocarcinoma demonstrating clear acinar gland differentiation. These amphophilic staining tumor cells were primarily arranged in nests or glandular paterns which were observed to be bound by alveolar septae. Mitotic figures were rarely observed in this tumor cell population. Less frequently observed within these masses were focal areas of primitive-appearing relatively small Primitive Tumor Cells with small to moderate amounts of pale basophilic staining cytoplasm, ovoid and variably vesicular nuclei, and moderate anisokaryosis. These Primitive Tumor Cells were observed to be growing randomly and in sheets. Increased numbers of mitotic figures and apoptotic bodies were noted most often in this basophilic Primitive Tumor Cell population. Inflammation, characterized by mixed inflammatory cell (predominately eosinophils, lymphocytes, foamy macrophages and the occasional giant cell) infiltration accompanied by interstitial fibrosis was commonly observed. Significant parenchymal necrosis was uncommon to absent.

[00200] The pathologist considered the presence of scalloping of the edges of the individual tumor masses characterized by gradual loss of tumor cells, to complete loss of tumor cells with residual fibrosis connective tissue scaffolding of the lung parenchyma and accompanied by invasion of foamy macrophages to be e vidence of Tumor Regression.

[00201] Compared to the positive control Grp. 1 and the Abraxane® treated comparative Grp. 2, there was a decreased overall lung tumor burden in the nPac treated groups (Gip. 3-6) characterized by a decrease in the severity of adenocarcinoma tumor masses and Primitive Tumor Cell population as well as evidence of Tumor Regression. No other treatment-related lesions or findings were observed. Extensive mononuclear cell infiltration was observed in the lungs of animals receiving nPac through inhalation. As the model used is T cell deficient, it is likely that the cells are B cells or NK cells.lt is hypothesized that the localized, likely higher concentration exposure of the tumor to nPac affected the tumors leading to an alteration in the environment to draw the mononuclear cellular infiltrate into the lung.

[QQ202] The objective of this study was to evaluate the efficacy of inhaled nPac formulation compared to a clinical reference dose of intravenous administered Abraxane® in reducing tumor burden in an orthotopic model of lung cancer.

MATERIALS AND METHODS Test System

Species/ Strain: NIH-rnu Nude Rats

Age of Animals at Study Start: 3-5 weeks old

Body Weight Range at Study Start: Approximately 150- 200 g

Number on Study/Sex: 127 Males (120 study animals and 7 spares)

Envigo

Identification: Permanent maker sail marking

Abraxane® Formulation

[QQ203] The clinical reference material used for IV formulation was the drug product Abraxane®. The drag product was reconstituted to 5.0 mg/mL with saline on the day of dosing and was stored per manufacturer's instructions.

nPac Fomulation

[00204] The 20.0 mg/ml nPac formulations for exposures were prepared per the sponsor recommendations. Specifically the nPac was reconstituted with 1% polysorbate 80. The vial was shaken by hand until all particles were wetted. Additional 0.9% sodium chloride for injection was added (to the desired concentration target) and the vial was shaken by hand for another minute. Shaking continued until no large clumps were visible and the suspension was properly dispersed.

[00205] Resultant formulations were left undisturbed for at least 5 minutes to reduce any air/foam in the vial before placing it in a nebulizer for aerosolization work. The final formulation was kept at room temperature and nebulized within 2 hours after reconstitution. The final 20.0 mg/mL was kept at room temperature and nebulized within 30 (+5) minutes after reconstitution.

Experimental Design

[00206] One hundred twenty-seven (127) animals were used for study. Prior to x- irradiation and dosing of tumor cells, 7 animals w'ere designated as spares (spare animals did not have irradiations or ceil line instillations). On Day -1 all study animals were x-irradiated to induce immunosuppression. On Day 0 animals were dosed with Calu3 tumor cells by intratracheal (IT) instillation. Animals underwent a growth period of three w'eeks. During the third week, animals were randomized by body weight stratification into the groups outlined in Table 1 below. Starting Week 4, animals in Group 2 received a once weekly target dose of Abraxane® by intravenous (IV) dosing (5 mg/kg). Animals in Groups 3 and 4 received once weekly (Monday) inhalation (1NH) target dose of nPac at low' (0.5mg/kg) and high (1.0 mg/kg) doses, respectively. Animals in Groups 5 and 6 received a twice weekly (Monday and Thursday) inhalation target dose of nPac at low (0.50 mg/kg) and high (1.0 mg/kg) respectively. Animals in Group 1 were left untreated as a control of normal tumor cell growth. All animals were necropsied during Week 8

Treatment occurred during Week 4-8 Necropsy occurred during Week 8

**Abraxane^®' target dose: 5.0 mg/kg based on bodyweight; target dose volume: not to exceed 250 gL, frequency: Day 1, 8, and 15 of each 21 day cycle beginning during Week 4.

Husbandry, Quarantine and Assignment to Study

[00207] After quarantine all animals were weighed and randomized to remove the 7 spares based on body weights. From Week 1 to Week 3 animals were identified by cage cards (LC numbers) and tad markings.

[00208] During Week 3, prior to beginning treatment, animals were weighed and randomized into the groups listed above by body weight stratification and assigned a Study ID. From this point forward, animals were identified by cage cards and sharpie tail marking. Immunosuppression and Irradiation [00209] On Day -1, animals underwent whole body x-ray exposure with -500 rads (Phillips RT 250 X-ray Therapy Unit, Phillips Medical Systems, Shelton, CT) set at 250 kVp, 15mA, and a source-to-object distance of 100cm. The animals were placed in a pie chamber unit, 2-3 animals per slice of pie. The irradiation process took -10-15 minutes.

Tumor Ceil Implantation

[00210] On Day 0, animals received tumor cells (Calu3) administered by IT. Briefly, after being anesthetized by 3-5% isoflurane in an induction chamber, the animal was placed with upper incisors hooked on an inclined hanging instillation platform. The animals tongue was gently secured while the stylet is inserted just past the larynx and into the trachea. A volume of cells in EDTA suspension (target dose volume: 500 pL; concentration: approximately 20x106 per 0.5 mL) was delivered to the lungs via intratracheal instillation. After the instillation, the animals’ breathing and movement was monitored carefully. Following tumor ceil implantation, animals underwent a tumor growth period of approximately 3 weeks prior to treatment to allow for tumor cell engraftment and the development of lung cancer.

Cabs 3 Growth and Preparation

[00211] Calu3 cells were grown at 37°C with 5% CQ2 m cell culture flasks. They were grown in Roswell Park Memorial Institute (RPMI) 1640 media with 10% fetal bovine serum (FBS) until 80% confluence. Cells were maintained until the day of instillation. Prior to instillation they were harvested by washing with PBS, then trypsin w'as added to remove cells from the flask. Tire cells were neutralized with RPMI 1640 media containing 10% FBS. They were then centrifuged at lOOxg for 5 minutes; the media was removed and the cells were resuspended to a concentration of 20 million cells in 450 pL of serum free RPMI. Prior to instillation, 50 mT of 70 mM EDTA was added to the cell suspension for a total IT dose volume of 500 pL per rat.

Body Weights and Daily Observations

[00212] Body weights were collected for randomization, weekly through Week 3, twice weekly beginning at Week 4 through the end of the study, and at necropsy.

[00213] Each animal on study was observed twice daily for any clinical signs of abnormality, morbidity or death. Technicians observed animals during dosing and body weight sessions.

Abraxane® Administration IV-Tail Vein Injections

[00214] Abraxane® (5 mg/mL, maximum dose volume of 250 mΐ,) was administered to animals in Group 2 by IV tail vein injection on Days 22, 29 and 36.

nPac Administration - Nose-only Aerosol Exposures Conditioning

[QQ215] Animals were conditioned to nose-only exposure tubes for up to 70 minutes. Three conditioning sessions occurred over three days prior to exposure, with the first session lasting 30 minutes, the second 60 minutes and the third 70 minutes. They were monitored closely throughout die conditioning periods and during exposures to assure that they did not experience more than momentary distress.

Exposure System

[00216] Aerosols were generated with two compressed air jet Hospitak at a nebulizer pressure of 20 psi. nPac suspension formulation of 20.0 mg/mL was used for low dose and high dose exposures. Aerosols were directed through a deliver} line into a 32-port nose-only exposure chamber. The rodent inhalation exposures were conducted for 33 or 65 minutes nPac suspension aerosol was generated with a set of two Hospitak compressed air jet nebulizers (used for up to 40 (± 1) minutes), then replaced with a second set of two Hospitak nebulizers for remaining exposure duration. Oxygen and temperature were monitored and recorded throughout each inhalation exposure

Concentration Monitoring

[00217] Aerosol concentration monitoring was conducted by collecting aerosols onto pre weighed GF/A 47-mm filters. Tire filters were sampled from animals breathing zones of the nose-only exposure chamber throughout each inhalation exposure. The aerosol sampling flow^¬ rate through GF/A filters was maintained at 1.0 ± 0.5 L/minute. Filters were collected throughout each exposure duration ever} 10-minutes except for the last filter. With the low- dose exposures (groups 3 and 5) lasting 33 minutes, tire final filter was collected after 13 minutes and with the high-dose exposures (groups 4 and 6) lasting 65 minutes, the final filter was collected after 15 minutes. After sample collection filters were weighed to determine the total aerosol concentration in the exposure system.

[00218] Post weighing, each filter w⁷as placed in a 7 mL glass vial. The filters in glass vials were extracted and analyzed by High Performance Liquid Chromatography (HPLC) to quantify the amount of Paelitaxei collected onto the filters. The total aerosol concentration and Paelitaxei aerosol concentrations were calculated for each filter by dividing the total amount of aerosols and Paelitaxei aerosols collected with total air flow⁷ through the filter. The average Paelitaxei aerosol concentration was used to calculate the achieved average deposited dose of Paelitaxei to the rodent lungs using Equation 1 as shown in the Determination of Dose section below.

Determination of Dose [00219] Deposited dose was calculated using Equation 1 same as in Example 4

Euthanasia and Necropsy

[00220] At scheduled necropsy, animals were euthanized by intraperitoneal injection of an overdose of a barbiturate-based sedative.

Blood and Tissue Collection

[00221] For all necropsies a terminal body weight and brain weight was collected. For scheduled euthanasia blood (for plasma) was collected by cardiac puncture into a K2EDTA tube. The lungs were removed and weighed. A section of lung tissue containing a tumor, a tracheobronchial lymph node, was frozen in liquid nitrogen for potential future analysis. The remaining lung was fixed for potential histopathology.

Histopathology

[00222] Fixed left lung lobes were trimmed in a“bread loaf’ manner and alternate sections were placed in 2 cassettes to yield 2 slides each with 3 representative sections of the left lung. Tissues were processed routinely, paraffin embedded, sectioned at ~4 pm, mounted, and stained with hematoxylin and eosin (H&E) for microscopic examination. Findings were graded subjectively, semi-quantitatively.

[00223] Sections of lung (l-4/animal) obtained from 60 out of the 120 treated nude rats on study, trimmed longitudinally, were processed to H & E stained glass slides for light microscopic evaluation.

[00224] During this review, the microscopic findings were recorded and then transferred to an electronic pathology reporting system (PDS-Ascentos- 1.2.0, V.1 2), which summarized the incidence and severities of the lung burden characteristics data and tabulated the results and generated the individual animal data. The lungs from the 60 nude rats were examined histologically: Group 1 [1001-1010], Group 2 [2001-2010], Group 3 [3001-3010], Group 4 [4001 -4010], Group 5 [5001-501 ] and Group 6 [6001-6010]). In order to assess the level of tumor burden in these lungs, the lungs were evaluated and scored during histopathologic examination. For each cumulative lung burden characteristic diagnosis: 1) Adenocarcinoma (undifferentiated and differentiated), 2) Primitive Tumor Cells (poorly differentiated pleomorphic cells) and 3) Tumor Regression, the lungs were graded semi-quantitatively using a 4-point grading scale indicating the percent involvement of tire overall lung tissue provided as follo 's: 0 = no evidence, 1 = minimal (~ 1 - 25% total area of lung sections involved), 2 = mild (~ 25 - 50% total area of lung sections involved), 3 = moderate (~ 50 - 75% total area of lung sections involved), and 4 = marked (~ 75 - 100% total area of lung sections involved). HistoMorphometry [00225] Histomorphometric analyses was performed using fixed left lung lobes of die first 10 animals from each group. Tissue was trimmed using a morphometry (“bread slice”) style trim. Briefly, trimming started at a random point between 2 and 4 mm from the cranial end of the lung. Each lung section was cut approximately 4 mm thick. Odd numbered sections were placed caudal side down in cassette 1 while even numbered sections were placed in cassette 2. Tissue sections were then processed, paraffin embedded, and sectioned at 4Dm and stained with hematoxylin and eosin (HE) for examination. Both slides (odd and even slices) were used to determine an average tumor fraction per animal .

[QQ226] Morphometric analysis was performed on the hematoxylin and eosin (HE) stained lung tissue from the designated animals by Lovelace Biomedical. Whole slides (2 per animal containing transverse sections of the entire left lung) were scanned using a Hamamatsu Nanozoomer. Scans were analyzed with Visiopharm Integrator System software (VIS, version 2017.2.5.3857). Statistical analysis of tumor area fraction was performed in Graph fad Prism 5 (version 5.04).

[00227] Computerized image quantification designed to quantify the amount of tumor area present on each slide was performed on ail left lung tissue using the whole slide scans. The Visiopharm Application for quantifying the area of lung metastases was used to differentiate tumor cells from normal lung tissue based on cell density, staining intensity, and size and staining intensity. It is noted that this quantitation based upon simple H&E staining will not be perfect (i.e. it is not capable of fully discriminating between types of tumor tissue, necrotic and viable tumor tissue, and some normal structures may he included as tumor). The value in application of this process to H&E sections is that it is an unbiased approach to tumor quantification. The area of the whole piece of lung is determined, and the area occupied by structures identified as metastases is then expressed as a percentage of the total area. Minor adjustment of the area to be analyzed to ensure extrapulmonary structures are excluded and the entire lung is included may be performed manually. Other manual manipulations are avoided in order to ensure consistency across all groups and remove potential for introduction of bias. If possible, development of specific immunohistochemical stains to identify only tumor tissue would increase specificity of this analysis.

Blood Collection and Processing

[QQ228] Blood collected at necropsy was processed to plasma by centrifugation at a minimum of 1300g at 4°C for 10 minutes. Plasma samples w ere stored at -70 to ~90°C until analysis or shipment to sponsor. ADDITIONAL MORPHOLOGIC AND IMMUNOHISTOCHEMICAL (IHC) STUDIES

[00229] A subset of 17 animals was chosen to review morphologic and immunohistochemical (IHC) features using slides prepared with Hematoxylin & Eosin, Masson’s Trichrome, AE1/AE3 (pan-keratin), and CD 11b (dendritic cells, natural killer cells and macrophages). This subset included Control animals (n=2) and Treated animals from each treatment group (n=3 per group). Rat lung blocks were sectioned at 4pm thickness and collected on positively charged slides.

Methods

[00230] H&E and Masson’s trichrome staining were performed according to standard protocols. For Anti-Pan Cytokeratin antibody [AE1/AE3], rat uterus was sectioned from a tissue bank as controls. Optimization was performed on formalin-fixed paraffin -embedded (FFPE) rat uterus tissue from the tissue bank using a Leica Bond automated immunostainer and a mouse Anti-Pan Cytokeratin [AE1/AE3] (Abeam, #ab27988, Lot #GR3209978-1) antibody at four different dilutions plus a negative control: no primary antibody, 1 :50, 1 : 100, 1 :200, and 1:400. Heat induced antigen retrieval was performed using Leica Bond Epitope Retrieval Buffer 1 (Citrate Buffer solution, pH6.0) for 20 minutes (ERl(20)) and Leica Bond Epitope Retrieval Buffer 2 (EDTA solution, pH9.0) for 20 minutes (ER2(20)). Non-specific background w'as blocked with Rodent Block M (Biocare, #RBM961H, Lot #062117).

[00231] Anti-pan Cytokeratin antibody [AE1/AE3] antibody was detected using Mouse- on-Mouse HRPPolymer (Biocare, #MM620H, Lot #062016) and visualized with 3’3~ diaminobenzidine (DAB; brown). A Hematoxylin nuclear counterstain (blue) was applied. Optimization slides were examined, and optimal staining conditions for sample slides were determined with Anti-Pan Cytokeratin antibody [AE1/AE3] at 1 :50 dilution with ER2(20).

[00232] For Anti-CD- 1 lb antibody, optimization was performed on formalin-fixed paraffin-embedded (FFPE) rat lymph nodes tissue from a tissue bank using a Leica Bond automated immunostainer and a rabbit anti-CD 11b antibody at four different dilutions plus a negative control: no primary antibody, 1 :250, 1 :500, 1 : 1000 and 1 :2000.

[Q0233] Heat induced antigen retrieval was performed using Leica Bond Epitope Retrieval Buffer 1 (Citrate Buffer, pH6.0) for 20 minutes (ER1(20)) or Leica Bond Epitope Retrieval Buffer 2 (EDTA solution, pH9.0) for 20 minutes (ER2(20)).

[00234] Anti-CDl lb antibody was detected using Novocastra Bond Refine Polymer Detection and visualized with 3’3-diaminobenzidine (DAB; brown). A Hematoxylin nuclear counterstain (blue) w^ras applied. Optimization slides were examined, and optimal staining conditions for FFPE tissue were determined with anti-CD 11b at 1 :2000 dilution with ER2(20). Rat lymph nodes controls were used alongside rat lung samples.

STUDY RESULTS

Clinical Observation, Survival, and Bodyweights

[00235] All animals survived to their designated necropsy timepoint. Clinical observations related to the model included skin rash and labored breathing. One animal was observed to have an upper abdominal hernia. Per vet recommendation the animal was switched with a Group 1 (Untreated Control) that would not undergo inhalation exposures therefore no exposure tube restraint would be necessary.

[00236] FIG. I shows the average body weights through the duration of the study FIG. 2 shows the percent change in average body weights from Day 0. All groups gained weight at about the same rate through the course of the study .

[00237] For the group receiving I V injections of Abraxane®, the average dose on Day 22, 29 and 36 was 4 94, 4.64 and 4 46 mg/ kg respectively.

nPac Exposures

Aerosol Concentrations and Deposited Dose

[00238] Total aerosol and Paclitaxel aerosol concentrations were measured by sampling of GF/A filters during each exposure. The inhalation exposure average Paclitaxel aerosol concentration for once weekly Low Dose and twice weekly Low Dose nPac groups was of 270.51 pg/L and 263.56 pg/L, respectively. The inhalation exposure average Paclitaxel aerosol concentration for once weekly High Dose and twice weekly High Dose nPac groups was of 244.82 pg/L and 245.76 pg/L, respectively. The oxygen and temperature levels were monitored throughout each exposure.

[00239] Doses were based on average aerosol paclitaxel concentration, most recent average group body weight, the assumed deposition fraction of 10% and an exposure duration of 33 or 65 minutes. During four weeks of treatment, the average achieved rodent deposited dose for the once weekly Low Dose nPac group and twice weekly Low Dose nPac group were 0.655 mg/kg and 0.640 mg/kg (1.28 mg/kg/week), respectively.

[00240] Hie average achieved rodent deposited dose for the once weekly High Dose nPac group and twice weekly High Dose nPac group were 1.166 mg/kg and 1.176 mg/kg (2.352 mg/kg/week), respectively.

Particle

[00241] The particle size distribution was determined in terms of Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) for each nPac formulation aerosols using cascade impactor. For the 20.0 mg/mL nPac aerosols the average MMAD was determined to be 2.01 mhi and a GSD of 1.87.

Necropsy Observations and Organ Weights

[00242] Ail animals survived to their designated necropsy timepoint. At necropsy animals from each group had tan nodules on the lungs and/or red or tan patchy discolorations of the lung. Other sporadic observations included an abdominal hernia in one animal and a nodule on the pericardiumin another animal. No other abnormal gross observations were noted at necropsy. One animal did not have any visible tumors (nodules) at the time of necropsy

[00243] Individual animal organ weight data is shown graphically in FIG. 3, FIG. 4 and FIG. 5. In Abraxane®) treated animal’s lung weights, lung to BW ratios and lung to brain weight ratios were significantly lower compared to Untreated Controls. The once weekly nPac High Dose group had similar weights to the Abraxane® group and significantly lower lung weights and lung to brain ratios compared to Untreated Controls. Hie once weekly Low' Dose, nPac twice weekly Low Dose and twice weekly High Dose nPac groups generally had similar average lung weights and ratios.

[00244] All treatment groups showed a decrease in average lung tumor fraction when compared to the control group; however, there was no statistically significant difference between groups. There was also no statistically significant difference between TV Abraxane® treatment and any of the nPac treatment regimens in regards to the tumor area fraction examined on cross sectional lung slides. As is typical of this model, there is wide variability between animals within all groups in the tumor fraction. These data should be considered in combination with other indicators of lung tumor burden in this model including lung to brain weight ratios and standard histopathology for final interpretation. It is important to note that morphometric analysis and histopathologic examination was performed on fixed lung tissue from the left lobe while other analyses on lung tissue may be performed on frozen tissue from the right lung lobes. Average tumor area is shown in FIG. 6 and FIG. 7.

PATHOLOGY RESULTS

[QQ245] As a result of the slide examination of the identified populations of neoplastic cells the pathologist determined: (1) There was a slight decrease in severity of an overall lung tumor burden of Adenocarcinoma (undifferentiated and differentiated cells) in all treated groups (Grp. 2 (1.7), Grp, 3 (1.8), Grp. 4 (1.7), Grp 5 (1.6) and Grp. 6 (1.6) compared to the untreated Control Grp. 1 (2.1). (2) There was reduction in the Primitive Tumor Cell population evident by a decrease in the severity m Grp. 3 (0.3), Grp. 4 (0.3), Grp 5 (0.2) and Grp 6 (0.2) compared to the corresponding control Grp 1 (0.9) and Grp 2 (1.0), and 3). There was Tumor Regression present in Grp 3 (0.6), Grp 4 (1.0), Grp 5 (0.8) and Grp 6 (1.0) compared to the corresponding control Grp 1 (0.0) and Grp 2 (0.1). The incidence and severities of the lung burden characteristics data are summarized in Table 2, and in FIG. 8. Photomicrographs of the slides are shown in FIGs. 9 to 42.

Table 2. Incidences and Severities of Cumulative Lung Burden

a. Severity Grade is based on a 4-point grading scale of 1 to 4: l=mimmal, 2=mild, 3=moderate,

4-tnarked

b. The presence of a (0) indicates that there in no evidence histopathologicaily of the lesion in question

HISTOLOGICAL OVERVIEW OF H&E STAINED LUNG CANCER TISSUE SLIDE PHOTOMICROGRAPHS in FIGs. 9 TO 43

Genera! Observations:

[00246] ( Control: Extensive levels of viable tumor with proliferating cells and little to no immune cell infiltration. [00247] Abraxane® IV: Many viable appearing tumor masses with some lymphocytic response along with some tumor regression.

[00248] nPac lx per week, High: Clearance of tumor from the lung with few viable tumor cells remaining. Masses remaining appear to be immune cell infiltrate and fibrosis.

[00249] nPac 2x per week, Low: Some remaining tumor nodules surrounded by immune ceil infiltrate including macrophages and mononuclear cells.

[00250] nPac 2x per week, High: Few tumor nodules with immune infiltrate and stromal fibrosis replacing tumor.

[00251] Extensive mononuclear tumoricidal cell infiltration was observed in the lungs of animals receiving nPac through inhalation. As the model used is T cell deficient, it is likely that the cells are B cells or NK cells, or both. B cells are responsible for the production of antibodies and can be involved in tumor cell killing through antibody-dependent cell mediated cytotoxicity (the antibodies bind to ceils expressing Fc Receptors and enhance the killing ability of these cells). NK cells are innate lymphoid cells that are crucial in the killing of tumor cells. In patients with tumors, NK cell activity is reduced allowing for the growth of the tumor. Along with T cells, NK cells are the target of some check point inhibitors to increase their activity.

[00252] By the use of a wide array of surface receptors capable of delivering either triggering or inhibitory signals, NK cells can monitor cells within their environment to ascertain if the cell is abnormal (tumor or virally infected) and should be eliminated through cytotoxicity.

[0Q253] Hie cytotoxicity and chemotaxis of NK cells can be modified by many pathological processes including tumor cells and their byproducts. In response to certain signals their functions are enhanced or potentiated. In response to several Pathogen Associated Molecular Patterns (PAMPs) by using different Toll Like Receptors (TLR); NK cells can increase cytokine production and/or cytolytic activity. Cytokines, including IL-2, IL-15, IL-12, IL-18, and IFNs a/b can also modify the activity of NK cells. NK cells are not simple cells that are only cytolytic effectors capable of killing different tumor cell targets; rather, they represent a heterogeneous population which can finely tune their activity in variable environmental contexts.

[QQ254] Hie tumor burden seems to be significantly reduced in the lungs of the animals treated with nPac and is lower than that for Abraxane® TV. Therefore, the localized administration of paciitaxel in the form of nPac provides additional potency. Hiis is likely- due to both the longer exposure to tire chemotherapy over time and the vigorous cellular infiltration to the site of the tumor. This latter response appeared to be dependent on the dose density (actual dose and dose frequency).

Observations of Specific Photomicrographs:

[00255] FIG. 9: Subject 1006 (Control) Adenocarcinoma-3, Primitive-!, Regression-0. Low-power magnification (2x) showing the general distribution of undifferentiated, pleomorphic, large, anaplastic tumor cells within alveolar spaces or lining the alveolar septae. The majority of cells do not have features of adenocarcinoma and appear in sheets of contiguous tumor. Many cells have basophilic staining cytoplasm, while others are large, anaplastic and contain pale amphophilic-staining. Note tire presence of a pre-existing resident population of alveolar macrophages and the absence of tumor regression.

[00256] FIG. 23: Subject 2003 (IV Abraxane®) Adenocarcinoma- 1 , Primitive-!,

Regression- 1. Low-power magnification (4x) showing the general distribution of tumor masses predominantly at the periphery as well as multiple smaller expansive tumor masses filling alveolar spaces. The tumor ceils are pleomorphic, large, anaplastic and have pale amphophilic-staining, varying from undifferentiated to differentiated patterns of adenocarcinoma. Evidence of tumor regression is present around tire periphery of the mass and primarily characterized by the infiltration of macrophages.

[00257] FIG. 29: Subject 2010 (IV Abraxane®) Adenocarcinoma-3, Primitive-I,

Regression-0. Low-power magnification (2x) showing the general distribution of large expansive tumor mass filling most alveolar spaces as well as neoplastic cells in the periphery. Most tumor cells are predominantly undifferentiated, pleomorphic, large, anaplastic with pale amphophilic-staining. The primitive cells are smaller, ovoid, and have more basophilic staining cytoplasm with variable, vesicular nuclei and moderate to marked anisokaryosis. Inflammatory cell infiltration are predominantly neutrophils and macrophages. This image demonstrates an absence of tumor regression

[00258] FIG. 32: Subject 4009 (IH nPac l /wk High) Adenocarcinoma-0, Primitive-0, Regression-4. Low'-power magnification (2x) showing the general distribution of previously populated tumor masses, the presence of multiple small areas of fibrous connective tissue, central collagenous stroma and fibrocytes are seen at the peripheral alveolar spaces as well as thickened alveolar septae supports evidence of tumor regression. In addition, the alveolar spaces are commonly filled with infiltrate of macrophages and lymphocytes together with additional evidence of tumor regression .

[00259] FIG. 35: Subject 5010 (IH nPac 2x/w'k Low') Adenocarcinoma- 1, Primitive-0, Regression-3. Low-pow'er magnification (2x) showing the general distribution of previously populated tumor masses. Regressing masses are variably small and randomly distributed. Fibrous connective tissue is seen filling/replacing alveolar spaces and suggests foci of regressing adenocarcinoma. Acute necrosis, fibrous connective scaffolding, mixed cell infiltration of macrophages, giant cells and lymphocytes in the epithelium as well as around tire stroma are signs of tumor regression.

[00260] FIG. 39: Subject 6005 (IH nPac 2x/wk High) Adenocarcinoma- 1, Primitive-0, Regression-4. Low-power magnification (2x) showing the general distribution of previously populated tumor masses in multiple small areas of fibrous connective tissue filling/replacing the alveolar spaces suggesting foci of previous infiltrates of adenocarcinoma cells. Tumor regression is evidenced by fibrosi s of previously populated tumor m asses, central collagenou s stromal core and fibrous connective tissue at the periphery filling/replacing the alveolar spaces, thickening of the septae as well as tire presence of fibrocytes filling the alveolar space infiltrated by lymphocytes and macrophages.

RESULTS OF THE ADDITIONAL MORPHOLOGIC AND (IHC) STUDIES

[00261] After a review' of H&E slides of ail 120 animals in the study, it was noted that a possible immune response was seen in treatment groups. To further investigate this finding, a subset of animals was chosen from each group for further immunohistochemical evaluation.

[00262] Firstly, the trend of tumor regression as evaluated by a pathologist reviewing all 120 animals was compared to a different pathologist reviewing a subset of 1 7 animals to show a similar trend between the sample sizes.

[00263] Initial evaluation of the degree of tumor regression on all 120 animals was done via a pathologist grading semi-quantitively using a 5-point scale indicating the percent of involvement of the overall lung tissue. Hie grading system is based on a grading scale of: 0=no evidence, 1= 1-25% total area of lung sections, 2= 25-50% total area of lung sections, 3= 50-75% total area of lung sections, 4=75-100% total area of lung sections. Tills evaluation showed the incidence of animals presenting with tumor regression scored as follows, 0% of non-treated controls, 10% of IV Abraxane®, 55% of IH nPac low-dose once weekly, 55% of IH nPac low-dose twice weekly, 55% of IH nPac high-dose once weekly and 65% of IH nPac high-dose twice weekly.

[00264] A review of the subset of 17 animals performed by a separate pathologist evaluating tumor regression using as similar semi-quantitative grading scale (0=no evidence, 1=1-19% total area of lung sections, 2=11-50% total area of lung section, 3=greater than 50% total area of lung sections, 4=complete regression). This evaluation showed tire incidence of animals presenting with tumor regression scored as follows: 0% of non-treated controls, between 65-69% of IV Abraxane®, 100% of IH nPac low-dose once weekly, 100% of IH nPac low-dose twice weekly, 100% of IH nPac high-dose once weekly and 100% of IH nPac high-dose twice weekly. This review (17 animals) presented a similar patern to the previous review (120 animals) with the inhaled groups showing the greatest percent of animals with tumor regression.

[00265] Upon histological review' of the subset of 17 animals from the study, interesting patterns w'ith respect to tumor regression and immune response were seen. TWO main features differed amongst the various groups, notably the presence and degree of tumor regression and the presence and intensity of an accompanying immune response. Below are the observations and remarks of the histological review- .

No Treatment Group

[00266] Observations: FIG. 44: Control cases. Top row-: H/E stained sections. Bottom row: Immunohistochemical staining.

Column 1 : (A) Poorly differentiated area of adenocarcinoma composed of sheets of large cells with pleomorphic nuclei, increased mitoses and lack of glandular differentiation. Note dense compact arrangement of tumor cells, sharp demarcation from surrounding normal lung in lower right comer and the lack of a fihrotie capsule surrounding the tumor. (D) Corresponding keratin immunostain from same area shown in A. This demonstrates sensitive and specific labeling of carcinoma cells with pancytokeratin (solid arrow).

Column 2: (B) Adenocarcinoma with focal rudimentary duct formation (dashed arrow at top right). Note the focal, limited immune cell component in the center, consisting of small lymphocytes and focal macrophages (solid arrow's in center) (E) CDl lb stain showing minimal numbers of NK ceils and macrophages at the periphery of a tumor cell nodule (solid arrow).

Column 3: (C) Adenocarcinoma growing adjacent to a focus of bronchial associated lymphoid tissue (BALT) that consists of densely packed small mature lymphocytes (marked with solid arrow). Note the close association of the BALT with the adjacent normal bronchial lining (dashed arrow top left comer). (F) Corresponding focus to that seen in C, stained with keratin, showing positive staining in carcinoma cells and lack of staining in the lymphoid cells.

[QQ267] Remarks: Both animals presented uniform growth of solid, densely packed collections of adenocarcinoma. The tumors had relatively well demarcated margins bordering the surrounding normal lung parenchyma with no evidence of tumor regression and unabated tumor cell growth. The lymphoid infiltrate in these animals was mild and tertiary lymphoid structures were sparse.

Intravenous (IV) Abraxane® Positive Treatment Control Group

[00268] Observations: FIG. 45: IV Abraxane® case (2003) showing a nodule of adenocarcinoma with tumor regression consisting of separation of the tumor towards the periphery of the nodule into progressively smaller tumor cell clusters and single tumor cells, with an associated increased immune cell infiltrate.

Column 1 : (A) Low power view of a nodule of invasive adenocarcinoma (highlighted by dashed arrows). Note the irregular peripheral border of tire nodule due to progressive separation of tumor cells at the periphery and increased immune cell response (solid arrows). (D) Corresponding keratin immunostain from same area shown in A . This clearly demonstrates the progressively smaller size of tumor ceil nodules toward the periphery (dashed arrow's) and the increased intervening stroma between them (solid arrow).

Column 2: (B) High power view of the area in image A, showing the progressively smaller clusters of tumor cells (dashed arrows). (E) Higher power view of the keratin stained area shown in D, highlighting the separated smaller tumor cell nodules. Note the progressive decrease in tumor cell cluster size moving from the top right comer toward the bottom left comer where the tumor is present as individual single tumor cells (dashed arrows). The solid arrow⁷ highlights the increased intervening stroma with immune cells.

Column 3: (C) Immune ceils (highlighted with solid arrow) seen within the center of a tumor nodule (dashed arrow's highlight the tumor cells). (F) Low power view of a CD l ib-stained section highlighting the same area seen in image A. This shows the increased density of immune cells (solid arrow's) at the periphery of the nodule and within the tumor nodule. Dashed arrows highlight residual carcinoma cells that are not labeled with the CD 11b antibody.

[00269] Remarks: All three animals presented tumor growth in densely packed collections of adenocarcinoma, however, two of the animals show'ed some features compatible with tumor regression. This regression was characterized by the presence of progressive separation and loss of tumor cell clusters at the peripher of the tumor nodules with ill-defined demarcated margins bordering the surrounding normal lung parenchyma. The lymphoid infiltrate in the areas showing tumor loss showed an increase in lymphoid infiltrate in the stroma.

Inhaled nPac treatment groups

[00270] Observations: FIG 46: Inhaled nPac cases. Top row: Low dose, lx/week (LD1 X) (case 3006). (A) H/E staining showing tumor regression with in a nodule with prominent separation and loss of tumor cells at the periphery (dashed arrows show residual tumor and solid arrows show intervening stroma with inflammation) (B) Keratin stain highlights the residual carcinoma (dashed arrows) with a large intervening area of tumor loss (solid arrows) composed of background stroma with lymphocytes and macrophages. (C) CD l ib immunostain highlights a marked iymphohistiocytic immune cell infiltrate in the areas where there is tumor cell dropout (solid arrows). Residual unstained carcinoma is highlighted with dashed arrow.

Second row: Low dose, 2x/week (LD2X) (case 4009). (D) H/E staining showed no residual viable adenocarcinoma. This case contained scattered foci such as this that were composed of collections of small lymphocytes and macrophages within background stroma. No diagnostic viable tumor cells were seen in these nodules, or elsewhere in the lung sections. (E) Keratin stain in the same area as D, showing lack of staining, thus adding immunohistochemical support for the interpretation of no residual viable carcinoma and complete regression. (F) CD! lb stain shows that this focus has a mild-moderate immune cell infiltrate.

Third row: High dose, lx/week (HD IX) (case 5008). (G) H/E staining showing tumor regression in a nodule with prominent separation and loss of tumor cells at the periphery (dashed arrows show residual tumor and solid arrows show intervening stroma with inflammation). (H) Keratin stain highlights the residual carcinoma (dashed arrows) and a large imstained area of tumor loss (solid arrows) composed of background stroma with lymphocytes and macrophages. (I) CDl lb immunostain highlights a marked immune cell infiltrate in the areas where there is tumor cell dropout (solid arrow). Residual pockets of unstained carcinoma are highlighted with dashed arro .

Fourth row: High dose, 2x/week (HD2X) (case 6005). (J) H/E staining showed numerous collections such as this one that contains cells with eosinophilic and foamy cytoplasm (low power). (K) Higher power of same area shows cells with spindled nuclei (solid arrow) and rare possible duct-like structures or regenerating small blood vessels (dashed arrow). (L) Masson trichrome stain shows blue staining of stroma consistent with early collagen fibrosis and organization .

Fifth row : High dose, 2x/week (HD2X) (case 6005 continued). (M) Keratin stain shows labeling of focal single cells and duct-like structures (dashed arrow). Intervening cells are negative for keratin (solid arrow⁷). (N) CDl lb immunostain highlights an immune cell infiltrate in the area where there is tumor ceil dropout (solid arrow⁷). The magnification in this image matches that in j. [00271] Remarks: Of the 12 animals one animal presented no residual adenocarcinoma and was interpreted as a complete responder (versus non-engraftment). One animal presented as difficult to classify as it contained rare instances of tumor positive staining that were difficult to differentiate as tumor or as regenerative small blood vessels and/or regenerative/atrophic non-neoplastic lung parenchyma. As such, this second case also was interpreted as extensive and near-complete responder. In these two cases, there were scattered foci of immune cells in areas presumed to previously contain solid clusters of adenocarcinoma. One case presented evidence of organization with deposition of fibrous collagen noted by Masson’s Trichrome staining. All remaining 10 animals presented tumor nodules w ith varying degrees of apparent tumor regression with 8 of the 10 animals presenting tumor regression in >50% of the tumor nodules. The inhaled nPac group presented with lymphoid infiltrate that varied from well-defined organized collections of densely packed mature lymphoid cells with well-defined lymphoid follicles and germinal centers and interfoil icuiar areas and paracortical zones. As well as smaller dense collections of lymphoid tissue at the periphery and focally within the center of the tumor nodules.

Observation of Tertiary Lymphoid Structures (TLSs)

[00272] Secondary lymphoid organs develop as past of a genetically preprogrammed process during embryogenesis and primarily serve to initiate adaptive immune response providing a location for interactions between rare antigen-specific naive lymphocytes and antigen-presenting cells draining from local tissue. Organogenesis of secondary lymphoid tissues can also be recapitulated in adulthood during de novo lymphoid neogenesis of tertiary lymphoid structures (TLS) and form in die inflamed tissue afflicted by various pathological conditions, including cancer. Organogenesis of mucosal-associated lymphoid tissue such as bronchial-associated lymphoid tissue is one such example. The term TLS can refer to structures of varying organization, from simple clusters of lymphocytes, to sophisticated, segregated structures highly reminiscent of secondary⁷ lymphoid organs. A notable difference between lymph nodes and TLS’s is tire that where lymph nodes are encapsulated, TLS’s represent a congregation of immune and stromal cells confined within an organ or tissue. [QQ273] Observations: FIG, 47: Lymphoid structures in treated and untreated cases.

Top row: Inhaled nPac case demonstrating tertiary lymphoid structures (TLSs) with follicular hyperplasia. High dose, 2x/week (HD2X) (case 6007). (A) H/E stain showing two adjacent TLSs (highlighted with solid arrows). In the lung these are referred to as bronchial associated lymphoid tissue (BALT). Note the organoid appearance of these TLSs in that they are composed of well-circumscribed collections of dense lymphoid tissue with distinct topology that includes lymphoid follicles with prominent germinal centers, interfollicular areas and paracortical zones. Dashed arrows highlight adjacent foci of tumor with irregular peripheral borders consistent with tumor regression. (B) Higher power image from area in A. The smaller TLS contains a lymphoid follicle with a prominent germinal center (paler area at tip of arrow). This process of germinal center formation in lymphoid follicles is referred to as follicular lymphoid hyperplasia and is indicative of lymphoid tissue that is activated and is the process of mounting an immune response to various antigens including foreign material and tumor debris. Germinal centers characteristically show polarization with light and dark zones of lymphoid ceils. In this image, the pale zone of the germinal center is pointing toward the adjacent tumor nodule. (C) Keratin stain showing the adjacent carcinoma nodules that have irregular peripheral borders. Solid arrow shows the TLS This TLS appears smaller in tliis section as this tissue section was from a deeper portion of the paraffin embedded tissue compared to that in the H/E stained section shown in A and B.

Second row: Comparison between control (D), IV Abraxane® (E) and nPac (F) cases to illustrate the differences in the number and density of smaller lymphoid collections associated with tumor nodules in the different groups. These three images are all at the same lower power magnification (4x objective). (D) Control case (1003) shows densely packed adenocarcinoma (dashed arrow ) without any discrete lymphoid collections. (E) TV Abraxane® case (2009) showing nodules of adenocarcinoma (dashed arrow) and only a single rare small lymphoid collection at the lower right (solid arrow). (F) nPac case, high dose 2x/week (HD2X) showing adenocarcinoma nodules (dashed arrow) w ith numerous associated small and medium sized collections of small lymphoid cells. These are arranged at the periphery of the tumor and also ideally within the tumor (solid arrows).

[00274] Remarks: The inhaled nPac groups showed increased numbers and density of TLSs (2 per low power field) compared to controls and the IV Abraxane® group (1 per low power field), and more of these TLSs showed increased size and activation with follicular lymphoid hyperplasia containing prominent germinal centers.

[00275] In summary , the sub-review⁷ of 17 animals presented some interesting patterns with respect to tumor regression and immune response. In particular, all of the animals treated with nPac showed at least some features compatible with tumor regression which includes two animals showing complete and/or near complete regression, while 8 of the remaining 10 animals in this group showed some features compatible with tumor regression in >50% of the tumor nodules. This was an increased response compared to the control groups wliere no animals showed a response, and die IV Abraxane® group where 2 of 3 animals showed tumor regression m 1-10% of the tumor nodules.

[00276] Evaluating the nPac groups with each other, a higher dose and increased frequency in dosage (2x/week versus lx/week) were both associated with a greater effect on tumor response. Hie data supports an immune based association with tumor regression, the nPac groups also showed increased numbers, and density of TLSs (2 per low power field) compared to controls and the IV Abraxane® group (1 per low power field), and more of these TLSs showed increased size and activation with follicular lymphoid hyperplasia containing prominent germinal centers. There was also a greater density of immune cells at tire periphery of tumor nodules and within tumor nodules in the nPac groups.

CONCLUSIONS

[00277] One hundred twenty-seven (127) NIH-mu Nude Rats were x-irradiated to induce immunosuppression on Day -1. On Day 0 animals were dosed with Calu3 tumor cells by intratracheal (IT) instillation. Animals underwent a growth period of three weeks. During the third week, animals were randomized by body weight stratification into the groups. Starting Week 4, animals in Group 2 received a once weekly dose of Abraxane© by intravenous (IV) dosing (5 mg/kg) on Days 22, 29 and 36 Animals in Groups 3 and 4 received once weekly (Monday) inhalation (INH) dose of nPac at low (0.5mg/kg) and high (1.0 mg/kg) target doses, respectively. Animals in Groups 5 and 6 received a twice weekly (Monday and Thursday) target inhalation dose of nPac at low (0.50 mg/kg) and high (1.0 mg/kg) doses respectively. Animals in Group 1 were left untreated as a control of normal tumor cell growth. All animals w'ere necropsied during Week 8.

[00278] All animals survived to their designated necropsy timepoint. Clinical observations related to the model included skin rash, labored breathing. All groups gained weight at about the same rate through the course of the study.

[00279] The inhalation exposure average Paclitaxel aerosol concentration for once weekly Low Dose and twice weekly Low Dose nPac groups was 270.51 pg/L and 263.56 pg/L, respectively. The inhalation exposure average Paclitaxel aerosol concentration for once weekly High Dose and twice weekly High Dose nPac groups was 244.82 pg/L and 245.76 pg/L, respectively.

[00280] Doses were based on average aerosol paclitaxel concentration, most recent average group body weight, assumed deposition fraction of 10% and exposure duration of 33 or 65 minutes. During four weeks of treatment, the average achieved rodent deposited dose for the once weekly Low Dose nPac group and twice weekly Low Dose nPac group were 0.655 mg/kg and 0.640 mg/kg (1.28 mg/kg/week), respectively. The average achieved rodent deposited dose for the once weekly High Dose nPac group and twice weekly High Dose nPac group were 1.166 mg/kg and 1 .176 mg/kg (2.352 mg/kg/week), respectively. For the group receiving IV injections of Abraxane ®, the average dose on Day 22, 29 and 36 was 4.94, 4.64 and 4.46 mg/ kg respectively.

[00281] At scheduled necropsy, the majority of animals from each group had tan nodules on tire lungs and/ or red or tan patchy discolorations of the lung. Other sporadic observations included an abdominal hernia in one animal and nodule on the pericardium of another animal. No other abnormal gross observations were noted at necropsy.

[00282] In Abraxane® treated animals, lung weights, lung to BW ratios and lung to brain weight ratios were significantly lower compared to Untreated Controls. Hie once weekly nPac High Dose group had similar weights to the Abraxane® group and significantly lower lung weights and lung to brain ratios compared to Untreated Controls.

[0Q283] Compared to the positive control Gip. 1 and the Abraxane® treated comparative Grp. 2, there was a therapeutic effect as measured by lower lung/brain weight ratio and lower overall lung tumor burden without apparent adverse events. Histological analysis of lung tumor burden treated with inhaled nPac showed a decrease in tumor mass, a decrease in primitive tumor cell population, and an increase in tumor regression. Extensive mononuclear ceil infiltration was observed in the lungs of animals receiving nPac through inhalation. As the model used is T cell deficient, it is likely that the cells are B cells or NK cells. It is hypothesized that the localized, likely higher concentration exposure of the tumor to nPac affected the tumors leading to an alteration in the environment to draw the mononuclear cellular infiltrate into the lung.

Example 2. Human Bladder Cancer (UM-UC-3) Mouse Xenograft Study

[00284] A study was conducted to evaluate the effect of 1 , 2, and 3 weekly intratumorai injection (IT) administrations (administration cycles) of nDoce (nanoparticle docetaxel as disclosed herein, approximately 99% docetaxel with a mean particle size (number) of 1.078 microns, a SSA of 37.2 m²/g, and a bulk density (not tapped) of 0.0723 g/cnf used in this example) suspension on growth of subcutaneous (SC) UM-UC-3 bladder cancer cell line (ATCC-CRL-1749) tumors in immunocompromised (Hsd:Athymic Nude-Foxnlnu nude) mice. Intratumorai injection administration of a vehicle and intravenous (IV) administration of docetaxel solution ware also incorporated into the study as control groups. [00285] Tumors were implanted with 1x10' cells (lOOpL volume) into right flank (PBS 1: 1 with matngel : BD356234). Tumor volume was determined with calipers. Formula: V~ (r length* r width * r height) *p* 4/3. Animals were weighed 2x/week. Tumor volumes were determined ever' 3 to 4 days following tumor implant (total of ~20 measurements) and on day of euthanasia. Photo images of tumors were obtained at 2, 3 and 4 weeks post implantation and on day of euthanasia. Animals were euthanized once the tumor reached a size of 3,000mm³ or up to the point of significant tumor ulceration. At the time of euthanasia, tumors were isolated and halved. One half of tire tumor was flash frozen in LN2 stored at -80°C and will subsequently be analyzed. The second half of the tumor w'as fixed in formalin. Two H&E stained slides/tumor were prepared (up to 4 tumor/group were processed).

[00286] At day 18 after tumor implant, when average tumor size was between 50-325 mm’, animals were sorted into five groups with equal average tumor sizes and were treated as shown in Table 25 below.

Table 25. Main Study Design

[QQ287] For IT administration (Vehicle/nDoce), injections (using 27G, ½” needle) were administered at three sites within the tumor (total calculated injection volume based on 40 mg/mL nDoce stock and 25 g mouse = 63 pL; split evenly across the three injection sites) to maximize distribution of the test formulation throughout the tumor. The second treatments (2^nd cycle) occurred 7 days following first treatment (1^st cycle) and third treatments (3^rd cycle) occurred 14 days following the first treatment. The docetaxel solution IV was administered via tire tail vein.

[QQ288] The test formulations were prepared as follows:

Vehicle (Control): Diluted 1 m3 of the 1% Polysorbate 80/8% Ethanol in normal saline (0.9% Sodium Chloride for Injection) reconstitution solution with 1 .5 mL of normal saline (0.9% Sodium Chloride for Injection, USP). The final concentration of polysorbate 80 was 0.4% and the final concentration of ethanol was 3.2% the V ehicie .

nDoce Suspension: Added 1 ml of the 1 % Polysorbate 80/8% Ethanol in normal saline (0.9% Sodium Chloride for Injection) reconstitution solution into die vial of nDoce particles powder (100 mg/60 cc vial). The mean particle size (number) of the nDoce particles powder was 1.0 micron. Vigorously hand shook the vial with inversions for 1 minute. Immediately after shaking, added 1.5 ml of normal saline solution (0.9% Sodium Chloride for Injection USP) to the vial and hand shook the vial for another 1 minute to make a 40 mg/mL suspension. Allowed the suspension to sit undisturbed for at least 5 minutes to reduce entrapped air and foam.

Docetaxel Solution: Prepared a 20 mg/mL docetaxel stock solution in 50% Ethanol/50% Polysorbate 80. Added normal saline solution (0.9% Sodium Chloride for Injection) to stock solution to make a final, 3 mg/mL docetaxel solution. Vortexed to mix.

Results:

[00289] Tumor volumes -were determined 2x/week for the duration of the study (61 days). The results of the study are shown in FIG, 48, FIG. 49, FIG. 50, FIG. 51, FIG. 52, FIG. 53, FIG. 54, FIG. 55, FIG. 56 & FIG. 57. As seen in FIG. 48, tumor volumes decreased and tumors were effectively eliminated for dosages of nDoce IT 2 cycles and nDoce IT 3 cycles. Tumor volumes decreased initially for dosages of nDoce IT 1 cycle and Docetaxel IV 3 cycles, but subsequently increased. These observations are also reflected in FIG. 49, FIG. 50, FIG. 51, FIG. 52, FIG. 53, FIG. 56 & FIG. 57

[00290] The scatter plot in FIG. 54 shows tumor volumes per animal on Day 1 of treatment vs. end of study (day of sacrifice). As can be seen in FIG. 54, the volume of the tumor in a given animal at the end of study was not dependent upon the initial size of the tumor of the same animal for the animals treated with nDoce IT 2 cycles and nDoce IT 3 cycles, as essentially all the tumors were effectively eliminated. However, for animals treated with Docetaxe! TV 3 cycles, the volume of the tumor at the end of the study was generally dependent upon the initial tumor volume for a given animal, i.e., the larger the initial tumor volume, the larger the tumor volume at the end of the study. The treatment with Docetaxel IV 3 cycles was somewhat effective at treating small tumors, but not very effective in treating large tumors. Administering nDoce IT (intratumorally) for 2 cycles or 3 cycles effectively treated the tumors regardless of the initial tumor size.

[00291] As can be seen in FIG. 55, the initial animal weight loss for animals treated with Docetaxel IV 3 cycles w¾s generally greater than that of animals treated with nDoce IT 1 cycle, nDoce IT 2 cycles, and nDoce IT 3 cycles. Weights eventually recovered to some degree in all treatments. This may suggest that the side effect of initial appetite loss is greater with Docetaxel IV administration than with nDoce IT administrations. It was also observed that animals treated with Docetaxel IV 3 cycles had greater signs of peripheral neuropathy than did those treated with nDoce IT 3 cycles, and no signs of peripheral neuropathy w'ere observed in those treated with nDoce IT 1 cycle or 2 cycles.

[00292] On the day of death or euthanasia, tumor tissues samples were collected and frozen in LN2 for docetaxel analysis, histology, and immunohistochemistry (IHC) observations. In the IV docetaxel control group, only 1 tumor (of 7 measured) had docetaxel levels above the limit of quantitation of the assay (1 ng/g). Measurable levels of docetaxel were found in all tumors from the IT nDoce groups with the nDoce 3 cycle group tending to have the highest concentrations of docetaxel remaining in the tumors (see FIG. 58). Photomicrographs of histology slides, H&E stain, are shown in FIG.s 59 to 69. Photomicrographs of IHC slides stained wnth F4/80 antibody stain are shown in FIG. 70, FIG.71, and FIG. 72.

[00293] Additional H&E and Immunohistochemical (IHC) evaluations were conducted on formalin-fixed tissue and are shown in FIG. 73 and FIG. 74.

Histological Overview of Photomicrographs in FIG.s 59 to 69

General Observations:

[QQ294] Control: Extensive levels of viable tumor with proliferating cells and little to no mononuclear immune cell infiltration, occasional macrophages noted.

[00295] Docetaxel Solution: many viable appearing tumor masses with some macrophage and occasional lymphocytic response along with some tumor necrosis. [00296] nDoee 2 cycles: Some remaining isolated tumor cells, small area of skin injury, scar/fibrosis seen, immune cell infiltrate including macrophages and mononuclear cells.

[00297] nDoee 3 cycles: Some remaining isolated tumor cells, small area of skin injury, scar/fibrosis seen, immune cell infiltrate including macrophages and mononuclear cells

[00298] Extensive mononuclear cell infiltration was observed at the site of tumor implantation in the subcutaneous space in animals receiving intratumoral injection of nDoee. With increased numbers of cycles, there is increased tumor response, but there is some skin injury, perhaps due to the small space and shallow area for injection on tire flank of a nude mouse (e.g., tumor right up against skin that is tightly drawn over the tumor). As the model used is T cell deficient, it is likely that the lymphocytic cells are B cells or NK cells. B cells are responsible for the production of cytotoxicity (the antibodies bind to cells expressing Fc Receptors and enhance the killing ability of these cells. NK cells are innate lymphoid cells that are crucial in the killing of tumor cells. In patients with tumors, NK cell activity is reduced allowing for the growth of the tumor. Along with T cells, NK cells are the target of some check point inhibitors to increase their activity. In all histological samples provided, macrophages were present in the tumor, but the number did not appear to significantly increase.

[00299] By the use of a wide array of surface receptors capable of delivering either triggering or inhibitory signals, NK cells can monitor cells within their environment to ascertain if the cell is abnormal (tumor or virally infected) and should be eliminated through cytotoxicity. The cytotoxicity and chemotaxis of NK cells can be modified by many pathological processes including tumor cells and their byproducts. In response to certain signals their functions are enhanced or potentiated. In response to several Pathogen Associated Molecular Patterns (PAMPs) by using different Toll Like Receptors (TLR); NK cells can increase cytokine production and/or cytolytic activity. Cytokines, including IL-2, IL-15, IL-12, IL-18, and IFNs a/b can also modify the activity of NK cells. NK cells are not simple cells that are only cytolytic effectors capable of killing different tumor cell targets; rather, they represent a heterogeneous population which can finely tune their activity_' in variable environmental contexts.

[00300] The tumor burden is significantly reduced in the site of xenograft injection in the animals treated with nDoee and the intratumoral injection is more effective than intravenous docetaxel . Therefore, the localized administration of docetaxel in the form of nDoee provides additional potency. This is likely due to both the longer exposure to the chemotherapy over time and the vigorous cellular infiltration to the site of the tumor. This latter response appeared to be dependent on the dose density (actual dose and dose frequency). Anatomically, macrophages are present at high numbers at the margins of tumors with decreasing frequency throughout the stroma moving deeper within the tumor.

Immunohistochemistry Overview of FIG. 70, FIG. 71, and FIG. 72

[00301] FIG. 70: Vast sheet of viable tumor cells and no mononuclear immune cells (no brown staining).

[00302] FIG. 71: Very little tumor cell destruction and few scatered mononuclear immune cells among vast number of viable tumor cells.

[00303] FIG. 72: Virtually no tumor cells left and vast numbers of mononuclear immune cells organized into distinct patterns (likely mostly macrophages).

Additional H&E and Inimunohistochemka! (IHC) Evaluation (see FIG. 73 and FIG. 74) [00304] Tumor tissue was fixed before H&E and IHC staining. Bladder tissue sections were deparaffmized and processed by standard H&E and IHC staining. At least four tumors per treatment group were processed.

[00305] Observations: FIG. 73 Control Cases:

Top row: H&E Stained Sections (A-C): (A) Bladder carcinoma composed of sheets of closely packed large pleomorphic tumor cells. (B) Higher power view showing large tumor cells with prominent nucleoli (solid arrows) and a marked increase in mitotic figures (dashed arrows). (C) Low power view showing a focus of geographic tumor cell necrosis with admixed degenerating tumor cells (dashed arrow) and adjacent viable carcinoma at botom and top of image (solid arrow).

Botom row : IT vehicle (D) and IV Docetaxel (E and F): (D) IT vehicle case (case A3). H&E stained section showing extensive necrosis in bottom half of image (dashed arrow) and viable carcinoma in top left (solid arrow). (E) IV docetaxel (case B l). H&E stained section showing viable carcinoma in top right portion of image that appeared similar to that in the control and IT vehicle cases (solid arrow). Note sharp demarcation from non-neoplastic fatty tissue in low'er left without a capsule surrounding the tumor (dashed arrow ). The fat contained a sparse immune cell infiltrate. (F) IV docetaxel (case B l). CD68 stain highlighting mild macrophage infiltrate in surrounding stroma in bottom half of image (dashed arrows). Viable carcinoma is at top of image (solid arrow).

[QQ306] Observations: FIG. 74 Intratumoral nDoce cases (representative images from all groups included: 1 cycle, 2 cycles and 3 cycles).

Top row: One cycle nDoce (lx) (case C4). (A) Low power H/E staining showing extensive geographic tumor cell necrosis consisting of homogeneous eosinophilic staining of non- viable necrotic material (dashed arrows). The necrosis spans from the overlying mouse skin surface in top right of image (two solid arrows) to the focal viable carcinoma in the bottom left comer (single solid arrow). (B) High power view of viable carcinoma at left (solid arrow) and necrosis at right (dashed arrow). (C) CD68 immunohistochemical stain showing mild macrophage infiltrate (solid arrow) in the surrounding non-neoplastic fatty tissue.

Second row: Two cycles of nDoce treatment (2x) (case D2). (D) Low power view showing a tertiary lymphoid structure (TLS) that measured 2 mm in maximum dimension (solid arrow). Note well-circumscribed border of TLS and demarcation from surrounding tissue with immune cell infiltrate. Note overlying ulcerated skin (dashed arrow). (E) CD45R immunostain (B-cell marker) showing extensive staining throughout the TLS, confirming that the majority of the lymphocytes in the TLS are B-cells. Note the organization into B-cell lymphoid follicles (solid arrow's) and focal unstained areas that represent interfollicular‘T- ceir zones (dashed arrow's). (F) Higher power view of same TLS. Note the organization of the TLS with a hilar region that contains medullary sinuses (dashed arrow) and a germinal center forming in one of the lymphoid follicles (solid arrow).

Third row : Two cycles of nDoce treatment (2x) (case D2), continued. (G) Higher power view of germinal center. Note the polymorphous lymphoid population in the germinal center that consists of a mixed population of small mature lymphocytes, intermediate sized centrocytes and occasional larger centroblasts (solid arrow⁷). Compare this with the adjacent homogenous population of small mature lymphocytes (dashed arrow). (G) Same case, showing separate area with ulcerated skin at left (dashed arrow) and necrotic tissue at right (solid arrow). No viable carcinoma is present. (H) Higher power view of the necrotic area showing homogenous eosinophilic amorphous necrotic material with no diagnostic viable carcinoma. Fourth row: Three cycles of nDoce treatment (3x) (case D2). (J) Low power view showing ulcerated skin surface at top with underlying necrosis (dashed arrow). Note adjacent TLS in lower right portion of image (solid arrow). (J) Low power view⁷ of CD45R-immunostained section showing dense population of B-cells in the TLS (solid arrow⁷). (L) High power view of the necrotic area beneath the skin ulceration showing amorphous necrotic material with no diagnostic viable carcinoma cells.

Histopathology:

[QQ307] Non-treated Control: On day of necropsy, the tumor volume in the non-treated control animal was measured and then tumor site tissues were dissected and approximately half the tumor was processed for docetaxel content and half was preserved for histological analysis. The non-treated control tumor contained an extensive diffuse proliferation of invasive carcinoma that measured up to 15 mm on the slides and consisted of sheets of tumor ceils that were closely packed together (FIG. 73 - Slide A). The tumor cells were large with pleomorphic nuclei that had vesicular chromatin and prominent eosinophilic nucleoli. The tumor cells had a moderate amount of lightly eosinophilic cytoplasm and they showed markedly increased mitotic activity (122 mitoses per 10 high power fields [400x hpf])( FIG. 73 - Slide B). Individually necrotic and apoptotic tumor cells were present within the tumor and there were also scattered areas of coagulative tumor cell necrosis that overall occupied 5- 10% of the tumor area. The foci of necrosis consisted of homogenous eosinophilic necrotic debns and this contained areas of admixed degenerating tumor ceils (FIG. 73 - Slide C). There was no significant lymphoid infiltrate within the tumor and in particular, there were no discrete small lymphoid collections or tertiary' lymphoid structures (TLS) in the tumor tissue or in the surrounding non-neoplastic stromal tissue. The surrounding stroma contained a patchy mild immune cell infiltrate. Immunohistochemicai staining for CD68 (marker of macrophages) highlighted a mild macrophage infiltrate within and around the tumor with increased density of staining within the foci of tumor necrosis, consistent with increased concentration of macrophages in areas containing increased cellular debris.

[00308] Non-treated Intratumoral vehicle group: On day of necropsy, tumor volumes in these IT vehicle animals were measured and then tumor site tissues were dissected and approximately half the tumor was processed for docetaxel content and half was preserved for histological analysis. The two intratumoral vehicle cases demonstrated similar findings at the morphologic and immunohistochemical level and both had a similar morphologic and immunohistochemicai appearance to that seen in tire above-mentioned control case. In particular, both cases contained extensive sheets of large carcinoma cells with an identical appearance to that seen in the control cases. The viable tumor measured up to 12 and 24 mm m maximum dimension on the slide in these two cases, respectively. Both cases also contained geographic areas of necrosis and this was fairly extensive in one case where it occupied > 50% of the tumor area (case A3) (FIG. 73 Slide D). There was very limited non neoplastic tissue for assessment in both cases although where present, this contained a mild immune cell infiltrate. There were no TLSs present

[00309] Intravenous Docetaxel: On day of necropsy, tumor volumes in the IV docetaxel animals were measured and then tumor site tiss ues were dissected and approximately half the tumor was processed for docetaxel content and half was preserved for histological analysis. The two IV docetaxel cases demonstrated similar findings at the morphologic and immunohistochemicai level and both had a similar morphologic and immunohistochemicai appearance to that seen in the above-mentioned control ease and the two IT vehicle cases. Specifically, both cases contained sheets of large viable carcinoma cells and interspersed areas of geographic tumor cell necrosis that occupied 1 1-50% (case Bl) and 50-90% (case B3) of the tumor area in the two cases, respectively (see Table 29 below; FIG. 73 - Slide E and FIG. 73 - Slide F). Both cases had tumor masses that measured > 10 mm in maximum dimension on the slide (11 mm and 15 mm) (see Table 26 below'). The surrounding stromal tissue contained a mild immune cell infiltrate. There were no TLSs present.

[00310] Intratumoral nDoce 1 cycle: All three animals in this group contained residual carcinoma that w¾s composed of similar pleomorphic ceils as seen in the control, IT vehicle and IV doeetaxel groups. However, the amount of residual carcinoma varied dramatically within this group. Specifically, two of the three cases (cases Cl and C6) contained extensive residual viable carcinoma that measured 16 mm and 19 mm in maximum dimension on the slide. These two cases also had geographic necrosis that occupied 11-50% of the tumor area. One of these two cases (case Cl) contained a small amount of non-neoplastic tissue with a mild immune cell infiltrate. Hie other case did not have any non-neoplastic tissue present to assess for a surrounding immune cell infiltrate (Case C6). By contrast, the third case (case C4) showed necrosis of 50-90% of the tumor and in this case there was only a small focus of residual viable carcinoma present that measured 2.5 mm in maximum cross-sectional dimension on the slide (FIG. 74 - Slide A and FIG. 74 Slide B). In this same case the surrounding non-neoplastic stroma contained a mild immune cell infiltrate (FIG. 74 - Slide C). In addition, in the deeper immunohistochemical-stained sections a TLS was noted in the adjacent non-neoplastic fatty tissue. Hie TLS measured approximately 1 mm in maximum dimension and consisted of a dense, well-circumscribed collection of small mature lymphocytes showing organization into lymphoid follicles and a hilar region. Staining for CD45R confirmed that the majority of the lymphocytes in the TLS were B-cells and that these were organized into B-cell follicles within the TLS. As in the non-treated and vehicle controls, on day of necropsy, tumor volumes in these animals were measured and then tumor site tissues were dissected and approximately half the tumor was processed for doeetaxel content and half was preserved for histological analysis.

[00311] Intratumoral nDoce 2 cycles: Four of the five animals in this group had the entirety of their tumor site tissue preserved for histological analysis. TWO of the five animals (cases D2 and D8) in this group contained no residual viable carcinoma and these animals also demonstrated extensive geographic tumor necrosis ( 100% of tumor necrotic; FIG. 74 - Slide H and FIG. 74 - Slide I). In two of the remaining three animals (cases D4 and D6) there was also extensive necrosis (> 90% of tumor) and in both cases there were only rare, tiny collections of detached tumor cells present, tire largest of which measured up to 0.1 mm in each case. The significance of these rare tiny detached tumor cell clusters was not certain and given their appearance and detached localization adjacent to the edge of the tissue and edge of necrosis, an artifact of sectioning could not be excluded. In each of these four cases there was a single TLS. Three of the TLSs measured 1 mm, lmm and 2 mm, while the fourth measured 0.1 mm (case D8). The TLSs were discretely located within non -neoplastic tissue and were generally in the vicinity of, or directly adjacent to the necrotic material (FIG. 74 - Slide D). The TLSs were well-circumscribed, but they lacked a fibrous capsule. The internal topology of the TLSs showed varying degrees of maturation but in the more mature- appearing TLSs there was a distinct resemblance to secondary_' lymphoid organs, with some of these having hilar regions with medullary sinuses that extended towards peripherally placed lymphoid follicles that were composed of homogenous small mature lymphocytes without visible nucleoli (FIG. 74 - Slide F and FIG. 74 - Slide G). The interfollicular areas also contained similar appearing small mature lymphocytes with occasional larger lymphoid cells consistent with immunoblasts. Focally, some of the lymphoid follicles contained germinal centers that were composed of a polymorphous lymphoid population that included small mature lymphocytes, intermediate-sized centrocytes and larger cells consistent with centroblasts (FIG. 74 - Slide G). Occasional tangible-body macrophages were also noted in germinal centers. Immunohistochemical staining for CD45R showed strong staining of B- cells in the TLSs. Specifically', this result highlighted the B-cells in the lymphoid follicles, including germinal centers and showed absence of staining in the interfollicular lymphoid cells (T-cell areas)(FIG. 74 - Slide E). The fifth case in this group (case D9) contained a residual focus of viable carcinoma that measured 8 mm in maximum dimension and also showed necrosis of 5-10% of the tumor area. This animal had approximately 50% of tumor site tissue preserved for histological analysis and 50% analyzed for docetaxel content. Staining for CD68 showed a moderate macrophage infiltrate in 1 of the 5 cases in this group (case D2) and a mild macrophage infiltrate in the remaining four cases (eases D4, D6, D8 and D9).

[QQ312] Intratumoral nDoce 3 cycles: None of the three animals (El, E7, E9) in this group contained residual diagnostic viable invasive carcinoma nodules and all three cases also demonstrated extensive necrosis (FIG. 74 - Slide L). All three animals in this group had the entirety of their tumor site tissues preserved for histological analysis. In two of these animals (El and E7) there was a large area of skin ulceration, subjacent to which was an area of necrosis that extended into surrounding non-neoplastic fibrofatty and muscular tissue. This was associated with regenerative changes in the surrounding epidermal lining that included areas of pseudoepitheiiomatous hyperplasia, as well as degenerative changes in muscular cells. Similarly, within and adjacent to the necrosis there were regenerative larger stromal cells including fibroblasts and endothelial cells. There were also rare admixed single larger ceils in the necrosis that had degenerating nuclei. These rare cells appeared to be in the process of necrosi s or completely necrotic and while it was difficult to definitively exclude that these may have represented rare dying tumor cells, these could also have represented reactive/regenerative stromal cells or degenerating muscle cells as definitive muscle cells elsewhere in the section showed similar degenerative nuclear features. As such, the exact significance of these rare cells was not certain, but they did not form cohesive nodules and they appeared to be either dying or necrotic. A pancytokeratin (AE1/AE3) immunostain was performed to further assess these cells; however, while tins showed lack of labeling of some of these larger cells, there was excessive background staining that made definitive assessment difficult in some areas. In addition, the pancytokeratin performed in this study overall was not reliable with lack of sensitivity in the control cases. As such, definitive assessment of these sections with the current keratin stain was not reliable and this will be deferred to review of slides stained with another keratin immunostain (keratin 7) which is currently pending. All three cases also contained a single, well-formed TLS and these measured 0.8 mm, 1.5 mm and 2 mm in maximum dimension in the three animals. The TLSs in this group (FIG. 74 - Slide I and FIG. 74 - Slide K) had a similar range of maturation and CD45R pattern of staining to that described in the nDoce 2 cycle group above. In particular, the TLS were well circumscribed and located in tire vicinity of the necrosis and ulceration. The TLSs in this group showed internal organization with lymphoid follicles that were composed of B-cells that strongly expressed CD45R and some of these lymphoid follicles contained germinal centers. CD68 staining highlighted a moderate macrophage infiltrate in all three animals.

[00313] Tables 26 and 27 below reflect the maximum cross-sectional dimension of the viable carcinoma, as measured in millimeters on the slide.

Table 26: Maximum size of viable invasive carcinoma on the slide in each group

_

Table 27 Comparison of the non-nDoce treatment groups with the IT nDoce groups

_

[00314] Table 26 shows the range of sizes of residual tumor in the six groups. Table 27 condenses this data to directly compare the size of the residual carcinoma nodules in the three non-nDoce groups (5 animals in total) with the three nDoce groups (11 animals in total). All five non-nDoce animals had residual viable carcinoma nodules that measured greater than 10 mm. By contrast, just under half (5/11) of the animals treated with IT nDoce had no diagnostic residua! viable carcinoma on the slide to measure (complete regression). In two of the remaining 5 animals in the IT nDoce group that had residual viable carcinoma, this consisted of rare tiny tumor cell collections where tumor measured up to 0.1 mm in maximum dimension. The significance of the tiny amount of tumor in these cases was not certain as the detached localization and small size also raised the possibility of sectioning artifact. In a third case the residual tumor measured 2.5 mm and in the remaining three cases the tumors measured 8 mm, 16 mm and 19 mm in maximum dimension on the slide.

[00315] Comparison of the three IT nDoce groups with respect to percentage of cases with no residual invasive carcinoma and the size of residual viable carcinoma nodules on the slide is shown in Table 28.

Table 28: Comparison of tumor size the three IT nDoce groups

[QQ316] With progressive increase in the number of cycles of IT nDoce from 1 cycle to 3 cycles, the percentage of cases with no residual carcinoma increased. Specifically, the IT nDoce I cycle group had 0% (0/3) of cases with compete regression, although one of these cases measured only 2.5 mm, while the other two measured 16 and 19 mm on the slide. By contrast, the group given 2 cycles of nDoce had complete regression in 40% of cases (2/5). However, of the remaining three cases in this group that had residual viable carcinoma, this was extremely minimal, with clusters measuring up to 0.1 mm that could possibly have represented an artifact. Finally, the group given 3 cycles had complete regression m 100% (3/3) of the animals, with no residual viable carcinoma to measure in the any of the three cases in the IT nDoce 3 cycle group.

[00317] The percentage of tissue showing necrosis is shown in Table 29.

Table 29: Percentage of tumor showing necrosis

[00318] All 16 animals in tins study contained geographic tumor cell necrosis and in tire non-nDoce-treated cases this included two cases with 50-90% tumor necrosis. However, overall the extent of tumor cell necrosis was significantly greater in the nDoce-treated group than in the non-nDoce-treated group. Specifically, 5 of the 1 1 nDoce-treated animals showed 100% tumor cell necrosis (complete regression) and 2 of the remaining 6 animals showed >90% tumor cell regression. By contrast, none of the 5 non-nDoce-treated animals showed >90% tumor cell necrosis. [QQ319] The macrophage infiltrate density in surrounding non-neoplastic tissue based on assessment of H&E and immimohistochemical staining with CD68, graded semi quantitatively is shown in ^"fable 30.

Table 30: Macrophage infiltrate density per treatment group

[QQ320] The intensity of the macrophage infiltrate in the surrounding non-neoplastic tissue in all animals was not striking; however, when the non-nDoce-treated group was compared to the nDoce-treated group, it was noted that the latter contained cases with a moderate degree of macrophage infiltrate while this was not seen in the non-nDoce-treated group. * One case in the IT nDoce-treated 1 cycle group did not contain surrounding non- neoplastic tissue for assessment.

[00321] The number of cases in each group that contained at least one TLS is shown in Table 31

Table 31: Number of cases with TLSs in each group

[00322] None of the 5 cases in the non-nDoce-treated group contained TLSs However, 8 of the 11 animals in the nDoce-treated group contained a TLS and in all but one of these 8 cases, the TLS measured at least 1 mm in maximum dimension. Of particular importance, the presence or absence of a TLS was closely linked with the presence or absence of residual carcinoma. Specifically, all cases that had either no diagnostic residual carcinoma (5 cases) or residual carcinoma that measured 2.5 mm or less (3 cases) also contained a TLS and these were the only cases that contained a TLS. By contrast, none of the remaining cases, ail of which had residual carcinoma measuring at least 8 mm on the slide, contained a TLS.

[00323] The comparison of necropsy volume to maximum tumor size as measured on the slide is shown in Table 32.

Table 32: Comparison of Necropsy volume to maximum tumor size as measured on the slide

[00324] When the tumor-site volume at necropsy was compared to the maximum carcinoma length on tire slide, the trend seen in the tumor length on the slide amongst the different treatment groups was also seen in the necropsy tumor volume, supporting that the tumor measurement on the slide was a representative assessment of the different responses to treatment in the different animals (see Table 32). In animals where a tiny volume of tumor site was recorded at necropsy and no carcinoma, or very minimal carcinoma, was seen on microscopic examination, the small volume noted at necropsy may have been predominantly or entirely due to necrotic or fibrotic tissue. Alternatively, a 1-2 mm TLS could also have been detected in the tumor site at the time of necropsy and its measurement may have contributed to some of the recorded tumor-site volumes.

Discussion:

[00325] The morphologic and immunohistochemica! features of a subset of 16 mice from the bladder carcinoma study aimed to assess the general safety and efficacy of intratumoral nDoce. The current subset of 16 animals included 1 non-treated control animal, 2 animals given intratumoral vehicle, 2 animals treated with intravenous docetaxel (3 cycles) and 11 animals treated with intratumoral nDoce. The nDoce group was separated into 3 groups based on the number of administered cycles: group 1 (1 cycle. 3 animals): group 2 (2 cycles. 5 animals); and group 3 (3 cycles. 3 animals).

[00326] The two main features that differed amongst the various groups were the presence and degree of tumor regression and the presence of tertiary lymphoid aggregates. In particular, there was prominent tumor regression in the ma_jority of the animals the intratumoral nDoce groups while there was no overt tumor regression in any of the animals in the other groups. Mirroring this finding, all the animals in the nDoce group with significant regression contained a TLS, whereas none of animals that had persistent tumors without overt regression contained a TLS.

[00327] In this microscopic review, the residual viable carcinoma maximum dimension on the slide was used to compare the degree of response in the different groups. The corresponding maximum tumor length at necropsy was not available for comparison; however, the tumor volume at necropsy was available. When the tumor volume at necropsy was compared to the tumor length on the slide, the trend seen in the tumor length on the slide amongst the different treatment groups was also seen in the necropsy tumor volume, supporting that the tumor measurement on the slide was a representative metric to use in order to compare the different responses to treatment in the different animals (Table 32). In the non-nDoce group, all five animals contained extensive residual viable carcinoma that measured at least 11 mm in maximum dimension on the slide (range: 11 mm - 24 mm). By contrast, just under half (5/11) of the animals treated with IT nDoce had no diagnostic residual viable carcinoma on the slide to measure (complete regression). In two of the remaining 5 animals in the IT nDoce group that had residual viable carcinoma, this consisted of rare tiny tumor cell collections where tumor measured up to 0.1 mm in maximum dimension . The significance of the tiny amount of tumor in both of these cases was not certain as the detached localization and small size also raised the possibility of sectioning artifact resulting in a false positive finding in these cases. In a third case the residual tumor measured 2.5 mm and in the remaining three cases the tumors measured 8 nun, 16 mm and 19 mm in maximum dimension on the slide (Tables 26 and 27).

[00328] Ail 16 animals in this study contained areas of geographic tumor cell necrosis that represented at least 5% of the tumor area. However, when all cases were taken together in both groups, the extent of tumor cell necrosis was significantly greater in the nDoce group than in the non-nDoce group. Specifically, 5 of the 11 nDoce animals showed 100% tumor cell necrosis (complete regression) and 2 of the remaining 6 animals in this group showed >90% tumor cell regression. By contrast, none of the 5 non-nDoce animals showed >90% tumor cell necrosis. Specifically, in non-nDoce group, 3 of the 5 cases had less than 50% necrosis while 2 of the 5 cases in the non-nDoce cases showed 50-90% tumor necrosis (Table 29)

[00329] When the three nDoce groups ( 1 cycle, 2 cycles, 3 cycles) were compared together, it was noted that a progressi ve increase in the number of cycles of IT nDoce from 1 cycle to 3 cycles, was associated with an increase in the percentage of cases that had no residual carcinoma. Specifically, the IT nDoce 1 cycle group had 0% (0/3) of cases with compete regression, although one of these cases the residual viable carcinoma nodule measured only 2.5 mm on the slide, while the other two cases had residual viable carcinoma that measured 16 and 19 mm on the slide. By contrast, the group given 2 cycles had complete regression in 2 of 5 cases (40%). In addition, in two of the remaining three cases in this group that had residual viable carcinoma, the size of the residual carcinoma was extremely minimal, with clusters measuring up to 0.1 mm in maximum dimension. Given the peripheral and detached localization of the tiny clusters in these two animals, these could possibly have represented an artifact of sectioning resulting in a false positive in these two animals, in which case the actual complete regression rate would have been 4/5 (80%) in the group given 2 cycles. The last animal in the 2 cycle group had residual carcinoma measuring 8 mm. Finally, the group given 3 cycles of nDoce had complete regression in 100% (3/3) of the animals, with no residual viable carcinoma available to measure in tire any of the three cases in the IT nDoce 3 cycle group (Table 28).

[QQ330] Another striking finding in this study was the presence of tertiar ' lymphoid structures (TLSs) in all of die nDoce animals that demonstrated a significant response to treatment. Specifically, a TLS was found in 8 animals and all of these were in the nDoce group. These 8 animals that contained a TLS included the 5 animals with no residual viable carcinoma; the two animals with rare detached clusters of carcinoma measuring up to 0.1 mm; and the animal with a residual carcinoma focus measuring 2.5 mm. None of the remaining animals, all of which had residual carcinoma nodules measuring at least 8 mm, had any TLSs. This finding demonstrated a very strong correlation between the presence of a TLS and a significant tumor response to therapy. In addition, a TLS was only seen in animals that received IT nDoce and within that group, a TLS was present in 8 of the 11 animals, including all three animals given 3 cycles of nDoce.

[00331] The TLSs in this study ranged in size from 0.1 up to 2 mm; however, 7 of the 8 TLSs were at least 1 mm in maximum dimension and two measured up to 2 ram. Given these sizes, the TLSs in most of these animals were easily appreciated by naked eye examination of the stained slides as a discrete nodule and in turn these may have been palpable in the in vivo state. All of the TLSs were well circumscribed, and they lacked a well -formed capsule. They showed varying stages of maturation with the most mature TLSs having well -formed peripheral lymphoid follicles composed of mature B-cells that labeled strongly with CD45R and intervening interfoliicular“T-celi areas” as well as medullary areas with sinuses. Some of the TLSs showed evidence of activation with lymphoid follicles containing germinal centers.

[00332] Finally, there was an associated macrophage infiltrate in the non-neoplastic tissue that generally correlated with the degree of tumor response to therapy. In particular, all of the animals in the non-nDoce group had a mild macrophage infiltrate vdnle the nDoce group included cases with a mild and a moderate immune cell infiltrate. All four cases with a moderate immune cell infiltrate had complete tumor regression and this included all three animals in the group given 3 cycles of IT nDoce.

Conculsions:

[00333] In conclusion, this study performed on a subset of 16 mice from the bladder carcinoma cohort clearly showed a strong association between IT nDoce therapy and tumor regression with 5 of 11 animals treated with IT nDoce showing complete tumor regression while a further 3 animals in this group had minimal residual tumor that measured 0.1 mm, 0.1 mm and 2.5 mm in maximum extent. Moreover, increasing cycles of IT nDoce (moving from 1 cycle to 3 cycles) resulted in a greater degree of tumor regression with all three animals the 3-cycle group showing complete tumor regression. Furthermore, a tertiary lymphoid structure (TLS) was seen in all 8 animals that demonstrated a significant tumor response w'hile a TLS was not seen in tiny of the animals drat did not show a significant tumor response. These findings suggest that in animals given IT nDoce there is significant interplay between the local drug effect on the tumor and the host animal’s immune system that results in formation of a robust local TLS adjacent to the tumor that in turn sets up a rapid feedback loop of adaptive and humoral immunity which further contributes to the significant tumor regression.

Claims

1. A method of producing tertiary lymphoid structures a subject with a malignant solid tumor, the method comprising locally_' administering a composition comprising antineoplastic particles to the tumor of the subject, wherein the antineoplastic particles reside at the tumor site after administration of the composition exposing the tumor to the antineoplastic particles for a sustained amount of time sufficient to stimulate the endogenous immune system of the subject resulting in the production of tertiary lymphoid structures, and infiltration of the tertiary lymphoid structures in and/or around the tumor site.

2. The method of claim 1, wherein the sustained amount of time is at least 4 weeks.

3. The method of any one of claims 1 or 2, wherein the administering comprises two or more separate administrations.

4. The method of any one of claims 1-3, wherein the administering comprises two or more separate administrations once a week for at least two weeks.

5. The method of any one of claims 1-3, wherein the administering comprises two or more separate administrations twice a week for at least one week, wherein the two or more separate administrations are separated by at least one day.

6. The method of any one of claims 1-5, wherein the administering is by pulmonary administration, by intratumoral injection administration, by intraperitoneaJ injection administration, by topical administration, by intravesical instillation, or by direct in_jection into tissue surrounding the tumor.

7. The method of any one of claims 1-6, wherein the antineoplastic particles have a mean particle size (number) of from 0.1 microns to 5 microns, or from 0.1 microns to 1.5 microns, or from 0.4 microns to 1.2 microns.

8. The method of any one of claims 1-7, wherein the antineoplastic particles are taxane particles.

9. The method of claim 8, wherein the taxane particles comprise paclitaxel particles, docetaxel particles, cabazitaxel particles, or combinations thereof.

I 0. The method of any one of claims 8 or 9, wherein the taxane particles comprise at least

95% of the taxane.

I I . The method of any one of claims 8-10, w'herein the taxane particles are paclitaxel particles.

12 The method of claim 1 1, wherein the paclitaxel particles have a specific surface area (SSA) of at least 18 m²/g, 20 m²/g, 25 m²/g, 30 m²/g, 32 m²/g, 34 m²/g, or 35 m²/g; or from about 18 m²/g to about 50 m²/g.

13 The method of any one of claims 11 or 12, wherein the paclitaxel particles have a bulk density (not-tapped) of 0.05 g/cm^J to 0.15 g/cm^J.

14. The method of any one of claims 8-10, wherein the taxane particles are docetaxel particles.

15. The method of claim 14, wherein the docetaxel particles have a specific surface area (SSA) of at least 18 m²/g, 20 m

from about 18 m²/g and about 6

16 The method of any one of claims 14 or 13, wherein the docetaxel particles have a bulk density (not-tapped) of 0.05 g/cm³ to 0.15 g/cm³.

17. The method of any one of claims 8-16, wherein, the taxane particles are not bound to, encapsulated in, or coated with one or more of a monomer, a polymer (or biocompatible polymer), a protein, a surfactant, or albumin.

18. The method of any one of claims 8-17, wherein the taxane particles are in crystalline form.

19 The method of any one of claims 1-18, wherein the stimulation of the endogenous immune system produces a cellular immune response.

20 The method of any one of claims 1-18, wherein the stimulation of the endogenous immune system produces a humoral immune response.

21 The method of any one of claims 6-20, wherein the administering is by pulmonary administration.

7? The method of claim 21, wherein the pulmonary administration comprises nebuhzation, wherein the composition further comprises a liquid carrier, wherein the antineoplastic particles are dispersed in the carrier, and wherein the nebulizing results in pulmonary delivery of aerosol droplets of the composition.

23. The method of claim 22, wherein the aerosol droplets have a mass median aerodynamic diameter (MMAD) of between about 0.5 pm to about 6 pm diameter, or between about 1 pm to about 3 pm diameter, or about 2 pm to about 3 pm diameter.

24. The method of any one of claims 21-23, wherein the tumor is a lung tumor.

25 The method of claim 24, wherein the lung tumor is non-small-cell lung carcinoma (NSCLC).

26 The method of claim 24, wherein the lung tumor is small-cell lung carcinoma.

27. The method of any one of claims 6-20, wherein the administering is by intratumoral injection.

28 The method of claim 27, wherein the tumor is a bladder tumor.