WO2018160794A1

WO2018160794A1 - Epha2-targeted docetaxel -generating liposomes in combination with an agent that impedes regulatory t cell activity for treating cancer

Info

Publication number: WO2018160794A1
Application number: PCT/US2018/020381
Authority: WO
Inventors: Daryl C. Drummond; Walid KAMOUN; Andrew J. SAWYER
Original assignee: Merrimack Pharmaceuticals, Inc.
Priority date: 2017-03-01
Filing date: 2018-03-01
Publication date: 2018-09-07

Abstract

Methods for treating a patient with cancer comprising administering to the patient an effective amount of a first agent that is an antibody targeted liposome-encapsulated taxane and a second agent that impedes regulatory T cell activity.

Description

EPHA2-TARGETED DOCETAXEL -GENERATING LIPOSOMES IN COMBINATION WITH AN AGENT THAT IMPEDES REGULATORY T CELL ACTIVITY FOR

TREATING CANCER

PRIORITY CLAIM

[0001] This application claims priority to United States Application Serial No. 62/465,750, filed

March 1, 2017,and United States Application Serial No. 62/479,571, filed March 31, 2017. The entire contents of the aforementioned applications are incorporated herein by reference.

SEQUENCE LISTING

[0002] This application incorporates by reference in its entirety the sequence listing entitled

"1143WO01_ST25.txt" created on February 26, 2018, at 2:47 pm, that is 38 KB, and filed electronically herewith.

FIELD

[0003] This disclosure relates to nanoliposomes that deliver docetaxel, useful in the treatment of cancer in combination with immunotherapies.

BACKGROUND

[0004] Ephrin receptors are cell to cell adhesion molecules that mediate signaling and are implicated in neuronal repulsion, cell migration and angiogenesis. EphA2 is part of the Ephrin family of cell-cell junction proteins highly overexpressed in several solid tumors. Ephrin receptor A2 (EphA2) is overexpressed in several solid tumors including prostate, pancreatic, ovarian, gastric and lung cancer, and is associated with poor prognosis in certain cancer conditions. The Eph receptors are comprised of a large family of tyrosine kinase receptors divided into two groups (A and B) based upon homology of the N-terminal ligand binding domain. The Eph receptors are involved several key signaling pathways that control cell growth, migration and differentiation. These receptors are unique in that their ligands bind to the surface of neighboring cells. The Eph receptors and their ligands display specific patterns of expression during development. For example the EphA2 receptor is expressed in the nervous system during embryonic development and also on the surface of proliferating epithelial cells in adults. EphA2 also plays an important role in angiogenesis and tumor vascularization, mediated through the ligand ephrin Al . In addition, EphA2 is overexpressed in a variety of human epithelial tumors including breast, colon, ovarian, prostate and pancreatic carcinomas. Expression of EphA2 can also be detected in tumor blood vessels stromal cells as well.

[0005] Immunotherapeutics have rapidly become an important option for patients and oncologists in the treatment of malignant diseases. Immunotherapy, at its most basic level, uses the immune system to fight cancer and disease. When the immune system is working properly, it is able to identify foreign and harmful components within the body and eliminate them. For example, when patients are infected with the flu virus or a bacterial infection, the immune system will recruit white blood cells and other immune cells to the site of the infection. Once there, the different components of the immune system work together to target the foreign viruses or bacteria and remove them from the body.

[0006] Cancer cells use a variety of mechanisms to remain invisible to the immune system. In some cases, the cancer cells down-regulate cell surface markers or proteins that would normally be used by the immune system to recognize the cells as being foreign and therefore targeted for destruction. Cancer cells and other cells within its microenvironment can also secrete soluble proteins, called cytokines or chemokines, which limit the function of immune cells. Furthermore, cancer cells can recruit other cells to make the immune system think they are normal or "host" cells that should not be destroyed.

[0007] One of the mechanisms that the body uses to prevent the immune system from attacking normal tissues and/or leading to autoimmune disease is to modulate the number of regulatory T cells ("Tregs") in any location. Genetic mutations leading to severe decreases in either the number or function of Tregs have been reported to lead to autoimmune disease.

However, cancer cells have modified this paradigm by recruiting Tregs to its microenvironment in order to make them invisible to the immune system. Many studies have noted that elevated levels of Tregs within the tumor microenvironment are associated with poor responses to therapy and poor overall survival. Immunotherapeutics targeting Tregs have led to improvements in clinical response rates, thus suggesting the importance of this pathway in a subset of cancer types.

[0008] Cancer immunotherapy, irrespective of treatment modality (i.e., adoptive cellular therapy, vaccines, monoclonal antibodies, and checkpoint inhibitors) and target (i.e., CD19, GD2, PD1, PD-L1, and Tregs), has shown promise within a subset of patients. The goal is to expand the clinical utility of these agents, either alone or in combination, in order to provide all patients with the optimal chance of being cured of their cancer. Thus, there is an unmet need to discover not only therapeutics that target cancer cells, but also those that target cells within the microenvironment that allow cancer cells to evade detection by the immune system, e.g., Tregs. The disclosure below addresses this need.

SUMMARY

[0009] Applicants have discovered novel EphA2 targeted nanoliposomes for delivering docetaxel to tumors, capable of leveraging organ specificity through enhanced permeability and retention, as well as leveraging cellular specificity through an EphA2 targeting moiety covalently bound to the nanoliposome membrane. With the goal of addressing the pharmacokinetic limitations of free docetaxel and the lack of cellular specificity, we developed novel docetaxel- based nanoliposomes, targeted against Ephrin receptor A2 (EphA2) which is overexpressed in a wide range of tumors. These EphA2 -targeted docetaxel-generating liposomes provide sustained release of docetaxel following accumulation in solid tumors. Preclinical models have

demonstrated that EphA2 -targeted docetaxel-generating liposomes leverage tumor-specific accumulation through the enhanced permeability and retention effect, and cellular specificity through active targeting of EphA2 with specific scFv antibody fragments conjugated to the surface of the liposomes.

[0010] Applicants have further discovered that the administration of the EphA2 targeted docetaxel-generating nanoliposomes and second agent that impedes regulatory T cell activity wherein the cancer is treated.

[0011] In one embodiment, the invention is a method of treating cancer in a patient, comprising administering to the patient an effective amount of a first agent that is a liposome- encapsulated taxane or taxane prodrug and a second agent that impedes regulatory T cell activity.

[0012] In some embodiments, the liposome is targeted to a tumor antigen, e.g., EphA2. In some embodiments, the liposome is an EphA2-targeted docetaxel-generating liposome.

[0013] In some embodiments, the EphA2-targeted docetaxel-generating liposome comprises a docetaxel prodrug. In some embodiments, the EphA2-targeted docetaxel-generating liposome comprising a docetaxel prodrug is encapsulated within a lipid vesicle comprising one or more lipids, a PEG lipid derivative and an EphA2 binding moiety on the outside of the lipid vesicle. In some embodiments, the docetaxel prodrug is a compound of Formula (I)

where Rl and R2 are each independently H or lower alkyl, and n is an integer 2-3. In some embodiments, Rl and R2 are C1-C3 alkyl, and n is 3.

[0014] In some embodiments, the EphA2 binding moiety is a scFv moiety comprising the CDRs of SEQ ID NO:40. In some embodiments, the EphA2 binding moiety is a scFv moiety comprising the sequence of SEQ ID NO:41 or SEQ ID NO: 46. [0015] In some embodiments, the lipid vesicle comprises sphingomyelin, cholesterol and PEG-DSG. In some embodiments, the lipid vesicle comprises sphingomyelin, cholesterol and PEG-DSG in a weight ratio of about 4.4: 1.6: 1.

[0016] In some embodiments, the EphA2 -targeted docetaxel-generating liposome is 46scFv- ILs-DTXp3 or 46scFv-ILs-DTXp6.

[0017] In some embodiments, the second agent is anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody. In some embodiments, the second agent is atezolizumab, durvalumab, or avelumab. In some embodiments, the second agent is selected from the group consisting of nivolumab, pembrolizumab, spartalizumab, camrelizuab, tiselizumab, cemiplimab and pidilizumab. In some embodiments, the second agent is ipilimumab, or tremelimumab, or lirilumab.

[0018] In some embodiments, the cancer is treated. In some embodiments, wherein the cancer is breast, esophageal, prostate, ovarian, bladder, gastric, pancreatic, or lung cancer. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a sarcoma.

[0019] In one embodiment, the invention is a method of treating cancer in a patient, comprising administering to the patient an effective amount of a first agent that is a taxane or taxane prodrug generating composition and a second agent that impedes regulatory T cell activity.

[0020] In some embodiments, the taxane or taxane prodrug generating composition has a taxane or taxane prodrug release half-life in mouse of 10 hours or greater (e.g., 20 hours or greater, 30 hours or greater, 40 hours or greater, 50 hours or greater, 60 hours or greater, 70 hours or greater, or 80 hours or greater).

[0021] In some embodiments, the taxane or taxane prodrug generating composition is a liposome-encapsulated taxane or taxane prodrug.

[0022] In some embodiments, the liposome is targeted to a tumor antigen, e.g., EphA2. In some embodiments, the liposome is an EphA2-targeted docetaxel-generating liposome. In some embodiments, the EphA2-targeted docetaxel-generating liposome comprises a docetaxel prodrug. In some embodiments, the EphA2-targeted docetaxel-generating liposome comprising a docetaxel prodrug is encapsulated within a lipid vesicle comprising one or more lipids, a PEG lipid derivative and an EphA2 binding moiety on the outside of the lipid vesicle.

[0023] In some embodiments, the docetaxel prodrug is a compound of Formula (I)

[0024] In some embodiments, the EphA2 binding moiety is a scFv moiety comprising the CDRs of SEQ ID NO:40. In some embodiments, the EphA2 binding moiety is a scFv moiety comprising the sequence of SEQ ID NO:41 or SEQ ID NO: 46.

[0025] In some embodiments, the lipid vesicle comprises sphingomyelin, cholesterol and PEG-DSG. In some embodiments, the lipid vesicle comprises sphingomyelin, cholesterol and PEG-DSG in a weight ratio of about 4.4: 1.6: 1.

[0026] In some embodiments, the EphA2-targeted docetaxel-generating liposome is 46scFv- ILs-DTXp3 or 46scFv-ILs-DTXp6.

[0027] In some embodiments, the EphA2-targeted docetaxel-generating liposome comprises a binding moiety that competes for binding to EphA2 with an scFv consisting of SEQ ID NO:40.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 A is a schematic of a docetaxel-generating liposome comprising an EphA2 binding moiety (anti-EphA2 scFv PEG-DSPE).

[0029] FIG. IB is a schematic showing the processes of docetaxel prodrug loading into a liposome comprising sucrose octasulfate (SOS) as a trapping agent, and the process of docetaxel generation. The insolubility of the salt in the liposome interior when combined with a low pH environment can stabilize the prodrug to reduce or prevent premature conversion to the active docetaxel.

[0030] FIG. 2A is a chemical reaction scheme for the synthesis of certain docetaxel prodrugs.

[0031] FIG. 2B is a chart showing selected examples of docetaxel prodrugs.

[0032] FIG. 3 A shows various CDR sequences useful in EphA2 binding moieties that can be used to prepare an EphA2 targeted docetaxel-generating nanoliposome composition.

[0033] FIG. 3B is an amino acid sequence and corresponding encoding DNA sequence for the scFv that can be used to prepare an EphA2 targeted docetaxel-generating nanoliposome composition. The DNA sequence further encodes an N-terminal leader sequence that is cleaved off by mammalian (e.g., human or rodent) cells expressing the encoded scFv. [0034] FIG. 3C is an amino acid sequence and corresponding encoding DNA sequence for the scFv that can be used to prepare EphA2 -targeted docetaxel-generating liposomes. The DNA sequence further encodes an N-terminal leader sequence that is cleaved off by mammalian (e.g., human or rodent) cells expressing the encoded scFv.

[0035] FIG. 3D is an amino acid sequence and corresponding encoding DNA sequence for the scFv that can be used to prepare EphA2 -targeted docetaxel-generating liposomes. The DNA sequence further encodes an N-terminal leader sequence that is cleaved off by mammalian (e.g., human or rodent) cells expressing the encoded scFv.

[0036] FIG. 3E is an amino acid sequence and corresponding encoding DNA sequence for the scFv EphA2 binding moiety in an EphA2 targeted docetaxel-generating nanoliposome composition EphA2-ILs, used in Examples 2-9.

[0037] FIG. 3F is an amino acid sequence and corresponding encoding DNA sequence for the scFv that can be used to prepare EphA2 -targeted docetaxel-generating liposomes. The DNA sequence further encodes an N-terminal leader sequence that is cleaved off by mammalian (e.g., human or rodent) cells expressing the encoded scFv.

[0038] FIG. 3G is an amino acid sequence and corresponding encoding DNA sequence for the scFv that can be used to prepare EphA2 -targeted docetaxel-generating liposomes. The DNA sequence further encodes an N-terminal leader sequence that is cleaved off by mammalian (e.g., human or rodent) cells expressing the encoded scFv.

[0039] FIG. 3H is an amino acid sequence and corresponding encoding DNA sequence for the scFv that can be used to prepare EphA2 -targeted docetaxel-generating liposomes. The DNA sequence further encodes an N-terminal leader sequence that is cleaved off by mammalian (e.g., human or rodent) cells expressing the encoded scFv.

[0040] FIG. 31 is an amino acid sequence and corresponding encoding DNA sequence for the scFv that can be used to prepare EphA2 -targeted docetaxel-generating liposomes. The DNA sequence further encodes an N-terminal leader sequence that is cleaved off by mammalian (e.g., human or rodent) cells expressing the encoded scFv.

[0041] FIGS 4A and 4B are graphs showing single tumor growth curves from all the experimental groups in EMT-6 tumor model. FIG. 4 A shows graphs showing saline control and 46scFv-ILs-DTXp3 and FIG. 4B shows graphs of PD-1 monotherapy and the combination of 46scFv-ILs-DTXp3 with PD-1 and also a rechallenge saline control.

[0042] FIG. 4C is a Kaplan-Meier plot illustrating time to regrowth for all the experimental groups in EMT-6 tumor model comparing 46scFv-ILs-DTXp3, PD-1 monotherapy to

combination with PD-1 [0043] FIG. 5A is a graph showing single tumor growth curves from all the experimental groups in EMT-6 tumor model comparing 46scFv-ILs-DTXp3, PD-1, docetaxel monotherapy to combination with PD-1

[0044] FIG. 5B is a graph showing maximum tumor regression from all the experimental groups in EMT-6 tumor model comparing 46scFv-ILs-DTXp3, PD-1, docetaxel monotherapy to combination with PD-1

[0045] FIG. 6A is a chart showing the percentage of CD8+ T cells, out of all CD45+ cells, in EMT-6 tumors from mice previously treated with 46scFv-ILs-DTXp3, PD-1, docetaxel, PD- l/46scFv-ILs-DTXp3 or PD-1 /docetaxel.

[0046] FIG. 6B is a chart showing the percentage of CD8+ifny+ T cells, out of all CD45+ cells, in EMT-6 tumors from mice previously treated with 46scFv-ILs-DTXp3, PD-1, docetaxel, PD-l/46scFv-ILs-DTXp3 or PD-1 /docetaxel.

[0047] FIG. 6C is a chart showing the ratio of CD8+ T cells to Tregs in EMT-6 tumors from mice previously treated with 46scFv-ILs-DTXp3, PD-1, docetaxel, PD-l/46scFv-ILs-DTXp3 or PD-1 /docetaxel.

[0048] FIG. 7A is a chart showing the TGI of EMT-6 tumors from mice treated with 46scFv- ILs-DTXp3 or docetaxel given weekly (DTX-MTD) or three times a week (DTX-DD).

[0049] FIG. 7B is a chart showing the percentage of CD8+ T cells, out of all CD45+ cells, in EMT-6 tumors from mice previously treated with 46scFv-ILs-DTXp3 or docetaxel given weekly (DTX-MTD) or three times a week (DTX-DD). ** pO.01, *** pO.001

[0050] FIG. 7C is a chart showing the percentage of CD8+ifny+ T cells, out of all CD45+ cells, in EMT-6 tumors from mice previously treated with 46scFv-ILs-DTXp3 or docetaxel given weekly (DTX-MTD) or three times a week (DTX-DD).

[0051] FIG. 7D is a chart showing the ratio of CD8+ T cells to Tregs in EMT-6 tumors from mice previously treated with 46scFv-ILs-DTXp3 or docetaxel given weekly (DTX-MTD) or three times a week (DTX-DD).

[0052] FIG. 7E is a chart showing the percentage of CD4+ifny+ T cells, out of all CD45+ cells, in EMT-6 tumors from mice previously treated with 46scFv-ILs-DTXp3 or docetaxel given weekly (DTX-MTD) or three times a week (DTX-DD). * p<0.05, ** p<0.01

[0053] FIG. 8 is a chart showing the in vivo efficacy of treatment of A/J mice implanted with a SAIN murine cancer cell line with saline, 46scFv-ILs-DTXp3, PD-1, and 46scFv-ILs-DTXp3 in combination with PD-1.

[0054] FIG. 9A is a chart showing the in vivo efficacy of treatment of C57BL6/J mice implanted with MC38 murine cancer cells with saline, 46scFv-ILs-DTXp3 were treated with four tail vein injections (every 7 days), and mice receiving PD-1, PD-Ll, 46scFv-ILs-DTXp3 + PD-1 or 46scFv-ILs-DTXp3 + PD-Ll (dosed IP twice weekly).

[0055] FIG. 9B is a chart showing the in vivo efficacy of treatment of C57BL6/J mice implanted with MC38 murine cancer cells with saline, 46scFv-ILs-DTXp3, and DTX were treated with four tail vein injections (every 7 days), and mice receiving PD-1, DTX+ PD-1, 46scFv-ILs-DTXp3 + PD-1 or 46scFv-ILs-DTXp3 + PD-Ll (dosed IP twice weekly).

[0056] FIG. 10A is a chart showing the ratio of CD8+ T cells to Tregs in BALB/c mice implanted with murine breast cancer cell line-EMT-6 after treatment with saline, 46scFv-ILs DTXp3, NT-ILs-DTXp3, and 46scFv-ILs dosed to match the lipid content of the 46scFv-ILs- DTXp3 group.

[0057] FIG. 10B is a chart showing the percent of NK cells of CD45+ in BALB/c mice implanted with murine breast cancer cell line-EMT-6 after treatment with saline, 46scFv-ILs DTXp3, NT-ILs-DTXp3, and 46scFv-ILs dosed to match the lipid content of the 46scFv-ILs- DTXp3 group. *p<0.05.

DETAILED DESCRIPTION

[0058] Disclosed herein are methods for providing treatment for a cancer in a human patient. These methods comprise administration to the patient of an effective amount of an EphA2 - targeted docetaxel-generating liposome comprising a docetaxel prodrug encapsulated within a lipid vesicle comprising one or more lipids, a PEG lipid derivative and an EphA2 binding moiety on the outside of the lipid vesicle and second agent that impedes regulatory T cell activity wherein the cancer is treated.

[0059] The efficacy of the current class of PD-1/PD-L1 antagonists can be limited by the immunogenicity of the tumor microenvironment. Studies showed that some chemotherapeutic agents including taxanes and anthracyclines can increase immunogenicity, resulting in therapeutic synergy with immune checkpoint inhibitors. In particular, treatment with a taxane has been shown to increase the recruitment of CTLs and decrease immunosuppressive cells such as MDSCs and T-regs. Additionally, the immune-modulatory activity of paclitaxel has been shown to increase with prolonged exposure of the taxane at the tumor level, achieved through metronomic dosing.

[0060] MM-310 an Ephrin Receptor A2 (EphA2)-targeted antibody-directed nanotherapeutic (ADN) encapsulates a docetaxel prodrug. Preclinically, the EphA2 leads to prolonged exposure of docetaxel at the tumor level, while lowering systemic exposure to bioavailable docetaxel, and thus decreasing not only dose-limiting neutropenia but also the killing of circulating lymphocytes potentially critical to anti-PDl/PDLl activity relative to free docetaxel. Definitions

[0061] "Combination therapy," "co-administration," "co-administered" or "concurrent administration" (or minor variations of these terms) refers to simultaneous administration of at least two therapeutic agents to a patient or their sequential administration within a time period during which the first administered therapeutic agent is still present in the patient (e.g., in the patient's plasma or serum) when the second administered therapeutic agent is administered.

[0062] "Effective amount," "effective amounts," effective dosage," "effective dosages,"

"effective doses," or "effective dose" refers to an amount (administered in one or more doses) of an antibody, protein or additional therapeutic agent, which amount is sufficient to provide effective treatment to a subject in need thereof. "Effective treatment" refers to a decrease or cessation in the symptoms of a disorder in the subject. In the case of cancer, effective treatment may comprise, but is not limited to, one or more of the following results: reduction in size of one or more tumors, reduction in number of tumors in a subject, reduction in number of cancerous cells in a subject, inhibition of tumor growth in a subject, or prolongation of survival time for an animal with cancer. An effective amount of the compositions of the present disclosure, for the treatment of the described conditions vary depending upon many different factors, including (but not limited to) means of administration, target site, physiological state of the patient, whether the patient is human or an animal, and other medications administered. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[0063] "EphA2" refers to Ephrin type-A receptor 2, also referred to as "epithelial cell kinase (ECK)," a receptor tyrosine kinase that can bind and be activated by Ephrin-A ligands. The term "EphA2" can refer to any naturally occurring isoforms of EphA2. The amino acid sequence of human EphA2 is recorded as GenBank Accession No. NP_004422.2.

[0064] As used herein, the term "mpk" refers to mg per kg body weight in a dose

administered to an animal.

[0065] As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0066] "Lower alkyl" means a Ci-C₆ linear or branched alkyl chain. EphA2-Targeted Liposomes for Delivery of Taxanes

[0067] Information regarding various EphA2-Targeted Liposomes for Delivery of Docetaxel including methods for making the targeted liposomes can be found in United States Patent Application Numbers 2018/0021294, 62/322,971 (WO2017161069), and 62/464,571 each of which are hereby incorporated by reference. [0068] As used herein, non-targeted liposomes can be designated as "Ls" or "NT-Ls." Ls (or NT-Ls) can refer to non-targeted liposomes with or without a docetaxel prodrug. "Ls-DTX"' refers to liposomes containing any suitable docetaxel prodrug, including equivalent or alternative embodiments to those docetaxel prodrugs disclosed herein. "NT -Ls-DTX" refers to liposomes without a targeting moiety that encapsulate any suitable docetaxel prodrug, including equivalent or alternative embodiments to those docetaxel prodrugs disclosed herein. Examples of non- targeted liposomes including a particular docetaxel prodrug can be specified in the format "Ls- DTXp[y]" or "NT-DTXp[y]" where [y] refers to a particular compound number specified herein. For example, unless otherwise indicated, Ls-DTXpl is a liposome containing the docetaxel prodrug of compound 1 herein, without an antibody targeting moiety.

[0069] As used herein, targeted immunoliposomes can be designated as "ILs." Recitation of "ILs-DTXp" refers to any embodiments or variations of the targeted docetaxel-generating immunoliposomes comprising a targeting moiety, such as a scFv. The ILs disclosed herein refer to immunoliposomes comprising a moiety for binding a biological epitope, such as an epitope- binding scFv portion of the immunoliposome. Unless otherwise indicated, ILs recited herein refer to EphA2 binding immunoliposomes (alternatively referred to as "EphA2-ILs"). The term "EphA2-ILs" refers herein to immunoliposomes enabled by the present disclosure with a moiety targeted to bind to EphA2. ILs include EphA2-ILs having a moiety that binds to EphA2 (e.g., using any scFv sequences that bind EphA2). Preferred targeted docetaxel-generating

immunoliposomes include ILs-DTXp3, ILs-DTXp4, and ILs-DTXp6. Absent indication to the contrary, these include immunoliposomes with an EphA2 binding moiety and encapsulating docetaxel prodrugs of compound 3, compound 4 or compound 6, respectively (Figure 2B).

EphA2-ILs can refer to and include immunoliposomes with or without a docetaxel prodrug (e.g., immunoliposomes encapsulating a trapping agent such as sucrose octasulfate without a docetaxel prodrug).

[0070] The abbreviation format "[x]scFv-ILs-DTXp[y]" is used herein to describe examples of immune-liposomes ("ILs") that include a scFv "targeting" moiety having the amino acid sequence specified in a particular SEQ ID NO:[x], attached to a liposome encapsulating or otherwise containing a docetaxel prodrug ("DTXp") having a particular Compound number ([y]) specified herein. Unless otherwise indicated, the scFv sequences for targeted ILs can bind to the EphA2 target.

[0071] In some embodiments, the EphA2-targeted docetaxel-generating liposome is 46scFv- ILs-DTXp3 or 46scFv-ILs-DTXp6. In some embodiments, the EphA2 -targeted docetaxel- generating liposome is 46scFv-ILs-DTXp3 (also known as MM-310). [0072] Figure 1 A is a schematic showing the structure of a PEGylated EphA2 targeted, nano- sized liposome (nanoliposome) encapsulating a docetaxel prodrug (e.g., having a liposome size on the order of about 100 nm). The immunoliposome can include an Ephrin A2 (EphA2) targeted moiety, such as a scFv, bound to the liposome (e.g., through a covalently bound PEG- DSPE moiety). The PEGylated EphA2 targeted liposome encapsulating a docetaxel prodrug can be created by covalently conjugating single chain Fv (scFv) antibody fragments that recognize the EphA2 receptor to pegylated liposomes, containing docetaxel in the form of a prodrug described herein, resulting in an immunoliposomal drug product. In one particular example of a PEGylated EphA2 targeted liposome encapsulating a docetaxel prodrug (herein designated "EphA2-ILs-DTX"), the lipid membrane can be composed of egg sphingomyelin, cholesterol, and 1,2-distearoyl-sn-glyceryl methoxypolyethylene glycol ether (PEG-DSG). The

nanoliposomes can be dispersed in an aqueous buffered solution, such as a sterile pharmaceutical composition formulated for parenteral administration to a human.

[0073] The EphA2 targeted nanoliposome is preferably a unilamellar lipid bilayer vesicle, approximately 110 nm in diameter, which encapsulates an aqueous space which contains a compound of disclosed herein in a gelated or precipitated state, as sucrosofate (sucrose octasulfate) salt.

[0074] The docetaxel prodrug can be stabilized in the liposomal interior during storage and while the intact liposome is in the general circulation, but is hydrolyzed rapidly (e.g., t½ = -10 h) to the active docetaxel upon release from the liposome and entering the environment of the circulating blood. A docetaxel prodrug can be loaded at mildly acidic pH and entrapped in the acidic interior of liposomes, using an electrochemical gradient where it is stabilized in a non- soluble form. Upon release from the liposome, the docetaxel prodrug is subsequently converted to active docetaxel by simple base-mediated hydrolysis at neutral pH.

Compounds

[0075] The PEGylated EphA2 targeted liposome encapsulating a Taxane or Taxane prodrug (e.g., a docetaxel prodrug) can encapsulate one or more suitable Taxane or taxane prodrugs (e.g., a docetaxel prodrugs). Preferably, a docetaxel prodrug comprises a weak base such as tertiary amine introduced to the 2' or 7 position hydroxyl group of docetaxel through ester bond to form a docetaxel prodrug. Preferred 2'- docetaxel prodrugs suitable for loading into a liposome are characterized by comparatively high stability at acidic pH but convert to docetaxel at

physiological pH through enzyme-independent hydrolysis.

[0076] The chemical environment of the 2'-ester bond can be tuned systematically to obtain docetaxel prodrugs that are stable at relatively low pH but will release free docetaxel rapidly at physiologic pH through hydrolysis. Docetaxel prodrugs are loaded into liposome at relatively low pH by forming stable complexes with trapping agents such as polysulfated polyols, for example, sucrose octasulfate. The trapping agent sucrose octasulfate can be included in the liposome interior, as a solution of its amine salt, such as diethylamine salt (DEA-SOS), or triethylamine salt (TEA-SOS). The use of amine salts of the trapping agents helps to create a transmembrane ion gradient that aids the prodrug loading into the liposome and also to maintain the acidic intraliposomal environment favorable for keeping the prodrug from premature conversion to docetaxel before the prodrug-loaded liposome reaches its anatomical target.

Encapsulation of docetaxel prodrugs inside liposome in such a way allows the practical application of pH triggered release of docetaxel upon release from the liposome within the body of a patient. Thus, the liposome that encapsulates docetaxel-prodrug can be called docetaxel nanogenerator.

[0077] Preferably, the docetaxel prodrug is a compound of formula (I), including

pharmaceutically acceptable salts thereof, where Rl, R2 and n are selected to provide desired liposome loading and stability properties, as well as desired docetaxel generation (e.g., as measured by the hydrolysis profile at various pH values, as disclosed herein). The docetaxel prodrug (DTX') compounds can form a pharmaceutically acceptable salt within the liposome (e.g., a salt with a suitable trapping agent such as a sulfonated polyol). In some examples, the compounds of formula (I) where Rl and R2 are independently H or lower alkyl (preferably Cl- C4 linear or branched alkyl, most preferably C2 or C3), and n is an integer (preferably 1-4, most preferably 2-3). Examples of docetaxel prodrugs also include docetaxel analog compounds having a 2' substituents -0-(CO)-(CH2)nN(Rl)(R2) in formula (I) that are substituted in the manner disclosed in formula (III) of U.S. Patent 4,960,790 to Stella et al. (filed March 9, 1989), incorporated herein by reference in its entiret .

[0078] The docetaxel prodrugs, including compounds of Formula (I), can be prepared using the reaction Scheme in Figure 2A. Specific Examples of docetaxel prodrugs are shown in Figure 2B. Other examples of docetaxel prodrugs include 2'-(2-(N,N'-diethylamino)propionyl)- docetaxel or 7-(2-(N,N'-diethylamino)propionyl)-docetaxel. [0079] Preferred docetaxel prodrug compounds of formula (I) include compounds where (n) is 2 or 3, to provide a rapid hydrolysis rate at pH 7.5 and a sufficiently high relative hydrolysis rate for the compound at pH 7.5 compared to pH 2.5 (e.g., selecting docetaxel prodrugs with maximum hydrolysis rate of the docetaxel prodrug to docetaxel at pH 7.5 compared to the hydrolysis rate at pH 2.5).

[0080] Additional prodrug compositions suitable for encapsulation by liposomes can be found in US 8,790,691, the entire contents of which is incorporated herein by reference.

Targeting Moieties

[0081] The docetaxel-generating liposome can comprise a tumor antigen targeting moiety, e.g., an EphA2 targeting moiety. Other exemplary tumor antigens include alpha-actinin-4, A3, antigen specific for A33 antibody, ART-4, B7, Ba 733, BAGE, bcl-2, bcl-6, BCMA, BrE3- antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX (CAIX), CASP-8/m, CCL19, CCL21, CD1, CDla, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123, CD126, CD132, CD133, CD 138, CD 147, CD 154, CD171, CDC27, CDK-4/m, CDKN2A, CEA, CEACAM5, CEACAM6, complement factors (such as C3, C3a, C3b, C5a and C5), colon-specific antigen- p (CSAp), c-Met, CTLA-4, CXCR4, CXCR7, CXCL12, DAM, Dickkopf-related protein (DKK), ED-B fibronectin, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, EpCAM, EphA2, EphA3, fibroblast activation protein (FAP), fibroblast growth factor (FGF), Fit- 1 , Fit- 3, folate binding protein, folate receptor, G250 antigen, gangliosides (such as GC2, GD3 and GM2), GAGE, GD2, gplOO, GPC3, GRO-13, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2, HER3, HMGB-1, hypoxia inducible factor (HTF- 1), HIF-la, HSP70-2M, HST-2, la, IFN-gamma, IFN- alpha, IFN-beta, IFN-X, IL-4R, IL-6R, IL-13R, IL13Ralpha2, IL-15R, IL-17R, IL-18R, IL-2, IL- 6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, ILGF, ILGF-1R, insulin-like growth factor-1 (IGF-1), IGF-1R, integrin ανβ3, integrin α5β1, KC4-antigen, killer-cell immunoglobulin-like receptor (KIR), Kras, KS-1- antigen, KS1-4, LDR/FUT, Lei, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART -2, mCRP, MCP-1, melanoma glycoprotein, mesothelin, MIP-1 A, MIP-1B, MIF, mucins (such as MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM- 1/2 and MUM-3), NCA66, NCA95, NCA90, NY-ESO-1, PAM4 antigen, pancreatic cancer mucin, PD-1, PD-L1, PD-1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, RS5, RANTES, SAGE, 5100, survivin, survivin-2B, T101, TAC, TAG-72, tenascin, Thomson-Friedenreich antigens, Tn antigen, TNF-alpha, tumor endothelial marker 8 (TEM8), tumour necrosis antigens, TRAG-3, TRAIL receptors, VEGF-A, VEGFR and WT-1. In certain embodiments, the EphA2 targeting moiety is a binding agent that competes for binding with the scFv D2-1 A7 (SEQ ID NO:41). The targeting moiety can be a single chain Fv ("scFv"), a protein that can be covalently bound to a liposome to target the docetaxel-producing liposomes disclosed herein. The scFv can be comprised of a single polypeptide chain in which a VH and a VL are covalently linked to each other, typically via a linker peptide that allows the formation of a functional antigen binding site comprised of VH and VL CDRs. An Ig light or heavy chain variable region is composed of a plurality of "framework" regions (FR) alternating with three hypervariable regions, also called "complementarity determining regions" or "CDRs". The extent of the framework regions and CDRs can be defined based on homology to sequences found in public databases. See, for example, "Sequences of Proteins of Immunological Interest," E. Kabat et al., Sequences of proteins of immunological interest, 4th ed. U.S. Dept. Health and Human Services, Public Health Services, Bethesda, MD (1987). All scFv sequence numbering used herein is as defined by Kabat et al.

[0082] As used herein, unless otherwise indicated, the term "anti-EphA2 scFv" refers to an scFv that immunospecifically binds to EphA2, preferably the ECD of EphA2. An EphA2-specific scFv does not immunospecifically bind to antigens not present in EphA2 protein.

[0083] In certain embodiments, an scFv disclosed herein includes one or any combination of VH FR1, VH FR2, VH FR3, VL FR1, VL FR2, and VL FR3 set forth in Table A. In one embodiment, the scFv contains all of the frameworks of Table A below.

Table A: Exemplary Framework Sequences

VH FR1 (SEQ ID NO: 1) Q VQL VQ S GGGL VQP GGSLRL S C A AS GF TF S

VH FR2 (SEQ ID NO: 2) WVRQAPGKGLEWVT

VH FR3 (SEQ ID NO: 3) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR

VH FR4 (SEQ ID NO: 4) WGQGTLVTVSS

VL FR1 (SEQ ID NO: 5) SSELTQPPSVSVAPGQTVTITC

VL FR2 (SEQ ID NO: 6) WYQQKPGTAPKLLIY

VL FR3 (SEQ ID NO: Ό GVPDRF SGS S SGTS ASLTITGAQ AEDEAD YYC

VL FR4 (SEQ ID NO: 8) FGGGTKLTVLG [0084] In certain aspects, an scFv disclosed herein is thermostable, e.g., such that the scFv is well-suited for robust and scalable manufacturing. As used herein, a "thermostable" scFv is an scFv having a melting temperature (Tm) of at least about 70°C, e.g., as measured using differential scanning fluorimetry (DSF).

[0085] A preferred anti-EphA2 scFv binds to the extracellular domain of EphA2 polypeptide, i.e., the part of the EphA2 protein spanning at least amino acid residues 25 to 534 of the sequence set forth in GenBank Accession No. NP_004422.2 or UniProt Accession No. P29317.

[0086] In certain embodiments, an anti-EphA2 scFv disclosed herein includes a VH CDRl, VH CDR2, VH CDR3, VL CDRl, VL CDR2, and VL CDR3 each with a sequence as set forth in Table B. Note that the VH CDR2 sequence (also referred to as CDRH2) will be any one selected from the 18 different VH CDR2 sequences set forth in Table B.

Table B: Complementary Determining Regions (CDRs)

VH CDRl (SEQ ID NO: 10) SYAMH

VH CDR2 (SEQ ID NO: 11) VISPAGNNTYYADSVK

VH CDR2 (SEQ ID NO: 12) VISPAGRNKYYADSVK

VH CDR2 (SEQ ID NO: 13) VISPDGHNTYYADSVKG

VH CDR2 (SEQ ID NO: 14) VISPHGRNKYYADSVK

VH CDR2 (SEQ ID NO: 15) VISRRGDNK YYAD S VK

VH CDR2 (SEQ ID NO: 16) VISNNGHNK YYAD S VK

VH CDR2 (SEQ ID NO: 17) VISPAGPNTYYADSVK

VH CDR2 (SEQ ID NO: 18) VISPSGHNTYYADSVK

VH CDR2 (SEQ ID NO: 19) VISPNGHNT YYAD S VK

VH CDR2 (SEQ ID NO: 20) AISPPGHNTYYADSVK

VH CDR2 (SEQ ID NO: 21) VISPTGANT YYAD S VK

VH CDR2 (SEQ ID NO: 22) VISPHGSNKYYADSVK

VH CDR2 (SEQ ID NO: 23) VISNNGHNT YYAD S VK

VH CDR2 (SEQ ID NO: 24) VISP AGTNT YYAD S VK VH CDR2 (SEQ ID NO: 25) VISPPGHNTYYADSVK VH CDR2 (SEQ ID NO: 26) VISHDGTNTYYADSVK VH CDR2 (SEQ ID NO: 27) VISRHGNNK YYAD S VK VH CDR2 (SEQ ID NO: 28) VIS YDGSNK YYAD SVKG VH CDR3 (SEQ ID NO: 29) ASVGATGPFDI VL CDR1 (SEQ ID NO: 30) QGDSLRSYYAS VL CDR2 (SEQ ID NO: 31) GENNRPS VL CDR3 (SEQ ID NO: 39) NSRDSSGTHLTV

[0087] In certain embodiments, an scFv disclosed herein is an internalizing anti-EphA2 scFv. Binding of such an scFv to the ECD of and EphA2 molecule present on the surface of a living cell under appropriate conditions results in internalization of the scFv. Internalization results in the transport of an scFv contacted with the exterior of the cell membrane into the cell-membrane- bound interior of the cell. Internalizing scFvs find use, e.g., as vehicles for targeted delivery of drugs, toxins, enzymes, nanoparticles (e.g., liposomes), DNA, etc., e.g., for therapeutic applications.

[0088] Certain scFvs described herein are single chain Fv scFvs e.g., scFvs or (scFv')2s. In such scFvs, the VH and VL polypeptides are joined to each other in either of two orientations (i.e., the VH N-terminal to the VL, or the VL N-terminal to the VH) either directly or via an amino acid linker. Such a linker may be, e.g., from 1 to 50, 5 to 40, 10 to 30, or 15 to 25 amino acids in length. In certain embodiments, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 100% of the residues of the amino acid linker are serine (S) and/or glycine (G).

Suitable exemplary scFv linkers comprise or consist of the sequence:

AS TGGGGS GGGGS GGGGS GGGGS (SEQ ID NO 32),

GGGGS GGGGS GGGGS GGGGS (SEQ ID NO 33),

GGGGSGGGGSGGGGS (SEQ ID NO 34),

ASTGGGGAGGGGAGGGGAGGGGA (SEQ ID NO 35),

GGGGAGGGGAGGGGAGGGGA (SEQ ID NO 36),

TPSHNSHQVPSAGGPTANSGTSGS (SEQ ID NO: 37), and GGS SRS S S S GGGGS GGGG (SEQ ID NO: 38).

[0089] An exemplary internalizing anti-EphA2 scFv is scFv TS1 (SEQ ID NO:40 (Figure 3 A)). In scFv TS1, and in certain other scFvs disclosed herein, the VH of the scFv is at the amino terminus of the scFv and is linked to the VL by a linker indicated in italics. The CDRs of the scFvs are underlined and are presented in the following order: VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3.

[0090] The docetaxel-generating EphA2 -targeted liposomes can also include one or more

EphA2 targeted scFv sequences shown Figure 3B (SEQ ID NO:41, designated "D2-1 A7", encoded by the DNA sequence of SEQ ID NO:42 designated "D2-1 A7 DNA"), or Figure 3C (SEQ ID NO:40, designated "TS1", encoded by the DNA sequence of SEQ ID NO:43 designated "TS1 DNA"), or Figure 3D (SEQ ID NO:44, designated "scFv2", encoded by the DNA sequence of SEQ ID NO:45 designated "scFv2 DNA"), or Figure 3E (SEQ ID NO:46, designated "scFv3", encoded by the DNA sequence of SEQ ID NO:47 designated "scFv3 DNA"), or Figure 3F (SEQ ID NO:48, designated "scFv8", encoded by the DNA sequence of SEQ ID NO:49 designated "scFv8 DNA"), or Figure 3G (SEQ ID NO:50, designated "scFv9", encoded by the DNA sequence of SEQ ID NO:51 designated "scFv9 DNA") or Figure 3H (SEQ ID NO:52, designated "scFv10", encoded by the DNA sequence of SEQ ID NO:53 designated "scFv10 DNA") or Figure 31 (SEQ ID NO:54, designated "scFvl3", encoded by the DNA sequence of SEQ ID NO: 9 designated "scFvl3 DNA").

[0091] Also provided are variants of scFv TS1 in which VH CDR2 is selected from any of the 18 different CDRH2 sequences set forth above in Table B.

[0092] Using the information provided herein, the scFvs disclosed herein may be prepared using standard techniques. For example, the amino acid sequences provided herein can be used to determine appropriate nucleic acid sequences encoding the scFvs and the nucleic acids sequences then used to express one or more of the scFvs . The nucleic acid sequence(s) can be optimized to reflect particular codon "preferences" for various expression systems according to standard methods.

[0093] Using the sequence information provided herein, the nucleic acids may be synthesized according to a number of standard methods. Oligonucleotide synthesis, is conveniently carried out on commercially available solid phase oligonucleotide synthesis machines or manually synthesized using, for example, the solid phase phosphoramidite triester method. Once a nucleic acid encoding an scFv disclosed herein is synthesized, it can be amplified and/or cloned according to standard methods. [0094] Expression of natural or synthetic nucleic acids encoding the scFvs disclosed herein can be achieved by operably linking a nucleic acid encoding the scFv to a promoter (which may be constitutive or inducible), and incorporating the construct into an expression vector to generate a recombinant expression vector. The vectors can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical cloning vectors contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the scFv. The vectors optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

[0095] The EphA2 Targeted scFv Amino Acid Sequence can be attached to the liposome using an EphA2 (scFv) to maleimide-activated PEG-DSPE. For example, the scFv-PEG-DSPE drug substance can be a fully humanized single chain antibody fragment (scFv) conjugated to maleimide PEG-DSPE via the C-terminal cysteine residue of scFv. In some examples, the EphA2 targeted scFv is conjugated covalently through a stable thioether bond to a lipopolymer lipid, Mal-PEG-DSPE, which interacts to form a micellular structure. Preferably, the scFv is not glycosylated.

Agents that impede regulatory T cell activity

[0096] In some embodiments, agents that impede regulatory T cell activity (Tregs) include small molecule immunomodulatory agents, radiation, anticancer vaccines, and

immunomodulatory antibodies that do not bind to human IGF-1. Tregs, formerly known as suppressor T cells, are a subpopulation of T cells which modulate the immune system, suppress immune responses against other cells and maintain tolerance to self-antigens. Generally, these cells suppress or downregulate the induction and proliferation of effector T cells. Therefore, when regulatory T cell activity is impeded, the immune response suppressive activities of these cells will be lessened or removed.

[0097] Immunomodulatory antibodies include, but are not limited to, a human cytotoxic T- lymphocyte antigen 4 (CTLA-4)-blocking antibody such as ipilimumab, and a human

programmed death receptor- 1 (PD-1) antibody such as nivolumab. Other Immunomodulatory antibodies include but are not limited to, the group consisting of (a) an agonistic anti-receptor antibody that immunospecifically binds human OX40, CD40,GITR, CD27, ICOS, or 4- IBB; (b) an antagonistic anti-receptor antibody that immunospecifically binds human CTLA-4 (cytotoxic

T-lymphocyte-associated protein 4, also known as CD 152), PD-1 (programmed cell death protein 1, also known as CD279), PD-Ll (programmed death-ligand 1, also known as CD274 or B7-H1), TIM-3, BTLA, VISTA, LAG-3, KIR, CD47,CD25, B7-H3, or B7-H4; and (c) an anti-ligand antibody that blocks the function of IL-6, IL-10, TGFP, angiopoetin-2, VEGF, IL-17, IL-23, or TNF alpha, and (d) atezolizumab, avelimumab, , pembrolizumab, tremelimumab and/or durvalumab. In one embodiment, the anti-CTLA-4 antibody is ipilimumab or tremelimumab. In another embodiment, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, spartalizumab, camrelizuab, tiselizumab, cemiplimab, and pidilizumab. In another embodiment, the anti-PD-Ll antibody is selected from the group consisting of atezolizumab, durvalumab, and avelumab. In one embodiment, the anti-KIR antibody is lirilumab.

[0098] The immunotherapy can include molecules that bind to CTLA4, PDL1, PD1, 41BB and/or OX40 including the publicly available compounds in the table below or other compounds that bind to the same epitope or have the same or similar biological functions.

[0099] In some embodiments, the agents that impede regulatory T cell activity are anticancer vaccines selected from, but is not limited to, the group consisting of OncoVex, MAGE- A3, PROSTVAC, GVAX, CDX110, CDX1307, CDX1401, CV9104, BIOVAXID, IMA 901, and ADXS 11-001.

Combination therapy

[00100] The use of a combination of an EphA2 -targeted docetaxel-generating liposome and an immunotherapy can be used for the treatment of cancer in a host in need thereof, in an amount and in a schedule of administration that is therapeutically synergistic in the treatment of the cancer. In some embodiments, the cancer is breast, esophageal, prostate, ovarian, bladder, gastric, pancreatic or lung cancer. In one embodiment the cancer is triple negative breast cancer (TNBC). In another embodiment the cancer is a sarcoma. In one embodiment the cancer is a solid tumor. In another embodiment the cancer is gastric cancer, gastroesophageal junction cancer, or esophageal carcinoma. In another embodiment the cancer is esophageal cancer. In yet another embodiment the cancer is non-small cell lung cancer, or small cell lung cancer. In another embodiment the cancer is ovarian cancer. In another embodiment the cancer is prostate cancer. In another embodiment, the cancer is ovarian cancer, endometrial carcinoma, or urothelial carcinoma. In another embodiment, the cancer is prostate adenocarcinoma. In another embodiment, the cancer is soft tissue carcinoma or squamous cell carcinoma of the head and neck (SCCHN). In another embodiment, the cancer is pancreatic ductal adenocarcinoma

(PDAC).

EXAMPLES

Example 1 Synergy of EphA2-targeted Docetaxel-Generating Liposomes with an anti-PD-1 antibody

[00101] To investigate the effect of PD-1 therapy in combination with 46scFv-ILs-DTXp3 we evaluated the potential combination of 46scFv-ILs-DTXp3 and a murine anti-PD-1 antibody in the treatment of several syngeneic mouse tumor models. The anti-PDl antibody, J43, is a monoclonal mouse anti-mouse PD1. {see International Immunology, Vol. 8, no. 5, pp. 765-772) The tumor lines EMT-6, CT-26, and LLC were selected to provide a range of sensitivity to both docetaxel and anti-PD-1. EMT-6 (ATCC CRL-2755) is a mouse mammary carcinoma cell line, CT-26 (ATCC CRL-2638) is a mouse colon carcinoma cell line and LLC (ATCC CRL-1642) is a lung carcinoma cell line. In vivo activity studies and immune-phenotype studies were performed comparing 46scFv-ILs-DTXp3 + anti-PD-1 combination to the monotherapies.

[00102] 46scFv-ILs-DTXp3 administration was initiated two days prior to anti-PD-1 therapy and consisted of four weekly doses, while anti-PD-1 was dosed twice weekly for four weeks. 46scFv-ILs-DTXp3 was administered intravenously at 50 mg/kg (docetaxel equivalent) Q7D x 4. The anti-PDl antibody was administered at 10 mg/kg twice weekly for four weeks. The response to 46scFv-ILs-DTXp3 or anti-PD-1 as monotherapies varied between the models. LLC was unresponsive to anti-PD-1 and poorly responsive to 46scFv-ILs-DTXp3, CT-26 was poorly responsive to both anti-PD-1 and 46scFv-ILs-DTXp3, while EMT-6 responded moderately to anti-PD-1 with tumor stasis and well to 46scFv-ILs-DTXp3, achieving tumor regression. In all models, however, 46scFv-ILs-DTXp3 given in combination with anti-PD-1 outperformed controls and both monotherapy arms in terms of growth inhibition and tumor regression rate. In the EMT-6 model, combination treatment resulted in durable complete regressions in 6/10 mice when compared to 2/10 and 0/10 for 46scFv-ILs-DTXp3 and anti-PD-1 monotherapies respectively. In 46scFv-ILs-DTXp3 and 46scFv-ILs-DTXp3 + anti-PD-1 groups, re-challenge of mice with the same tumor cells, at 10 weeks post treatment interruption, did not lead to tumor growth, suggesting that treatment with anti-PDl + 46scFv-ILs-DTXp3 promotes the development of a memory response against the tumor antigen. These data show that the combination of an Ephrin Receptor A2 (EphA2) targeted liposomal form of a docetaxel pro-drug with an anti-PD-1 antibody, is highly active in syngeneic tumor models, and represents a promising strategy for the treatment of cancer.

Example 2 In vivo antitumor efficacy of 46scFv-ILs-DTXp3 in combination with a murine PD-1 antibody in EMT-6 murine breast cancer tumor model in mice

[00103] Immunocompetent mice of strain BALB/c were implanted with murine breast cancer cell line EMT-6 (3.6x10^5 cells/mouse). Animals were randomized into the following experimental groups: Saline, anti-PD-1 murine antibody at 10 mg/kg twice a week, 46scFv-ILs- DTXp3 at 50 mg/kg, and PD-l/46scFv-ILs-DTXp3. For the combination group doses used for the monotherapy arms were combined and PD-1 dosing was initiated 2 days following 46scFv- ILs-DTXp3.

[00104] Animals receiving saline or 46scFv-ILs-DTXp3 received four tail vein injections, at intervals of 7 days. Mice receiving PD-1 were dosed IP twice a week. Tumor size was monitored once to twice weekly. The tumor progression was monitored by palpation and caliper measurements of the tumors along the largest (length) and smallest (width) axis twice a week. The tumor sizes were determined twice weekly from the caliper measurements using the formula (Geran, R.I., et al., 1972 Cancer Chemother. Rep. 3 : 1-88):

Tumor volume (TV)= [(length) x (width)2] / 2

[00105] Maximum response was calculated using the following formula where TV is tumor volume:

Max tumor regression = [(TVmin - TVdayO) / TVdayO] x 100

[00106] Maximum tumor regression was classified as complete tumor regression (100% regression with no palpable tumor), partial tumor regression (max tumor regression more than 30%) or no tumor regression.

[00107] Animals were monitored until tumor regrowth or end of monitoring period (>120 days). Time to regrowth was defined as time for tumor to double its volume. Animals sacrificed prior to tumor volume doubling are censored.

[00108] The growth curves shown below illustrate the treatment effect when 46scFv-ILs- DTXp3 is combined with PD-1 (Figure 4A). Time to regrowth for all groups is illustrated in Figure 4B.

[00109] PD-1 showed minimal growth arrest and no tumor regression when given as monotherapies. 46scFv-ILs-DTXp3 did induce partial regression in most animals with complete regression in 20% (2/10) animals. However, a combination of 46scFv-ILs-DTXp3 and PD-1 significantly increased the tumor doubling time, in addition to inducing complete regression in 60% (6/10) of treated animals. Time to regrowth shows that the complete regression seen in the animals treated with 46scFv-ILs-DTXp3 monotherapy or 46scFv-ILs-DTXp3 combined with PD-1 was durable with no regrowth up to day 98.

[00110] After a 60 day observation period, mice that had completely regressed tumors were rechallenged with a inoculation of EMT-6 cells (3.6X10^5 cells), identical to the initial induction dose. Additionally, a cohort of naive littermate mice were also given an injection of EMT-6 cells. Mice from the littermate control group rapidly grew tumors. However, both mice from the 46scFv-ILs-DTXp3 monotherapy group and 5 of the 6 mice in the PD-l/46scFv-ILs-DTXp3 group were resistant to rechallenge. The resistance to rechallenge indicates immune involvement and potential immunity.

Example 3 In vivo antitumor efficacy of 46scFv-ILs-DTXp3 when compared to free docetaxel monotherapy and in combination with a murine PD-1 antibody in EMT-6 murine breast cancer tumor model in mice

[00111] Immunocompetent mice of strain BALB/c were implanted with murine breast cancer cell line EMT-6 (3.5x10^5). Animals were randomized into the following experimental groups: Saline, anti-PD-1 murine antibody at 10 mg/kg twice weekly, free docetaxel at 10 mg/kg qlw, 46scFv-ILs-DTXp3 at 50 mg/kg qlw, PD-l/docetaxel, and PD-l/46scFv-ILs-DTXp3. For the combination group doses used for the monotherapy arms were combined and PD-1 dosing was initiated 2 days following docetaxel or 46scFv-ILs-DTXp3.

[00112] Animals receiving saline, docetaxel or 46scFv-ILs-DTXp3 received four tail vein injections, at intervals of 7 days. Mice receiving PD-1 were dosed IP twice a week. Tumor size was monitored once to twice weekly. The tumor progression was monitored by palpation and caliper measurements of the tumors along the largest (length) and smallest (width) axis twice a week. The tumor sizes were determined twice weekly from the caliper measurements using the formula (Geran, R.I., et al., 1972 Cancer Chemother. Rep. 3 : 1-88):

Tumor volume (TV)= [(length) x (width)2] / 2

[00113] Maximum response was calculated using the following formula where TV is tumor volume:

Max tumor regression = [(TVmin - TVdayO) / TVdayO] x 100

[00114] Maximum tumor regression was classified as complete tumor regression (100% regression with no palpable tumor), partial tumor regression (max tumor regression more than 30%)) or no tumor regression. [00115] The growth curves (Figure 5 A) illustrate the treatment effect when 46scFv-ILs- DTXp3 was combined with PD-1 when compared to free docetaxel combination with PD-1. Time to regrowth for all groups is illustrated in Figure 5B.

[00116] PD-1 and DTX showed minimal growth arrest and no tumor regression when given as monotherapies. In combination, PD-1 and docetaxel showed improved activity, but limited tumor regression. In contrast, 46scFv-ILs-DTXp3 did induce partial regression in most animals. Additionally, a combination of 46scFv-ILs-DTXp3 and PD-1 significantly increased the tumor doubling time, in addition to inducing partial regression in 60% of treated animals and complete regression in 20% (2/10) of treated animals. This study shows that 46scFv-ILs-DTXp3 has superior in vivo activity in comparison to an equitoxic dose of docetaxel both in monotherapy or in combination with PD-1 when evaluated against EMT-6 tumors.

Example 4 In vivo antitumor efficacy of 46scFv-ILs-DTXp3 in combination with a murine PD-1 antibody in murine models of lung and colon cancer in mice

[00117] Immunocompetent mice of strains C57BL6/J and BALB/c were implanted with murine cancer cell lines LLC (5x 10^5 cells/mouse, lung cancer) or CT26 (lx 10^6 cells/mouse, colon cancer) respectively. Animals were randomized into the following experimental groups: Saline, anti-PD-1 murine antibody at 10 mg/kg twice a week, 46scFv-ILs-DTXp3 at 50 mg/kg, and PD-l/46scFv-ILs-DTXp3. For the combination group doses used for the monotherapy arms were combined and PD-1 dosing was initiated 2 days following 46scFv-ILs-DTXp3.

[00118] Animals receiving saline or 46scFv-ILs-DTXp3 received four tail vein injections, at intervals of 7 days. Mice receiving PD-1 were dosed IP twice a week. Tumor size was monitored once to twice weekly. The tumor progression was monitored by palpation and caliper

measurements of the tumors along the largest (length) and smallest (width) axis twice a week. The tumor sizes were determined twice weekly from the caliper measurements using the formula (Geran, R.I., et al., 1972 Cancer Chemother. Rep. 3 : 1-88):

Tumor volume (TV)= [(length) x (width)2] / 2

[00119] Animals were monitored until tumor regrowth or end of monitoring period (>120 days). Time to regrowth was defined as time for tumor to double its volume. Tumor growth inhibition (TGI) is defined as the average tumor size relative to the control group at the last time point before any mouse was sacrificed due to tumor burden. Animals sacrificed prior to tumor volume doubling are censored.

[00120] Tumor growth inhibition of each of the treatment groups for both cell lines is shown in the table below. In both models the PD-l/46scFv-ILs-DTXp3 groups showed increased activity when compared to the saline and monotherapy groups. The increase in growth inhibition also translated to an increase in survival for mice treated with PD-l/46scFv-ILs-DTXp3.

[00121] The activity of the monotherapy groups varied between the two models. In the LLC study, PD-1 inhibition had a negative effect on tumor growth, and 46scFv-ILs-DTXp3 had a moderately positive effect. In contrast, PD-1 inhibition was able to inhibit the growth of CT26 tumors, and the effect of 46scFv-ILs-DTXp3 was smaller in comparison. PD-l/46scFv-ILs- DTXp3 was superior in both models, which illustrates the activity of the combination in multiple models regardless of the activity of the monotherapies.

Example 5 Effects of 46scFv-ILs-DTXp3, free docetaxel monotherapy and PD-1 combination on immune cell infiltrate in EMT-6 mure breast tumor model

[00122] Immunocompetent mice of strain BALB/c were implanted with murine breast cancer cell line EMT-6 (3.5x10^5). Animals randomized into the following experimental groups: Saline, anti-PD-1 murine antibody at 10 mg/kg twice weekly, free docetaxel at 10 mg/kg qlw, 46scFv- ILs-DTXp3 at 50 mg/kg qlw, PD-l/docetaxel, and PD-l/46scFv-ILs-DTXp3. For the

combination group doses used for the monotherapy arms were combined and PD-1 dosing was initiated 2 days following docetaxel or 46scFv-ILs-DTXp3. Animals receiving saline or 46scFv- ILs-DTXp3 received two tail vein injections, at an interval of 7 days. Mice receiving PD-1 were dosed IP twice a week. All mice completed two treatment cycles before the tumors were harvested and prepared for analysis using fluorescence-activated cell sorting (FACS).

48 hourse after the second dose of either saline, docetaxel, or 46scFv-ILs-DTXp3 the mice were euthanized and the tumors were removed. The tumors were homogenized using a combination of mechanical and enzymatic cell dissociation before being filtered into a suspension of single cells. Antibodies were used to identify total immune cells (CD45), T cells (CD3), T cell subsets (CD8, CD4, and FOXP3), as well as the inflammatory capacity of the T cells (IFNy). It was observed that mice treated with 46scFv-ILs-DTXp3, either alone or in combination with PD-1, had increased levels of CD8+ T cells, as a fraction of all CD45+ cells, in their tumors (Figure 6A). CD8+ T cells are also referred to as cytoxic T cells and are primarily responsible for cell killing. When T cells also express interferony (IFNу) it indicates that they are responding to antigen-specific immune signals and, in this context, acting to kill tumor cells. When analyzed via FACS, more CD8 and IFNу double-postive T cells were observed in the tumors of mice that received 46scFv-ILs-DTXp3 (Figure 6B).

[00123] An additional measure that indicates the creation of an antitumor immune response is the ratio of CD8+ T cells to regulatory T cells (Tregs). Increases in the ratio show that the balance is shifted towards antitumor immunity and away from immune protection. The ratio of CD8+ T cells to Tregs is increased in mice that received 46scFv-ILs-DTXp3, indicating the enhancement of an antitumor immune response (Figure 6C).

Example 6 In vivo antitumor efficacy and immune cell infiltrate of

46scFv-ILs-DTXp3 when compared to dose-dense and standard dosing of free docetaxel in EMT-6 murine breast tumor model

[00124] Immunocompetent mice of strain BALB/c were implanted with murine breast cancer cell line EMT-6 (3.5x10^5). Animals were randomized in cohorts of 10 into the following experimental groups: Saline, docetaxel 10mg/kg once a week (DTX-MTD), docetaxel 3.33mg/kg three times a week (DTX-DD), and 46scFv-ILs-DTXp3 at 50 mg/kg.

[00125] Animals received three weeks of treatment with all drugs delivered via tail vein injections. Tumor size was monitored twice weekly. The tumor progression was monitored by palpation and caliper measurements of the tumors along the largest (length) and smallest (width) axis twice a week. The tumor sizes were determined twice weekly from the caliper measurements using the formula (Geran, R.I., et al., 1972 Cancer Chemother. Rep. 3 : 1-88):

Tumor volume (TV)= [(length) x (width)2] / 2

[00126] Animals were monitored until the end of the study. Tumor growth inhibition (TGI) is defined as the average tumor size relative to the control group at the end of the three treatment cycles.

[00127] 48 hours after the third dosing cycle of either saline, docetaxel, or 46scFv-ILs-DTXp3 the mice were euthanized and the tumors were removed. The tumors were homogenized using a combination of mechanical and enzymatic cell dissociation before being filtered into a suspension of single cells. Antibodies were used to identify total immune cells (CD45), T cells (CD3), T cell subsets (CD8, CD4, and F0XP3), as well as the inflammatory capacity of the T cells (ifny).

[00128] Figure 7 A shows the tumor growth inhibition for all groups in this study. Mice receiving 46scFv-ILs-DTXp3 showed more growth inhibition than mice receiving docetaxel in both dosing schedules. DTX-DD improved growth inhibition relative to DTX-MTD, but it did not recapitulate the inhibition observed in 46scFv-ILs-DTXp3 treatment.

[00129] FACS was used to compare the immune response generated after treatment with 46scFv-ILs-DTXp3 or docetaxel given in two different dosing schemes. As described in Example 5, treatment with 46scFv-ILs-DTXp3 increased CD8+ T cell and CD8+ ifny+ T cell populations in the tumor, in addition to increasing the CD8+/Treg ratio (Figure 7B, 7C, and 7D). In this dosing schedule, treatment with 46scFv-ILs-DTXp3 also increased CD4+∑FNy+ T cells in the tumor (Figure 7E). Neither the dose-dense (DTX-DD) nor the weekly (DTX-MTD) docetaxel treatment groups were observed to affect these intratumoral immune cell populations in this study.

Example 7 In vivo antitumor efficacy of 46scFV-ILs-DTXp3 in combination with a murine PD-1 antibody in a murine model of sarcoma cancer in mice

[00130] Immunocompetent mice of strain A/J were implanted with the SAIN murine cancer cell line (2x10^6 cells/mouse, sarcoma). Animals were randomized into the following experimental groups: Saline (n = 8), anti-PD-1 murine antibody at 10 mg/kg twice a week (n = 8), 46scFv-ILs-DTXp3 at 50 mg/kg (n = 9), and PD-l/46scFv-ILs-DTXp3 (n = 9). For the combination group doses used for the monotherapy arms were combined and PD-1 dosing was initiated 2 days following 46scFv-ILs-DTXp3.

[00131] Animals receiving saline or 46scFv-ILs-DTXp3 received four tail vein injections, at intervals of 7 days. Mice receiving PD-1 were dosed IP twice a week. Tumor size was monitored once to twice weekly. The tumor progression was monitored by palpation and caliper

Tumor volume (TV)= [(length) x (width)2] / 2

[00132] Maximum response was calculated using the following formula where TV is tumor volume:

Max tumor regression = [(TVmin - TVdayO) / TVdayO] x 100 [00133] Maximum tumor regression was classified as complete tumor regression (100% regression with no palpable tumor), partial tumor regression (max tumor regression more than 30%) or no tumor regression.

[00134] Animals were monitored until tumor regrowth or end of monitoring period (>120 days). Time to regrowth was defined as time for tumor to double its volume. Tumor growth inhibition (TGI) is defined as the average tumor size relative to the control group at the last time point before any mouse was sacrificed due to tumor burden. Animals sacrificed prior to tumor volume doubling are censored.

[00135] Tumor growth inhibition is shown in the table below. The PD-l/46scFv-ILs-DTXp3 group showed increased activity when compared to the saline and monotherapy groups. PD-1 inhibition and treatment with 46scFv-ILs-DTXp3 as a monotherapy showed similar growth inhibition.

[00136] The increase in growth inhibition also translated to an increase in survival for mice treated with PD-l/46scFv-ILs-DTXp3. A 33% complete response rate was observed in this study for the PD-l/46scFv-ILs-DTXp3 group (Figure 8). The mice that were free of tumor burden at the end of the 140 day monitoring period were resistant to rechallenge with an identical bolus of SAIN cells.

Example 8 In vivo antitumor efficacy of 46scFV-ILs-DTXp3 when compared to free docetaxel and in combination with murine PD-1 and PD-L1 antibodies in a murine model of colon cancer in mice

[00137] Immunocompetent C57BL6/J mice were implanted with MC38 murine cancer cells (3x10^5 cells/mouse, colon cancer). Animals were randomized into the following experimental groups: Saline (n = 8), anti-PD-1 murine antibody at 10 mg/kg twice a week (n = 9), anti-PD-Ll murine antibody at 10 mg/kg twice a week (n = 9), 46scFv-ILs-DTXp3 at 50 mg/kg (n = 9), PD- l/46scFv-ILs-DTXp3 (n = 9), PD-Ll/46scFv-ILs-DTXp3 (n = 9), docetaxel (DTX) at 10mg/kg (n = 9), and PD-l/DTX (n = 9). For the combination group, doses used for the monotherapy arms were combined, and PD-l/PD-Ll dosing was initiated 2 days following 46scFv-ILs-DTXp3 or docetaxel.

[00138] Animals receiving saline, DTX or 46scFv-ILs-DTXp3 received four tail vein injections, at intervals of 7 days. Mice receiving PD-1 or PD-L1 were dosed IP twice a week. Tumor size was monitored once to twice weekly. The tumor progression was monitored by palpation and caliper measurements of the tumors along the largest (length) and smallest (width) axis twice a week. The tumor sizes were determined twice weekly from the caliper measurements using the formula (Geran, R.I., et al., 1972 Cancer Chemother. Rep. 3 : 1-88):

Tumor volume (TV)= [(length) x (width)2] / 2

[00139] Animals were monitored until tumor regrowth or end of monitoring period (>75 days). Tumor growth inhibition (TGI) is defined as the average tumor size relative to the control group at the last time point before any mouse was sacrificed due to tumor burden. Animals sacrificed prior to tumor volume doubling are censored.

[00140] Tumor growth inhibition is shown in the table below. The PD-l/46scFv-ILs-DTXp3 group showed increased activity when compared to the saline and all monotherapy groups.

46scFv-ILs-DTXp3 treatment both alone and in combination with PD-1 improved growth inhibition relative to DTX and PD-l/DTX groups. The greatest amount of growth inhibition was observed in the PD-Ll/46scFv-ILs-DTXp3 group

[00141] The increase in growth inhibition resulted in an increase in survival for all 46scFv- ILs-DTXp3 containing groups (Figure 9A). Median survival in the PD-l/46scFv-ILs-DTXp3 and PD-Ll/46scFv-ILs-DTXp3 was superior to the PD-1 and PD-L1 monotherapy groups.

Combinations with 46scFv-ILs-DTXp3 and PD-1 or PD-L1 also improved survival relative to PD-l/46scFv-ILs-DTXp3 monotherapy. The PD-Ll/46scFv-ILs-DTXp3 treatment resulted in a 10% complete response rate.

[00142] 46scFv-ILs-DTXp3 monotherapy and PD-l/46scFv-ILs-DTXp3 combination therapy was superior to DTX based treatment groups (Figure 9B). Median survival for the DTX and PD- 1/DTX groups was 16 days after treatment initiation. In contrast, median survival was improved to 27 days for 46scFv-ILs-DTXp3 and to 34 days with PD-l/46scFv-ILs-DTXp3.

Example 9 In vivo antitumor efficacy and immune cell infiltrate of

46scFv-ILs-DTXp3 when compared to NT-ILs-DTXp3 and 46scFv-ILs in EMT-6 murine breast tumor model

[00143] Immunocompetent mice of strain BALB/c were implanted with murine breast cancer cell line EMT-6 (3.5x10^5). Animals were randomized in cohorts of 10 into the following experimental groups: Saline, 46scFv-ILs-DTXp3 at 50 mg/kg, NT-ILs-DTXp3 at 50 mg/kg, and 46scFv-ILs dosed to match the lipid content of the 46scFv-ILs-DTXp3 group. The NT-ILs- DTXp3 group is identical to 46scFv-ILs-DTXp3 but is lacking the antibody fragment. The 46scFv-ILs group is identical to the 46scFv-ILs-DTXp3 but does not contain the docetaxel prodrug.

[00144] Animals received three weeks of treatment with all drugs delivered via tail vein injections. Tumor size was monitored twice weekly. The tumor progression was monitored by palpation and caliper measurements of the tumors along the largest (length) and smallest (width) axis twice a week. The tumor sizes were determined twice weekly from the caliper measurements using the formula (Geran, R.I., et al., 1972 Cancer Chemother. Rep. 3 : 1-88):

Tumor volume (TV)= [(length) x (width)2] / 2

[00145] Animals were monitored until the end of the study. Tumor growth inhibition (TGI) is defined as the average tumor size relative to the control group at the end of the three treatment cycles.

[00146] 48 hours after the third dosing cycle of either saline, docetaxel, or 46scFv-ILs-DTXp3 the mice were euthanized and the tumors were removed. The tumors were homogenized using a combination of mechanical and enzymatic cell dissociation before being filtered into a suspension of single cells. Antibodies were used to identify total immune cells (CD45), T cells (CD3), T cell subsets (CD8, CD4, and FOXP3).

[00147] The table below shows the tumor growth inhibition for all groups in this study. The growth inhibition observed with either 46scFv-ILs-DTXp3 or NT-ILs-DTXp3 was nearly identical. No growth inhibition was observed in the 46scFv-ILs group.

[00148] FACS was used to compare the immune response generated after treatment with 46scFv-ILs-DTXp3, NT-ILs-DTXp3, or 46scFv-ILs. As described in Example 5, treatment with 46scFv-ILs-DTXp3 increased CD8+ T cell and CD8+ ifny+ T cell populations in the tumor, in addition to increasing the CD8+/Treg ratio (see Figures 7B, 7C, and 7D). In this study the increase in CD8+/Treg ratio was observed only in the 46scFv-ILs-DTXp3 and the NT-ILs- DTXp3 groups (Figure 10A). An increase the in natural killer T cell (NK) population was also only observed in the 46scFv-ILs-DTXp3 and the NT-ILs-DTXp3 groups (Figure 10B).

Claims

What is claimed is:

1. A method of treating cancer in a patient, comprising administering to the patient an effective amount of a first agent that is a liposome-encapsulaLed taxane or taxane prodrug and a second agent that impedes regulatory T cell activity.

2. The method of claim L, wherein the liposome is targeted to EphAl

3. The method of claim 1, wherein the liposome is an EphA2-targetcd docctaxel-generating liposome.

4. The method of claim 3, wherein the E phA2-targeted docetaxel-generating liposome comprises a docetaxel prodrug.

5. The method of claim 4, wherein the EphA2-targetsd docetaxel-generaring liposome comprising a docetaxel prodrug, is encapsulated within a. lipid vesicle comprising one or more lipids, a PEG lipid derivative and an £phA2 binding moiety on the outside of the lipid vesicle.

6. The method of claim 4 or claim 5, wherein the docetaxel prodrug is a compound of Formula (I)

where R1 end R2 are each independently H or lower ulkyl, and n is an integer 2-3.

7. The method of claim 6. wherein R 1 and R2 are C1-C3 alkyl, and n is 3.

8. The method according to any one of claims 5-7, wherein the EphA2 binding moiety is a scFv moiety comprising the CDRs of SEQ ID NO:40.

9. The method according to anyone of claims 5-7, wherein the EphA2 binding moiety is a scFv moiety comprising the sequence of SEQ ID NO:41 or SEQ ID NO:46.

10. The method according to any one of claims 5-8, wherein die lipid vesicle comprises sphingomyelin, cholesterol and PEG-DSG.

11. The method of claim 10, wherein the lipid vesicle comprises sphingomyelin, cholesterol and PEG-DSG in a weight ratio of about 4,4: 1.6; 1.

12. The method of claim 3, wherein the EphA2 -targeted docetaxel-generating liposome is 46seFv-II.,s-DTXp3 or 46scFv-ILs-DTXp6.

13. The method according to any one of claims 1 -I 1 , wherein the cancer is treated.

14. The method of claim .13, wherein the second agent is aatirPD-1 antibody, an anti-PD-L 1 antibody, or an.anti-CTLA-4 antibody.

13. The method of claim 13, the second agent is atezolizumab, durvalumab, or avelumab.

16. The method of claim 13, the second agent is selected from the group consi sting of nivolumab, pembrolizumab and pidilizumab.

17 The method of claim 13, the second agent is ipilimumab, or tremelimumab, or lirilomab.

18. The method according to any one of claims 1-17, wherein the cancer is breast, esophageal, prostate, ovarian, bladder, gastric, pancreatic, or lung cancer.

19. The method according to any one of claims 1-17, wherein the cancer is triple negative breast cenccr.

20. The method according to any one of claims 1-17, wherein the cancer is non-small cell lung cancer.

21. The method according to any one of claims 1-17, wherein the cancer is a solid tumor..

22. The method according to any one of claims 1-17, wherein the cancer is a sarcoma.

23. A method of treating cancer in a patient, comprising administering to the patient an effective amount of a first agent that is a taxanc or taxane prodrug generating composition and a second agent that impedes regulatory T cell activity.

24. The method of claim 23, wherein the taxane or taxane prodrug generating composition has a taxane or taxane prodrug release half-life in mouse of 10 hours or greater (e.g., 20 hours or greater, 30 hours or greater, 40 hours or greater, 50 hours or greater, 60 hours or greater, 70 hours or greater, or 80 hours or greater).

25. The method of claim 23 or 24, wherein the taxane or taxane prodrug generating composition is a liposome-encapsulated taxane or taxanc prodrug.

26. The method of claim.25, wherein the liposome, is targeted to Eph A2.

27. The method of claim 26, wherein the liposome is an Eph.A2-targeted docetaxel- generuting liposome.

28. The method of claim 27, wherein the EphA2-targeted docetaxel-generaring liposome comprises a docetaxel prodrug.

29. The method of claim 28, wherein the EphA2 targeted docetaxel-generating liposome comprising a. docetaxel prodrug is encapsulated within a lipid vesicle comprising one or more lipids, a PEG lipid derivative and an EphA2 binding moiety on the outside of the lipid vesicle.

30. The method of claim 28 or 29, wherein the docetaxel prodrug is a compound of Formula (I)

where R1 and R2 are each independently H or lower alkyl. and a is an integer 2-3.

3.1. The method of claim 30, wherein. R1 and R2 are C1-C3 alkyl, and n is 3.

32. The method according to any one of claims.29-31, wherein the EphA2 binding moiety is a scFv moiety comprisiag the CDRs of.SEQ ID N^'O;40.

33. The method according to any one of claims 29-31 _> wherein the EphA2 binding moiety is a scFv moiety comprising the sequence of SEQ ID NO:4l or SEQ ID NO:46.

34. The method according to any one of claims 29-33, wherein the lipid vesicle comprises sphingomyelin, cholesterol and PEG-DSG.

35. The method of claim 34, wherein the iipid vesicle comprises sphingomyelin, cholesterol and PEG-DSG In a weight ratio of about

36. The method of claim 27, wherein the EphA2-targeted docetaxel-generating liposome is 46scFv-lLs-DTXp3 or 46scFv-iLs,DTXp6.

37. The method ucconling to any one of claims 23-36, wherein the cancer is treated.

38. The method according to any one of claims 23-37, wherein the second agent is anti-PP- 1, antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody.

39. The method of claim 38, wherein the second agent is atezolizumab, dirrvahimab, or avelumab.

40. the method of claim 38, wherein the second agent, is selected from the group consisting of nivolumab. pembrolizumab, and pidilizumab.

41. The method of claim 38, wherein the second agent is ipilimumab or tremelimumab or lirilumab.

42. The method according to any one of claims 23-41, wherein the cancer is breast, esophageal, prostate, ovarian, bladder, gastric, pancreatic or lung cancer.

43. The method according to any one of claims 23-41 , wherein the cancer is triple negative breast cancer.

44. The method according to any one of claims 23-41, wherein the cancer is non-small cell lung cancer.

45. The method according to any one of claims 23-41. wherein che cancer is a solid tumor.

46. The method of any one of claims 23-40, wherein the cancer is a sarcoma.

47. The method of claim 3 or 25, wherein the. EphA2-targeted docetaxel-generating liposome comprises a binding moiety that, competes for binding to EphA2 with an scFv consisting of SEQ ID NO:40.