WO2017004167A1

WO2017004167A1 - Methods and compositions for treating cancer

Info

Publication number: WO2017004167A1
Application number: PCT/US2016/040039
Authority: WO
Inventors: David Munn; Andrew Mellor
Original assignee: Augusta University Research Institute, Inc.
Priority date: 2015-07-01
Filing date: 2016-06-29
Publication date: 2017-01-05

Abstract

The present disclosure relates to methods for treating a tumor or cancer by administering to a subject a PTEN inhibitor and a chemotherapeutic agent. The subject may be pre-treated with a vaccine that targets or comprises a tumor or cancer antigen or another therapeutic that enhances immune responses. The disclosure also provides compositions related to coadministration of a PTEN inhibitor, a chemotherapeutic and, optionally, a vaccine.

Description

METHODS AND COMPOSITIONS FOR TREATING CANCER

[001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 62/187,438, filed on July 1 , 2015. The contents of this application are herein

incorporated by reference in their entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[002] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename:NEWL_024_01WO_SeqList_ST25, date recorded: June 23, 2016, file size 7, 147 bytes).

STATEMENT REGARDING GOVERNMENT SUPPORT

[003] The invention described herein was made in part with government support under Grant Nos. CA112431 , CA103320 and CA096651 awarded by the National Institutes of Health. The United States government has certain rights in the invention.

FIELD OF THE INVENTION

[004] The field of the invention generally relates to methods and compositions for treating cancers and tumors.

BACKGROUND OF THE INVENTION

[005] Many chemotherapeutic agents have adverse side effects associated with the toxicity of the chemotherapeutic dose required to achieve efficacy. Furthermore, the efficacy is limited by the toxicity of the drug because tumors often regrow before the patient can tolerate another dose of the chemotherapeutic agent. Thus, there is a need for treatments that would allow reducing the chemotherapeutic dose and, optionally, increasing the frequency of the chemotherapeutic dose while maintaining or improving the efficacy of the chemotherapeutic agent.

[006] Many chemotherapeutics kill tumor cells and release antigens from the tumor.

Immune response to these antigens would be beneficial, and would increase the efficacy of chemotherapy, but such immune responses are hindered by suppression of the immune response in the tumor microenvironment. T-cell anergy and the existence of regulatory T- cells contribute to tumor evasion of immune surveillance. In particular, the tumor

microenvironment is profoundly suppressive for anti-tumor T-cell responses, which has generally been considered an intrinsic property of the tumor milieu. Although

immunotherapy offers a promising approach to activating the immune system against cancer, problems with efficacy and toxicity have hindered its practical application.

Therefore, there remains a need for improved approaches to immunotherapy.

SUMMARY OF THE INVENTION

[007] It is an object of the invention to provide compositions and methods for treating a cancer or a tumor in a subject in need thereof. When combined with a chemotherapeutic agent, a PTEN inhibitor allows a reduction in dose of the chemotherapeutic agent, thus decreasing adverse side effects associated with toxicity. A reduced dose of the

chemotherapeutic agent may be adminstered more frequently.

[008] The invention encompasses a method of reducing tumor volume in a subject in need thereof, the method comprising administering to the subject a PTEN inhibitor and a chemotherapeutic agent given at the standard dose used to reduce the volume of that tumor, wherein the reduction in tumor volume by the combination is greater than the reduction in tumor volume produced by the chemotherapeutic agent administered without any PTEN inhibitor. The efficacy of the combination (e.g., as measured by reduction in tumor volume) may be increased at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% compared to the efficacy of the chemotherapeutic agent administered alone at the same dose. The invention further encompasses a method of reducing tumor volume in a subject in need thereof, the method comprising administering a PTEN inhibitor and a

chemotherapeutic agent to the subject, wherein the chemotherapeutic agent is administered at a reduced dose compared to the standard dose used to reduce tumor volume when the chemotherapeutic agent is administered without any PTEN inhibitor. The PTEN inhibitor and the chemotherapeutic agent may be administered to the subject concurrently or sequentially. The chemotherapeutic agent may be administered at a dose reduced at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% compared to the standard dose used to reduce tumor volume when the chemotherapeutic agent is administered without any PTEN inhibitor. The PTEN inhibitor and the reduced dose of chemotherapeutic agent may be administered to the subject in multiple cycles. In such multiple cycles, the reduced dose of chemotherapeutic agent may be administered more frequently than the standard dose is administered. For example, the PTEN inhibitor and the chemotherapeutic agent may be administered to the subject about every two weeks, about every ten days, about every one week, about every six days, about every five days, about every four days, about every three days, about every two days or about every one day. [009] The invention also encompasses a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a PTEN inhibitor and a chemotherapeutic agent given at the standard dose used to treat that cancer, wherein the efficacy in treating cancer by the combination is greater than the efficacy in treating cancer produced by the chemotherapeutic agent administered without any PTEN inhibitor. The efficacy of the combination (e.g., as measured by reduction in a symptom of the cancer) may be increased at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% compared to the efficacy of the chemotherapeutic agent administered alone at the same dose. The invention further encompasses a method of treating cancer in a subject in need thereof, the method comprising administering a PTEN inhibitor and a chemotherapeutic agent to the subject, wherein the chemotherapeutic agent is administered at a reduced dose compared to the standard dose used to treat cancer when the chemotherapeutic agent is administered without any PTEN inhibitor. The PTEN inhibitor and the chemotherapeutic agent may be administered to the subject concurrently or sequentially. The

chemotherapeutic agent may be administered at a dose reduced at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% compared to the standard dose used to treat cancer when the chemotherapeutic agent is administered without any PTEN inhibitor.

[0010] In one aspect of the invention, a vaccine that targets or comprises a tumor antigen is administered to a subject in combination with at least one PTEN inhibitor and a

chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is

administered after the vaccine. A PTEN inhibitor may be administered concurrently with the vaccine or concurrently with the chemotherapeutic agent. In certain embodiments, a PTEN inhibitor is administered both concurrently with the vaccine and, later, concurrently with the chemotherapeutic agent. Administration of the vaccine and the chemotherapeutic agent may be separated by 24 hrs to 72 hrs, 48 hrs to 96 hrs, or 72 hrs to 120 hrs, or by at least one week, at least about two weeks, at least about three weeks or at least about four weeks.

[0011] The invention encompasses a method of reducing tumor volume in a subject in need thereof, the method comprising: (i) administering a vaccine that targets or comprises a tumor antigen to the subject; and (ii) after step (i), administering a PTEN inhibitor and an antineoplastic chemotherapeutic agent to the subject.

[0012] The invention relates to a method of treating cancer in a subject in need thereof, the method comprising: (i) administering a vaccine that targets or comprises a tumor antigen to the subject; and (ii) after step (i), administering a PTEN inhibitor and an antineoplastic chemotherapeutic agent to the subject. [0013] In any of the disclosed methods, step (ii) may be performed at least about one week, at least about two weeks, at least about three weeks or at least about every four weeks after step (i). In some embodiments, step (ii) is performed 24 hrs to 72 hrs after step (i), 48 hrs to 96 hrs after step (i) or 72 hrs to 120 hrs after step (i). The PTEN inhibitor, the vaccine and the chemotherapeutic agent may be administered to the subject concurrently or sequentially.

[0014] In any of the disclosed methods, step (i) may further comprise administering a PTEN inhibitor to the subject. In such embodiments, the same PTEN inhibitor may be administered to the subject in steps (i) and (ii).

[0015] The vaccine of any of the disclosed methods may target or comprise a tumor antigen from a lung tumor, a breast tumor, an ovarian tumor, a brain tumor, a pancreatic tumor, a colon tumor or a melanoma tumor. In one embodiment, the vaccine targets or comprises human gp100, NY-ESO-1 , Mud or EGFR-vlll.

[0016] The PTEN inhibitor of any of the disclosed methods may be a small molecule, a nucleic acid or a protein. In certain embodiments, the PTEN inhibitor may be N-(9, 10- Dioxo-9, 10-dihydrophenanthren-2-yl)-2,2-dimethylpropionamide; or 3,4-Dephostatin, ethyl-. In some embodiments, the PTEN inhibitor is a vanadium complex (e.g., VO-OHpic, also known as [V(=0)(H₂0)(OHpic)₂], see, e.g., Mak et al., J. Chem. Biol. 3: 157-163 (2010)).

[0017] The disclosed methods may be used to treat a refractory tumor or a tumor with particularly low immunogenicity.

[0018] In some embodiments, the tumor treated by the disclosed methods is a lung tumor, a breast tumor, an ovarian tumor, a brain tumor, a pancreatic tumor, a colon tumor or a melanoma tumor. In some embodiments, the cancer treated by the disclosed methods is lung cancer, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, colon cancer or melanoma.

[0019] Optionally, the disclosed method further comprises (iii) at least about two weeks after step (ii), administering the vaccine that targets or comprises a tumor antigen to the subject; and (iv) after step (iii), administering a PTEN inhibitor and the chemotherapeutic agent to the subject. Step (iv) may be performed at least about one week or at least about two weeks after step (iii). The PTEN inhibitor and the chemotherapeutic agent of step (iv) may be administered to the subject concurrently or sequentially. Step (iii) may further comprise administering a PTEN inhibitor to the subject. In such embodiments, the same PTEN inhibitor may be administered to the subject in steps (iii) and (iv). The combination of a PTEN inhibitor, a chemotherapeutic agent, and, optionally, a vaccine may be administered to a subject in multiple cycles (e.g., steps (iii) and (iv) may be repeated). The number of cycles may be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10. In some cases, a subject may be treated with such cycles indefinitely, as long as the treatment remains effective.

[0020] These and other embodiments and/or other aspects of the invention will become evident upon reference to the following detailed description of the invention and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1A shows the results of an experiment studying the effects of vaccine treatment and pharmacologic inhibition of PTEN on B16F10 tumor volume in wild-type mouse hosts. The upper part of the figure shows the sequence of treatment with pmel-1 T- cells, hgp100 vaccine and the VO-OHpic PTEN inhibitor (also known as

[V(=0)(H₂0)(OHpic)₂], see, e.g., Mak et al., J. Chem. Biol. 3: 157-163 (2010)). Human gp100₂₅-33 had the sequence of KVPRNQDWL (SEQ ID NO:3). The lower part of the figure is a graph showing the changes in tumor volume (mm³) in groups that were treated with (1) pmel-1/vaccine; (2) pmel-1 /vaccine and VO-OHpic; or (3) nothing.

[0022] Figure 1 B shows the results of an experiment studying the effects of vaccine treatment and pharmacologic inhibition of PTEN on E.G7 tumor volume in wild-type mouse hosts. The upper part of the figure shows the sequence of treatment with OT-I T-cells, OVA vaccine and the VO-OHpic PTEN inhibitor. The lower part of the figure is a graph showing the changes in tumor volume (mm³) in groups that were treated with (1) OT-l/vaccine; (2) OT-l/vaccine and VO-OHpic; or (3) nothing.

[0023] Figure 1C shows the results of an experiment studying the effects of vaccine treatment and pharmacologic inhibition of PTEN on the intratumoral milieu in a B16F10 mouse tumor model. The figure includes FACS analysis scatter plots showing loss of Fox03a expression in regulatory T-cells (Tregs) (top panel); proliferation of

carboxyfluorescein succinimidyl ester (CFSE)-labeled effector cells (pmel-1 cells) within the tumor (second panel from the top); and emergence of activated Ly6c⁺CD11 b⁺ myeloid DCs (dendritic cells) (third panel from the top) and an inflammatory milieu with DCs expressing high levels of CD86 and low levels of PD-L1 (fourth panel from the top) in mice treated with pmel-1/vaccine and VO-OHpic. The pie charts in the bottom panel summarize the CD86 and PD-L1 expression data directly above. The figure is representative of 5 experiments using both B16F10 and E.G7 tumors. [0024] Figure 1 D shows the results of an experiment comparing endogenous tumor antigen presentation by DCs (dendritic cells) in B16-OVA mouse tumor models treated with (1) pmel- 1 T-cells, hgp100 vaccine and VO-OHpic or (2) pmel-1 T-cells and hgp100 vaccine. Ly6c⁺ and Ly6c^NEG cells were sorted and the SIINFEKL (SEQ ID NO: 1) peptide was added as shown. The DCs were tested for ability to present endogenous OVA antigen to OT-I responder cells in vitro. DCs from mice treated with pmel-1 T-cells, hgp100 vaccine and VO- OHpic were able to robustly cross-present endogenous tumor antigen acquired in vivo. DCs from mice treated with pmel-1 T-cells and hgp100 vaccine without VO-OHpic were suppressive and did not cross-present endogenous tumor antigens effectively.

[0025] Figure 2A shows the results of experiments studying the effects of pharmacologic inhibition of PTEN and chemotherapy on B16F10 tumor volume in wild-type mouse hosts. The upper part of the figure shows the sequence of treatment with CTX (cyclophosphamide) and the VO-OHpic PTEN inhibitor. The lower part of the figure is a graph showing the changes in tumor volume (mm³) in groups that were treated with (1) CTX; (2) VO-OHpic; or (3) CTX and VO-OHpic. The figure shows pooled data from 5 independent experiments; n=10-16 tumors in each group; bars show standard deviation.

[0026] Figure 2B shows the results of a FACS analysis of cells in B16F10 tumors treated with (1) CTX (cyclophosphamide); (2) CTX and VO-OHpic; or (3) nothing, analyzed four days after treatment with CTX. Addition of VO-OHpic to CTX treatment abrogated Fox03a expression in Tregs, down-regulated PD-L1 , and increased the number of inflammatory Ly6c⁺CD11 b⁺ CD1 1c⁺ DCs. Call-out shows gated CD11 c⁺ DCs from the CTX+ VO-OHpic group showing co-expression of CD103. The pie charts in the bottom panel summarize the CD86 and PD-L1 expression data directly above. The figure shows pooled data from 5 independent experiments; n=10-16 tumors in each group; bars show standard deviation.

[0027] Figure 2C shows the results of an experiment studying the contribution of the adaptive immune response to B16F10 tumor regression in mice. Rag1-KO hosts (lacking an adaptive immune system) or WT (wild-type) B6 controls were treated with CTX

(cyclophosphamide) plus concurrent VO-OHpic. The figure shows a mean of 16 tumors from 2 independent experiments. Bars show standard deviation; bars in WT group are smaller than the symbols.

[0028] Figure 3 shows the results of an experiment studying the effects of vaccine treatment, pharmacologic inhibition of PTEN and chemotherapy on Lewis Lung Carcinoma tumor cells stably transfected with the gp100 nominal tumor antigen (LLC-gp100). Tumor volume is shown in wild-type mouse hosts. The upper part of the figure shows the sequence of treatment with hgp100 vaccine, the VO-OHpic PTEN inhibitor and CTX (cyclophosphamide). The lower part of the figure is a graph showing the changes in tumor volume (mm³) in groups that were treated with (1) vaccine and CTX; (2) vaccine and VO- OHpic; (3) vaccine, VO-OHpic and CTX; or (4) VO-OHpic and CTX. CpG-1826

(phosphorothioate oligo 5'-TCCATGACGTTCCTGAGCTT-3' (SEQ ID NO:2)) was synthesized from the published sequence (Chu et al., J. Exp. Med. 186, 1623-1631 (1997)).

[0029] Figure 4A is a graph showing a hypothetical model of increased chemotherapy efficacy at a lower dose when combined with a PTEN inhibitor. The combination of both drugs creates a greater anti-tumor effect than could be achieved even by a maximum tolerated dose (MTD) of chemotherapy; and this increased efficacy occurs at a low dose of chemotherapy, which by itself would have minimal effect.

[0030] Figure 4B shows the results of an experiment studying the effects of pharmacologic inhibition of PTEN and chemotherapy on B16F10 tumor volume in wild-type mouse hosts. Mice with B16F10 tumors were treated with 0 or 10 mg/kg VO-OHpic (PTEN inhib.) at days 9, 10, 1 1 , 12, and 13 and with 0, 25, 50, or 150 mg/kg cyclophosphamide (CTX;

chemotherapeutic agent) at day 10, as shown in the table in Fig. 4B. The combination of a PTEN inhibitor (VO-OHpic) and a chemotherapeutic agent (cyclophosphamide (CTX)) given at a dose of 50 mg/kg created a greater anti-tumor effect than could be achieved even by a higher dose (150mg/kg) of the chemotherapeutic agent used alone.

[0031] Figure 5A is a graph showing the growth of B16F10 tumors in PTEN^Tre9-KO hosts and WT B6 hosts. Tumor volume (mm³) is shown on the y-axis. Pooled data from 4 experiments, n=6-8 tumors per time-point. *p<.05 vs. WT, and all points thereafter.

[0032] Figures 5B, 5C, and 5D show the results of experiments analyzing tumor-infiltrating immune cells in B16F10 tumors after 10 days of tumor growth in either PTEN^Tre9-KO or parental Foxp3-GFP-Cre hosts. (Figure 5B) Tregs; (Figure 5C) CD8⁺ T cells (Figure 5D) CD1 1c⁺ DCs. Representative of a total of 9 experiments on days 10, 15 and 22. Intracellular cytokines were measured after 4 hr activation with PMA/ionomycin.

[0033] Figures 6A, 6B, and 6C show the results of experiments analyzing the inflammatory intra-tumoral milieu in E.G7 lymphoma tumors grown in PTEN^Tre9-KO hosts. The changes in Tregs (Figure 6A), CD8⁺ cells (Figure 6B) and DCs (Figure 6C) in these tumors were similar to those seen in B16F10 tumors (see Figures 5B, 5C, and 5D).

DETAILED DESCRIPTION OF THE INVENTION

[0034] The disclosure provides methods for treating a subject in need thereof with a combination of a PTEN inhibitor and a chemotherapeutic agent. A PTEN inhibitor increases the efficacy of a chemotherapeutic agent and surprisingly allows a reduction in the dose of the chemotherapeutic agent. The reduced dose provides the advantage of decreased side effects associated with toxicity of a chemotherapeutic agent and may be administered more frequently. In the case of tumors and cancers, more frequent doses are advantageous because they may be given before the tumor or cancer regrows.

[0035] A PTEN inhibitor may also be administered in combination with a chemotherapeutic agent and a vaccine directed to or comprising a tumor or a cancer antigen. A PTEN inhibitor surprisingly improves the efficacy of the chemotherapeutic agent and the vaccine.

[0036] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited herein, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated documents or portions of documents define a term that contradicts that term's definition in the application, the definition that appears in this application controls. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0037] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. As used herein, "about" means ± 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components unless otherwise indicated. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include" and "comprise" are used synonymously.

[0038] As used herein, the terms "treat," "treating," "treatment" and "therapeutic use" refer to the elimination, reduction or amelioration of one or more symptoms of a disease or disorder. As used herein, a "therapeutically effective amount" refers to that amount of a therapeutic agent sufficient to mediate a clinically relevant elimination, reduction or amelioration of such symptoms. An effect is clinically relevant if its magnitude is sufficient to impact the health or prognosis of a recipient subject. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g. , delay or minimize the spread of cancer. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

[0039] As used herein, the term "cancer" refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells. As used herein, cancer explicitly includes, leukemias and lymphomas. The term "cancer" refers to a disease involving cells that have the potential to metastasize to distal sites and exhibit phenotypic traits that differ from those of non-cancer cells, for example, formation of colonies in a three-dimensional substrate such as soft agar or the formation of tubular networks or web-like matrices in a three-dimensional basement membrane or extracellular matrix preparation. Non-cancer cells do not form colonies in soft agar and form distinct sphere-like structures in three-dimensional basement membrane or extracellular matrix preparations.

[0040] As used herein, an "immune cell" refers to any cell from the hemopoietic origin including, but not limited to, T-cells, B cells, monocytes, dendritic cells, and macrophages.

[0041] As used herein, the terms "immunologic," "immunological" or "immune" response is the development of a beneficial humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T-cells or their secretion products) response directed against a peptide in a recipient patient. Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T-cells. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen-specific CD4+ T helper cells and/or CD8+ cytotoxic T-cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4+ T-cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T-cells from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.

[0042] As used herein, an "immunogenic agent" or "immunogen" is capable of inducing an immunological response against itself on administration to a mammal, optionally in conjunction with an adjuvant.

[0043] As used herein, the terms "individual," "host," "subject" and "patient" are used interchangeably, and refer to a mammal, including, but not limited to, humans, non-human primates, rodents, such as mice and rats, and other laboratory animals. [0044] As used herein, the term "polypeptide" refers to a chain of amino acids of any length, regardless of modification (e.g., phosphorylation or glycosylation). The term polypeptide includes proteins and fragments thereof. The polypeptides can be "exogenous," meaning that they are "heterologous," i.e., foreign to the host cell being utilized, such as human polypeptide produced by a bacterial cell. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M),

Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).

[0045] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0046] Modifications and changes can be made in the structure of the polypeptides of the disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity.

Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.

[0047] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);

cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0048] It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

[0049] Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamnine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an

immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

[0050] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (lie: Leu, Val), (Leu: lie, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: lie, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide of interest. [0051] The term "percent (%) sequence identity" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0052] For purposes herein, the % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given sequence C that has or comprises a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:

100 times the fraction W/Z,

[0053] where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program's alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C.

[0054] As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.

[0055] Typically, the disclosed methods of reducing tumor volume or treating cancer include administering to a subject in need thereof an effective amount of a PTEN inhibitor in combination with a chemotherapeutic agent and, optionally, a vaccine (e.g., a vaccine directed to or comprising a tumor antigen or a cancer antigen). The disclosed methods may increase the efficacy of a chemotherapeutic (e.g., an antineoplastic) agent.

[0056] The invention encompasses a method of reducing tumor volume in a subject in need thereof, the method comprising administering to the subject a PTEN inhibitor and a chemotherapeutic agent given at the standard dose used to reduce the volume of that tumor, wherein the reduction in tumor volume by the combination is greater than the reduction in tumor volume produced by the chemotherapeutic agent administered without any PTEN inhibitor. The efficacy of the combination (e.g., as measured by reduction in tumor volume) may be increased at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% compared to the efficacy of the chemotherapeutic agent administered alone at the same dose. The invention further encompasses a method of reducing tumor volume in a subject in need thereof, the method comprising administering a PTEN inhibitor and a

chemotherapeutic agent to the subject, wherein the chemotherapeutic agent is administered at a reduced dose compared to the standard dose used to reduce tumor volume when the chemotherapeutic agent is administered without any PTEN inhibitor. The PTEN inhibitor and the chemotherapeutic agent may be administered to the subject concurrently or sequentially. The chemotherapeutic agent may be administered at a dose reduced at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% compared to the standard dose used to reduce tumor volume when the chemotherapeutic agent is administered without any PTEN inhibitor. The PTEN inhibitor and the reduced dose of chemotherapeutic agent may be administered to the subject in multiple cycles. In such multiple cycles, the reduced dose of chemotherapeutic agent may be administered more frequently than the standard dose is administered. For example, the PTEN inhibitor and the chemotherapeutic agent may be administered to the subject about every two weeks, about every ten days, about every one week, about every six days, about every five days, about every four days, about every three days, about every two days or about every one day.

[0057] The invention also encompasses a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a PTEN inhibitor and a chemotherapeutic agent given at the standard dose used to treat that cancer, wherein the efficacy in treating cancer by the combination is greater than the efficacy in treating cancer produced by the chemotherapeutic agent administered without any PTEN inhibitor. The efficacy of the combination (e.g., as measured by reduction in a symptom of the cancer) may be increased at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% compared to the efficacy of the chemotherapeutic agent administered alone at the same dose. The invention further encompasses a method of treating cancer in a subject in need thereof, the method comprising administering a PTEN inhibitor and a chemotherapeutic agent to the subject, wherein the chemotherapeutic agent is administered at a reduced dose compared to the standard dose used to treat cancer when the chemotherapeutic agent is administered without any PTEN inhibitor. The PTEN inhibitor and the chemotherapeutic agent may be administered to the subject concurrently or sequentially. The

chemotherapeutic agent may be administered at a dose reduced at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% compared to the standard dose used to treat cancer when the chemotherapeutic agent is administered without any PTEN inhibitor. As described above, the PTEN inhibitor and the reduced dose of chemotherapeutic agent may be administered to the subject in multiple cycles. In such multiple cycles, the reduced dose of chemotherapeutic agent may be administered more frequently than the standard dose is administered. For example, the PTEN inhibitor and the chemotherapeutic agent may be administered to the subject about every two weeks, about every ten days, about every one week, about every six days, about every five days, about every four days, about every three days, about every two days or about every one day.

[0058] In one aspect of the invention, the chemotherapeutic agent is administered to a subject concurrently with a vaccine that is directed to or comprises a tumor antigen or a cancer antigen and a PTEN inhibitor. In some embodiments, the chemotherapeutic agent is administered after the vaccine. A PTEN inhibitor may be administered concurrently with the vaccine or concurrently with the chemotherapeutic agent. In certain embodiments, a PTEN inhibitor is administered both concurrently with the vaccine and, later, concurrently with the chemotherapeutic agent. In one embodiment, the invention encompasses a method of reducing tumor size or treating cancer in a subject in need thereof, the method comprising: (i) administering a vaccine that is directed to or comprises a tumor antigen or a cancer antigen to the subject; and (ii) after step (i), administering a PTEN inhibitor and a

chemotherapeutic (e.g., an antineoplastic) agent to the subject. In another embodiment, the invention encompasses a method of reducing tumor size or treating cancer in a subject in need thereof, the method comprising: (i) administering a vaccine that is directed to or comprises a tumor antigen or a cancer antigen and a PTEN inhibitor to the subject; and (ii) after step (i), administering a chemotherapeutic (e.g., an antineoplastic) agent to the subject.

[0059] The methods can be used in vivo or ex vivo in cancer treatment applications. In general, the combination of a PTEN inhibitor, a chemotherapeutic agent and a vaccine can be used for treating a subject having or being predisposed to any tumor-related or cancer- related antigen. The methods and compositions of the invention allow a reduction in chemotherapeutic agent dose compared to the dose of the same chemotherapeutic agent used as a standard of care without a PTEN inhibitor. The reduced dose exhibits decreased adverse side effects and may be administered more frequently.

[0060] The disclosed methods and compositions can be used to treat cancer or reduce tumor volume in a subject in need thereof. In some embodiments, the invention relates to a method of reducing tumor volume or treating cancer in a subject in need thereof, the method comprising: (i) administering a chemotherapeutic (e.g., an antineoplastic) agent to the subject; and (ii) after step (i), administering a PTEN inhibitor and a vaccine that is directed to or comprises a tumor antigen or a cancer antigen to the subject. In some embodiments, the invention relates to a method of reducing tumor volume or treating cancer in a subject in need thereof, the method comprising: (i) administering a vaccine that is directed to or comprises a tumor antigen or a cancer antigen to the subject; and (ii) after step (i), administering a PTEN inhibitor and a chemotherapeutic (e.g., an antineoplastic) agent to the subject. In some embodiments, the invention relates to a method of reducing tumor volume or treating cancer in a subject in need thereof, the method comprising: (i) administering a chemotherapeutic (e.g., an antineoplastic) agent and a PTEN inhibitor to the subject; and (ii) after step (i), administering a vaccine that is directed to or comprises a tumor antigen or a cancer antigen to the subject. In other embodiments, the invention relates to a method of reducing tumor volume or treating cancer in a subject in need thereof, the method

comprising: (i) administering a vaccine that is directed to or comprises a tumor antigen or a cancer antigen and a PTEN inhibitor to the subject; and (ii) after step (i), administering a chemotherapeutic (e.g., an antineoplastic) agent to the subject. In further embodiments, a PTEN inhibitor is administered in both step (i) and step (ii).

[0061] The disclosed methods are useful for treating refractory tumors or tumors that exhibit particularly low immunogenicity. The types of cancer and tumor that may be treated with the provided compositions and methods include, but are not limited to, prostate, colorectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin, melanoma, basal carcinoma, mesothelial lining, white blood cells, lymphoma, leukemia, and other hematological cancers, esophagus, breast, muscle, esophageal, nasopharangeal, uterine, connective tissue, lung, small-cell lung carcinoma, non-small-cell lung carcinoma, adrenal gland, thyroid, kidney, or bone; glioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma, sarcoma, choriocarcinoma, cutaneous basocellular carcinoma, and testicular seminoma.

[0062] Malignant tumors which may be treated are classified herein according to the embryonic origin of the tissue from which the tumor is derived. Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands. Sarcomas, which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage. The leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors may show up at numerous organs or tissues of the body to establish a cancer.

[0063] Many pathogens are associated with inducing cancer via chronic infection and inflammation (e.g., HCV, HPV and bacteria such as H. pylori). Accordingly, in some embodiments, the cancer is caused by a pathogen, and/or the result of an infection or inflammation. In a particular embodiment, the cancer is hepatocellular carcinoma.

[0064] Adoptive T-cell therapy may be used with the methods of the invention. The disclosed methods can be used to treat T-cells ex vivo. One embodiment provides a method for treating cancer by administering an inhibitor of PTEN in combination with a vaccine and a chemotherapeutic agent to overcome T-cell exhaustion and/or T-cell anergy. The adoptive T-cell transfer can be administered to the subject prior to or following administration of the agent.

[0065] Antigen-specific T-cell lines can be generated by in vitro stimulation with antigen followed by nonspecific expansion (for example, on CD3/CD28 beads). The ability to expand antigen-specific T-cells can be assessed using IFN-gamma and granzyme B enzyme-linked immunosorbent spot. The phenotype of the resultant T-cell lines can be evaluated by flow cytometry, for example, by monitoring for the presence of FOXP3-expressing CD4(+) T- cells. Amplification of antigen-specific T-cell populations from Peripheral Blood Mononuclear Cells (PBMCs) is usually performed through repeated in vitro stimulation with optimal length antigenic peptides in the presence of a cytokine (e.g., IL-2). Low doses of IL-2 (between 10 and 50 U/ml) have been used traditionally to avoid the activation/expansion of lymphokine- activated killer cells, as revealed in chromium release assays that were commonly employed to monitor specific T-cell expansion. Concentrations of antigenic peptides can be 0.1-10 μΜ.

[0066] In the methods and compositions of the invention, the active agents (e.g., PTEN inhibitors, vaccines, and chemotherapeutic agents) are typically administered to a subject in need thereof in an effective amount. For example, the active agents can be administered in a dosage sufficient reduce or prevent a least one, two, three, or more symptoms of the cancer, or to otherwise provide a desired pharmacologic and/or physiologic effect. The symptom may be physical or biological. For example, in the context of cancer treatment, the symptom may be physical, such as tumor burden, or biological such as proliferation of cancer cells. In some embodiments, the amount is effective to increase the killing of tumor cells or inhibit proliferation or metastasis of tumor cells. In some embodiments, the amount is effective to reduce tumor burden. In some embodiments, the amount is effective to reduce or prevent at least one comorbidity of a cancer.

[0067] In some embodiments, the effect of the active agent on a subject is compared to a control. For example, the effect of the active agent on a particular symptom, pharmacologic, or physiologic indicator can be compared to an untreated subject, or the condition of the subject prior to treatment. In some embodiments, the symptom, pharmacologic, or physiologic indicator is measured in a subject prior to treatment, and again one or more times after treatment is initiated. In some embodiments, the control is a reference level, or average determined based on measuring the symptom, pharmacologic, or physiologic indicator in one or more subjects that do not have the disease or condition to be treated (e.g., healthy subjects).

[0068] In some embodiments, the effect of the treatment is compared to a conventional treatment that is known in the art, such as one of those discussed herein. Co-administration of a PTEN inhibitor allows a reduction of the dose of the chemotherapeutic agent used as the standard of care (e.g., for human patients) or the maximum tolerated dose (MTD) without a PTEN inhibitor. Standard dose ranges for specific chemotherapy drugs are known to those skilled in the art. Doses of different chemotherapeutic agents vary, depending on the tumor type and on concomitant administration of other chemotherapy drugs, but

recommended doses are usually strictly followed. These doses are taught by published literature, based on controlled clinical trials, and also by sources such as the approved dose on package inserts approved for marketing in various countries. Because chemotherapy drugs almost always have a strong dose-response relationship, the standard-of-care doses are chosen to be as high as possible within the limitation of unacceptable toxicity to the patient. Lower doses than the standard-of-care doses would be expected to produce lower efficacy. However, the disclosed methods allow a reduction in the chemotherapeutic dose while maintaining, or even improving, the chemotherapeutic agent efficacy. Even modestly lower doses of chemotherapy often produce much lower toxicity in the patient (threshold effect). Thus, in a given clinical setting, the ability to reduce the standard dose of a chemotherapy drug by even a modest percentage, while maintaining equal or superior efficacy, is of great value to the patient. The chemotherapeutic agent may be administered at a dose reduced at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% compared to the standard dose used to reduce tumor volume (or to treat cancer) or the MTD when the chemotherapeutic agent is administered without any PTEN inhibitor. The reduced dose of the chemotherapeutic agent may maintain the same efficacy (or an improved efficacy) in reducing tumor volume or treating cancer as the standard of care dose.

[0069] The precise dosage of a PTEN inhibitor, a chemotherapeutic agent and a vaccine will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, clinical symptoms etc.). In general, the doses may range from about 1 ng/kg to 100 mg/kg for a typical subject, with exemplary shorter ranges being 1 to 50 mg/kg and 10 to 20 mg/kg. Such doses may be repeated. The dose will be correlated with the identity of the mammal receiving said dose. Doses in the above-recited mg/kg ranges are convenient for mammals, including rodents, such as mice and rats, and primates, especially humans.

[0070] The timing of the administration of various components of the disclosed methods and compositions will depend on the formulation and/or route of administration used. In some embodiments, administration is given as a long-term treatment regimen whereby

pharmacokinetic steady state conditions will be reached.

[0071] The data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration. For any compound, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography (HPLC).

[0072] The invention encompasses a treatment regimen comprising multiple cycles of administering a PTEN inhibitor, a chemotherapeutic agent, and, optionally, a vaccine that targets or comprises a tumor antigen to a subject in need thereof. The number of cycles may be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10. In some cases, a subject may be treated with such cycles indefinitely, as long as the treatment remains effective. The cycles may be separated by 24 hrs to 72 hrs, 48 hrs to 96 hrs, or 72 hrs to 120 hrs, or by at least one week, at least about ten days, at least about two weeks, at least about three weeks or at least about four weeks.

[0073] In some embodiments, the disclosed method comprises (i) administering a vaccine that targets or comprises a tumor antigen to the subject; and (ii) after step (i), administering a PTEN inhibitor and a chemotherapeutic (e.g., antineoplastic) agent to the subject. In further embodiments, the disclosed method comprises (i) administering a vaccine that targets or comprises a tumor antigen and a PTEN inhibitor to the subject; and (ii) after step (i), administering a chemotherapeutic (e.g., antineoplastic) agent to the subject. In some embodiments, the disclosed method comprises (i) administering a vaccine that targets or comprises a tumor antigen and a PTEN inhibitor to the subject; and (ii) after step (i), administering a PTEN inhibitor and a chemotherapeutic (e.g., antineoplastic) agent to the subject. In such embodiments, the same PTEN inhibitor may be administered to the subject in steps (i) and (ii). In step (i) of these multistep regimens, the PTEN inhibitor and the vaccine may be administered to the subject concurrently or sequentially. If the PTEN inhibitor and the vaccine are administered sequentially, either the PTEN inhibitor or the vaccine may be administered first. Step (ii) may be performed at least about 1 week, at least about 2 weeks, at least about 3 weeks or at least about 4 weeks after step (i). In step (ii), the PTEN inhibitor and the chemotherapeutic agent may be administered to the subject concurrently or sequentially. If the PTEN inhibitor and the chemotherapeutic agent are administered sequentially, either the PTEN inhibitor or the chemotherapeutic agent may be administered first.

[0074] The PTEN inhibitor, vaccine, and chemotherapeutic agent (and optional additional active agents) can be administered as part of a therapeutic regimen. A treatment regimen of the combination therapy can include one or multiple administrations of the PTEN inhibitor. In some embodiments, the vaccine, PTEN inhibitor and chemotherapeutic agent may be administered to the patients in multiple rounds. These rounds (e.g., administration of the vaccine and the chemotherapeutic agent) may be separated by 24 hrs to 72 hrs, 48 hrs to 96 hrs, or 72 hrs to 120 hrs, or by at least one week, at least about ten days, at least about two weeks, at least about three weeks or at least about four weeks. For example, the disclosed method may comprise (i) administering a vaccine that targets or comprises a tumor antigen to the subject; and (ii) after step (i), administering a PTEN inhibitor and a chemotherapeutic (e.g., antineoplastic) agent to the subject; (iii) at least about two weeks after step (ii), administering the vaccine to the subject; and (iv) after step (iii), administering a PTEN inhibitor and the chemotherapeutic (e.g., antineoplastic) agent to the subject. In some embodiments, the disclosed method may comprise (i) administering a vaccine that targets or comprises a tumor antigen to the subject; and (ii) after step (i), administering a PTEN inhibitor and a chemotherapeutic (e.g., antineoplastic) agent to the subject; (iii) administering the vaccine and a PTEN inhibitor to the subject; and (iv) after step (iii), administering a PTEN inhibitor and a chemotherapeutic (e.g., antineoplastic) agent to the subject. In such embodiments, the same PTEN inhibitor may be administered to the subject in steps (iii) and (iv). In step (iii), the PTEN inhibitor and the vaccine may be administered to the subject concurrently or sequentially. If the PTEN inhibitor and the vaccine are administered sequentially, either the PTEN inhibitor or the vaccine may be administered first. Step (iv) may be performed at least about 1 week, at least about 10 days, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks after step (iii). In step (iv), the PTEN inhibitor and the chemotherapeutic agent may be administered to the subject concurrently or sequentially. If the PTEN inhibitor and the chemotherapeutic agent are administered sequentially, either the PTEN inhibitor or the chemotherapeutic agent may be administered first.

[0075] In some embodiments, the disclosed methods include administering a subject in need thereof an effective amount of one or more additional active agents, for example, a PD- 1 or CTLA4 antagonist or other immunomodulator in either step (i), (ii), (iii) and/or (iv). In some embodiments, the PTEN inhibitor and the one or more additional active agents are administered to the subject separately, but simultaneously. The PTEN inhibitor and the additional active agent(s) can also be administered as part of the same composition. In other embodiments, the PTEN inhibitor and the additional active agent(s) are administered separately and at different times, but as part of the same treatment regimen. If the PTEN inhibitor is administered first, the additional active agent(s) can be administered second. Likewise, if the additional active agent(s) is administered first, the PTEN inhibitor can be administered second.

[0076] Dosage regimens or cycles of the agents can be completely, or partially overlapping, or can be sequential. For example, in some embodiments, all such administration(s) of the PTEN inhibitor occur before or after administration of the chemotherapeutic agent.

Alternatively, administration of one or more doses of the PTEN inhibitor can be temporally staggered with the administration of the chemotherapeutic agent to form a uniform or nonuniform course of treatment whereby one or more doses of PTEN inhibitor are administered, followed by one or more doses of the chemotherapeutic agent, followed by one or more doses of PTEN inhibitor; or one or more doses of the chemotherapeutic agent are administered, followed by one or more doses of PTEN inhibitor, followed by one or more doses of the chemotherapeutic agent; etc., all according to whatever schedule is selected or desired by the researcher or clinician administering the therapy.

[0077] An effective amount of each of the agents can be administered as a single unit dosage (e.g., as dosage unit), or sub-therapeutic doses that are administered over a finite time interval. Such unit doses may be administered on a daily basis for a finite time period, such as up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days or up to 20 days or up to 25 days, are all specifically contemplated by the invention.

[0078] The invention relates to methods of treatment that comprise administering to a subject a pharmaceutical composition comprising an effective amount of at least one PTEN inhibitor. Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase ("PTEN" and also referred to as MMAC1 , TEP1 , phosphatase and tensin homolog, and phosphatase and tensin homolog deleted on chromosome 10) is a dual- specificity protein phosphatase, dephosphorylating tyrosine-, serine- and threonine- phosphorylated protein. PTEN is also a lipid phosphatase that removes the phosphate in the D3 position of the inositol ring from phosphatidylinositol 3,4,5-trisphosphate,

phosphatidylinositol 3,4-diphosphate, phosphatidylinositol 3-phosphate and inositol 1 ,3,4,5- tetrakisphosphate.

[0079] Amino acid and nucleic acid sequences for PTEN are known in the art. See, for example, UniProt Accession No. P60484, which provides the canonical amino acid sequence including:

MTAI IKEIVSRNKRRYQEDGFDLDLTYIYPNI IAMGFPAERLEGVYRNNIDDVVRFLDSK HKNHYKIYNLCAERHYDTAKFNCRVAQYPFEDHNPPQLELIKPFCEDLDQWLSEDDNHVA AIHCKAGKGRTGVMICAYLLHRGKFLKAQEALDFYGEVRTRDKKGVTIPSQRRYVYYYSY LLK HLDYRPVALLFHKMMFETIPMFSGGTCNPQFVVCQLKVKIYSSNSGPTRREDKFMY FEFPQPLPVCGDIKVEFFHKQNKMLKKDKMFHFWVNTFFIPGPEETSEKVENGSLCDQEI DSICSIERADNDKEYLVLTLTKNDLDKANKDKA RYFSPNFKVKLYF KTVEEPSNPEAS SSTSVTPDVSDNEPDHYRYSDTTDSDPENEPFDEDQHTQITKV

(SEQ ID NO:4) (UniProt Accession No. P60484 (PTEN_HUMAN)), which can be encoded by the nucleic acid sequence:

GAATTCGGCACGAGGTGAGGCGAGGCCGGGCTCAGGCGAGGGAGATGAGAGACGGCGGCG GCCGCGGCCCGGAGCCCCTCTCAGCGCCTGTGAGCAGCCGCGGGGGCAGCGCCCTCGGGG AGCCGGCCGGCCTGCGGCGGCGGCAGCGGCGGCGTTTCTCGCCTCCTCTTCGTCTTTTCT AACCGTGCAGCCTCTTCCTCGGCTTCTCCTGAAAGGGAAGGTGGAAGCCGTGGGCTCGGG CGGGAGCCGGCTGAGGCGCGGCGGCGGCGGCGGCACCTCCCGCTCCTGGAGCGGGGGGGA GAAGCGGCGGCGGCGGCGGCCGCGGCGGCTGCAGCTCCAGGGAGGGGGTCTGAGTCGCCT GTCACCATTTCCAGGGCTGGGAACGCCGGAGAGTTGGTCTCTCCCCTTCTACTGCCTCCA ACACGGCGGCGGCGGCGGCGGCACATCCAGGGACCCGGGCCGGTTTTAAACCTCCCGTCC GCCGCCGCCGCACCCCCCGTGGCCCGGGCTCCGGAGGCCGCCGGCGGAGGCAGCCGTTCG GAGGATTATTCGTCTTCTCCCCATTCCGCTGCCGCCGCTGCCAGGCCTCTGGCTGCTGAG GAGAAGCAGGCCCAGTCGCTGCAACCATCCAGCAGCCGCCGCAGCAGCCATTACCCGGCT GCGGTCCAGAGCCAAGCGGCGGCAGAGCGAGGGGCATCAGCTACCGCCAAGTCCAGAGCC ATTTCCATCCTGCAGAAGAAGCCCCGCCACCAGCAGCTTCTGCCATCTCTCTCCTCCTTT TTCTTCAGCCACAGGCTCCCAGACATGACAGCCATCATCAAAGAGATCGTTAGCAGAAAC AAAAGGAGATATCAAGAGGATGGATTCGACTTAGACTTGACCTATATTTATCCAAACATT ATTGCTATGGGATTTCCTGCAGAAAGACTTGAAGGCGTATACAGGAACAATATTGATGAT GTAGTAAGGTTTTTGGATTCAAAGCATAAAAACCATTACAAGATATACAATCTTTGTGCT GAAAGACATTATGACACCGCCAAATTTAATTGCAGAGTTGCACAATATCCTTTTGAAGAC CATAACCCACCACAGCTAGAACTTATCAAACCCTTTTGTGAAGATCTTGACCAATGGCTA AGTGAAGATGACAATCATGTTGCAGCAATTCACTGTAAAGCTGGAAAGGGACGAACTGGT GTAATGATATGTGCATATTTATTACATCGGGGCAAATTTTTAAAGGCACAAGAGGCCCTA

GATTTCTATGGGGAAGTAAGGACCAGAGACAAAAAGGGAGTAACTATTCCCAGTCAGAGG CGCTATGTGTATTAT A AGCTACCTGTTAAAGAATCATCTGGAT A AGACCAGTGGCA CTGTTGTTTCACAAGATGATGTTTGAAACTATTCCAATGTTCAGTGGCGGAACTTGCAAT CCTCAGTTTGTGGTCTGCCAGCTAAAGGTGAAGATATATTCCTCCAATTCAGGACCCACA CGACGGGAAGACAAGTTCATGTACTTTGAGTTCCCTCAGCCGTTACCTGTGTGTGGTGAT ATCAAAGTAGAGTTCTTCCACAAACAGAACAAGATGCTAAAAAAGGACAAAATGTTTCAC TTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAAT GG AAGT C AT GT GAT C AAGAAAT C GAT AGC AT T T GC AGT AT AGAGC GT GC AG AT AAT G AC AAGGAATAT C T AGT AC T T AC T T T AAC AAAAAAT G AT CT T G AC AAAGC AAAT AAAGAC AAA GCCAACCGATACTTTTCTCCAAATTTTAAGGTGAAGCTGTACTTCACAAAAACAGTAGAG GAGCCGTCAAATCCAGAGGCTAGCAGTTCAACTTCTGTAACACCAGATGTTAGTGACAAT GAACCTGATCATTATAGATATTCTGACACCACTGACTCTGATCCAGAGAATGAACCTTTT GATGAAGATCAGCATACACAAATTACAAAAGTCTGAATTTTTTTTTATCAAGAGGGATAA AAC AC C AT G AAAAT AAAC T T G AAT AAAC T G AAAAAAAAAAAAAAAAAAA

(SEQ ID NO:5) (ENA Sequence No. U96180.1 (Human protein tyrosine phosphatase (TEP1) mRNA, complete cds)).

[0080] As used herein, "PTEN inhibitor" refers to agents that directly reduce, block, inhibit, prevent or suppress expression or activity of PTEN and/or agents that reduce, block, inhibit, prevent or suppress the downstream effects of PTEN activation. The agent can be a small molecule, or a biomacromolecule, such as a protein, polypeptide, or nucleic acid. In some embodiments, at least two PTEN inhibitors are administered to the subject. In one embodiment, at least one of the agents directly reduces, blocks, inhibits, prevents or suppresses expression or activity of PTEN. In particular embodiments, the composition includes, or the subject is otherwise administered, two PTEN inhibitors in combination, wherein one inhibitor directly inhibits PTEN and the other PTEN inhibitor reduces or prevents the downstream effects of PTEN activation in the subject.

[0081] A PTEN inhibitor may reduce, block, inhibit, prevent, or suppress the activity of PTEN with a particular potency. For example, a PTEN inhibitor may have an IC50 of less than about 100 μΜ, less than about 50 μΜ, less than about 25 μΜ, less than about 10 μΜ, less than about 5 μΜ, less than about 1 μΜ, less than about 500 nM, less than about 250 nm, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, or less than about 1 nM. PTEN inhibition assays for general screening (to identify and confirm alternative, suitable inhibitors) and IC50 determinations are described in the working examples provided herein and/or known in the art, e.g. U.S. 2007/0203098 and WO 2005/097119, each of which is specifically incorporated by reference herein in its entirety. For instance, a PTEN inhibition assay and IC50 determination may be performed as described in Examples 3 and 4 of U.S. 2007/0203098. Briefly, free phosphate, which is a product of the PTEN dephosphorylation reaction, can be detected by a

colorimetric reaction with a commercially available malachite green solution. Various volumes of the test inhibitor candidates can be mixed with PTEN solution and then substrate can be added. The reaction mix can be incubated for a suitable time. Subsequently, an aliquot of malachite green buffer can be added to develop the color in the dark. A

spectrophotometer can be used to measure the optical density at, for example, 650 nanometers. The activity of PTEN can be measured in vivo by quantifying cellular

PI(3,4,5)P₃ levels after treatment with a PTEN inhibitor.

[0082] In some embodiments, the PTEN is a PTEN inhibitor such as N-(9, 10-Dioxo-9, 10- dihydrophenanthren-2-yl)-2,2-dimethylpropionamide; or 3,4-Dephostatin, ethyl-.

[0083] Formulas for non-limiting examples of PTEN inhibitor compounds are disclosed in U.S. 2007/0203098, WO2005/0971 19, U.S. 201 1/0002877, and U.S. 2011/0189308, each of which is specifically incorporated by reference herein in its entirety. In particular

embodiments, the PTEN inhibitor is a vanadium complex including, for example, vanadate (VO) or bisperoxovanadate (bpV) complexed to one or more organic ligands. Non-limiting examples of ligands include 1-isoquinoline (isoqu), phenanthroline (phen), phenylbiguanide (biguan), 3-hydropicolinate (OH-pic), bipyridine (bipy) and picolinato (pic). See examples of such small molecules disclosed in Rosivatz, et al., ACS Chem. Biol., 1 (12):780-90 (2006); which is herein incorporated by reference. PTEN inhibitors including bpV(bipy), bpV(OHpic), bpV(phen), bpV(pic) are available from commercial vendors.

[0084] In some embodiments, a PTEN inhibitor is a vanadium-based PTEN inhibitor described in U.S. 2007/0292532 or U.S. 7,692,012, each of which is specifically incorporated by reference herein in its entirety. For example, a PTEN inhibitor may be a vanadium- containing compound of the formula:

wherein L-L' is:

and L' is COO, CONR⁵, CONHR⁶, CH₂NR⁵R'

or wherein L and L' together form a group:

wherein L" is O, S or NH;

R¹, R², R³, R⁴, R⁵ and R⁶ are independently H, hydroxyl, Ci_-6 alkyl, optionally substituted by hydroxy or NR⁷R⁸, 0₃.₆ cycloalkyi, optionally substituted by hydroxy or NR⁷R⁸, phenyl, optionally substituted by Ci_-3 alkyl, hydroxy, NR⁷R⁸ or S0₃, (OCHzCHzMNHCHzCI-yn, an amino acid or a peptide consisting of 2 to 5 amino acids; and

R⁷ and R⁸ are independently H or Ci_-6 alkyl; or a pharmaceutically acceptable salt thereof.

[0085] In another embodiment, a PTEN inhibitor may be a vanadium-containing compound of the formula:

wherein L-L' is:

and L' is COO, CONR⁵, CONHR⁶, CH₂NR⁵R'

or wherein L and L' together form a group:

or a group:

wherein L" is O, S or NH;

R¹, R², R³, R⁴, R⁵ and R⁶ are independently H, hydroxyl, Ci_-6 alkyl, optionally substituted by hydroxy or NR⁷R⁸, C₃.₆ cycloalkyl, optionally substituted by hydroxy or NR⁷R⁸, phenyl, optionally substituted by Ci.₃ alkyl, hydroxy, NR⁷R⁸ or S0₃, (OCH₂CH2)n(NHCH₂CH2)n, an amino acid or a peptide consisting of 2 to 5 amino acids; and

[0086] Additional non-limiting examples of PTEN inhibitors include potassium bisperoxo (bipyridine) oxovanadate (bpV(bipy), potassium bisperoxo(1 ,10-phenanthroline)oxovanadate (pV(phenanthroline)), potassium bisperoxo (piconlinate) oxovanadate (pV(pic)), potassium bisperoxo(phenylbiguanide)oxovanadate (pV(biguan)), pV(phenbig) [dipotassium

bisperoxo(phenylbiguanide)oxovanadate], bpV(HOpic) [dipotassium bisperoxo(5- hydroxypyridine-2-carboxyl)oxovanadate], VO-OHpic [V(=0)(H₂0)(OHpic)₂], bpV(pic), bpV(phen). [0087] The PTEN inhibitor can be a functional nucleic acid selected from the group consisting of antisense molecules, siRNA, shRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, or external guide sequences that target SEQ ID NO:5, or gene editing compositions such as CRISPR/Cas, zinc finger nuclease, or TALEN compositions that target the PTEN gene and reduce or otherwise modify its expression.

[0088] Methods for designing functional nucleic acids to suppress or inhibit expression of a nucleic acid, such as a nucleic acid encoding PTEN, are known in the art. For example, in some embodiments, the composition includes a functional nucleic acid or polypeptide designed to target and reduce or inhibit expression or translation of PTEN mRNA; or to reduce or inhibit expression, reduce activity, or increase degradation of PTEN protein. In some embodiments, the composition includes a vector suitable for in vivo expression of the functional nucleic acid.

[0089] In some embodiments, a functional nucleic acid or polypeptide is designed to target a segment of the nucleic acid sequence of SEQ ID NO:5, or the complement thereof, or variants thereof having a nucleic acid sequence at least about 65%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO:5.

[0090] In some embodiments, a functional nucleic acid or polypeptide is designed to target a segment of a the nucleic acid encoding the amino acid sequence of SEQ ID NO:4, or the complement thereof, or variants thereof having a nucleic acid sequence at least about 65%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to a nucleic acid encoding the amino acid sequence of SEQ ID NO:4.

[0091] In some embodiments, the functional nucleic acid hybridizes to the nucleic acid of SEQ ID NO:5, or a complement thereof, for example, under stringent conditions. In some embodiments, the functional nucleic acid hybridizes to a nucleic acid sequence that encodes SEQ ID NO:4, or a complement thereof, for example, under stringent conditions.

[0092] According to the invention, a PTEN inhibitor may be administered with one or more additional therapeutic agents. Additional therapeutic agents useful for the treatment of cancer are known to the skilled artisan. Additional therapeutic treatments include, but are not limited to, surgical resection, radiation therapy, hormone therapy, antibody-based therapies, whole body irradiation, bone marrow transplantation, peripheral blood stem cell

transplantation, vaccines, the administration of chemotherapeutic agents (also referred to herein as "antineoplastic chemotherapy agent," "antineoplastic agents," or "antineoplastic chemotherapeutic agents"), cytokines, antiviral agents, immune enhancers, tyrosine kinase inhibitors, signal transduction inhibitors, antibiotic, antimicrobial agents, a TLR agonists, such as for example, bacterial lipopolysaccharides (LPS), ligands that bind to Toll-Like Receptors such as CpG oligonucleotides (ODN), metabolic breakdown products of tryptophan, inhibitors of a GCN2 kinase, adjuvants, radionuclides, enzymes, anti-parasites (helminths, protozoans), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and bioactive fragments thereof (including humanized, single chain, and chimeric antibodies), antigen and vaccine formulations (including adjuvants), peptide drugs, molecules that mobilize and optimize the adaptive immune system, other molecules that activate or up-regulate the action of cytotoxic T lymphocytes, natural killer cells and helper T- cells, and other molecules that deactivate or down-regulate suppressor or regulatory T-cells.

[0093] Exemplary cytokines include, but are not limited to, IL-1 a, IL- β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-19, IL-20, IFN-a, IFN-β, IFN-γ, tumor necrosis factor (TNF), transforming growth factor-β (TGF-β), granulocyte colony stimulating factor (G- CSF), macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), and Flt-3 ligand.

[0094] Some embodiments include a cell based therapy such as sipuleucel-T treatment. In one embodiment, a PTEN inhibitor is administered to a subject after sipuleucel-T

(PROVENGE^®), preferably to treat metastatic castration-resistant prostate cancer.

[0095] Some additional therapeutic agents suitable for use in the methods and compositions of the invention are described in more detail below.

[0096] In some aspects, the invention relates to methods of treatment that comprise administering a vaccine to a subject in combination with a PTEN inhibitor. In some embodiments, a vaccine targets or comprises a tumor antigen. For example, a vaccine may be directed to or comprise an antigen from a lung tumor, a breast tumor, an ovarian tumor or a melanoma tumor. In some embodiments, the vaccine targets or comprises human gp100, NY-ESO-1 , Mud or EGFR-vlll.

[0097] The vaccine may be a tumor-specific cancer cell line that stimulates the subject's immune system to attack the subject's cancer cells. In one embodiment, the vaccine is a HyperAcute™ immunotherapy. A cell-based vaccine may be genetically modified to express alpha-gal carbohydrates on cell surface molecules. The vaccine may be any of the vaccines disclosed in U.S. 7,763,461 , U.S. 8,551 ,474, U.S. 8,535,658, U.S. 2014/0037692 or U.S. 2014/0072597, each of which is specifically incorporated herein by reference in its entirety. Non-limiting examples of cell-based vaccines that may be used in the methods and compositions of the invention are Algenpantucel-L, Tergenpumatucel-L and Dorgenmeltucel- L. [0098] A vaccine used in the methods and compositions of the invention may be formulated or administered with an adjuvant. The role of the adjuvant is to increase the immune system activation in the presence of target antigens. Non-limiting examples of immunostimulatory components of adjuvants and specific adjuvants include aluminum salts (e.g. , alum, aluminum phosphate, aluminum hydroxide), squalene-in-water emulsions (e.g. , AS03 or MF59 (4.3% w/v squalene, 0.5% w/v polysorbate 80, 0.5% w/v sorbitan trioleate)), MPL^® (Monophosphoryl Lipid A), 3DMPL (3-O-deacetylated MPL^®), AS04 (M PL^® and alum), MPL^® and QS-21 (e.g. , MPL^® and formulations such as AS01 and AS02), flagellin from S.

typhimurium (e.g. , flagellin or flagellin-Ag fusion proteins), imidazoquinoline derivatives (e.g., imiquimods), synthetic phophorothioate-linked DNA oligonucleotides with optimized CpG motifs (e.g. , CpG oligodeoxynuceotides and formulations (IC31 , QB10)), trehalose dimycolate (cord factor) (e.g, CAF01), saponins (e.g. , ISCOMS ((see, e.g., Sjolander et al., J. Leukocyte Biol. 64:713 (1998); WO 90/03184, WO 96/1 171 1 , WO 00/48630, WO 98/36772, WO 00/41720, WO 06/134423 and WO 07/026190)) and ISCOMATRIX), surfactant plus mineral or paraffin oil (e.g. , incomplete Freund's adjuvant (I FA) (and Montanide formulations)), I FA plus peptidoglycan and trehalose dimycolate (e.g., complete Freund's adjuvant (CFA)), QS-21 (saponin adjuvant), LT/CT mutants, poly(D, L-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukins, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor- muramyl-L-alanyl-D-isoglutamine (CGP 1 1637, referred to as nor-MDP), N-acetylmuramyl-L- alanyl-D-isoglutaminyl-L-alanine-2-(T-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)- ethylamine (CGP 19835A, referred to as MTP-PE), RIBI , which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL^®+TDM+CWS) in a 2% squalene/polysorbate 80 emulsion, synthetic derivatives of dsRNA (e.g. , Poly-IC or Poly-ICLC), and ligands for toll-like receptors (TLR), natural or synthesized (see, e.g. Kanzler et al. , Nature Med. 13: 1552-1559 (2007)), including TLR3 ligands such as polylC and similar compounds such as Hiltonol and Ampligen. Examples of suitable adjuvants are also found in Coffman et al., Immunity 33, 492-503 (2010).

[0099] Administration is not limited to the treatment of an existing tumor or cancer but can also be used to prevent or lower the risk of developing such diseases in an individual, i.e. , for prophylactic use. Potential candidates for prophylactic vaccination include individuals with a high risk of developing cancer, i.e. , with a personal or familial history of certain types of cancer.

[00100] Therefore, a PTEN inhibitor can be administered in conjunction with, or as a component of a vaccine composition. A PTEN inhibitor can be administered prior to, concurrently with, or after the administration of a vaccine. In one embodiment, a PTEN inhibitor is administered at the same time as administration of a vaccine. [00101] A PTEN inhibitor can be administered in conjunction with prophylactic vaccines, which confer resistance in a subject to subsequent exposure to cancer-causing molecules or events, or in conjunction with therapeutic vaccines, which can be used to initiate or enhance a subject's immune response to a pre-existing antigen, such as a tumor antigen in a subject with cancer, or a viral antigen in a subject infected with a virus.

[00102] The desired outcome of a prophylactic, therapeutic or de-sensitized immune response may vary according to the disease, according to principles well known in the art. For example, an immune response against a tumor-related or cancer-related antigen may completely prevent tumor or cancer presence in the patient, with an absence of any disease symptoms. However, a vaccine treatment against tumor-related or cancer-related antigens may be considered effective if it reduces the number, severity or duration of symptoms; if it reduces the number of individuals in a population with symptoms.

[00103] Similarly, immune responses against cancer may completely treat a disease, may alleviate symptoms, or may be one facet in an overall therapeutic intervention against a disease. For example, the stimulation of an immune response against a cancer may be coupled with surgical, chemotherapeutic, radiologic, hormonal and other immunologic approaches in order to affect treatment.

[00104] The invention includes methods of treatment comprising administering to a subject a PTEN inhibitor and a chemotherapeutic agent (in some cases, after administering a vaccine to the subject). In one embodiment, the chemotherapeutic agent is an

antineoplastic chemotherapeutic agent.

[00105] A chemotherapeutic agent may be, for example, a cytotoxic chemotherapy agent, such as, for example, epidophyllotoxin, procarbazine, mitoxantrone, platinum coordination complexes such as cisplatin and carboplatin, leucovorin, tegafur, paclitaxel, docetaxol, vincristine, vinblastine, methotrexate, cyclophosphamide, gemcitabine, estramustine, carmustine, adriamycin (doxorubicin), etoposide, arsenic trioxide, irinotecan, epothilone derivatives, navelbene, CPT-1 1 , anastrazole, letrazole, capecitabine, reloxafine, ifosamide, temozolomide and droloxafine.

[00106] A chemotherapeutic agent may be, for example, an alkylating agent, such as, for example, nitrogen mustards (such as chlorambucil, cyclophosphamide, ifosfamide, temozolomide, echlorethamine, melphalan, and uracil mustard), aziridines (such as thiotepa), methanesulphonate esters (such as busulfan), nitroso ureas (such as carmustine, lomustine, and streptozocin), platinum complexes (such as cisplatin and carboplatin), and bioreductive alkylators (such as mitomycin, procarbazine, dacarbazine and altretamine), ethylenimine derivatives, alkyl sulfonates, triazenes, pipobroman, temozolomide, triethylene- melamine, and triethylenethiophosphoramine.

[00107] A chemotherapeutic agent may be an antimetabolite, such as, for example, a folate antagonist (such as methotrexate and trimetrexate), a pyrimidine antagonist (such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, gemcitabine, and floxuridine), a purine antagonist (such as mercaptopurine, 6-thioguanine, fludarabine, and pentostatin), a ribonucleotide reductase inhibitor (such as hydroxyurea), and an adenosine deaminase inhibitor.

[00108] A chemotherapeutic agent may be a DNA strand-breakage agent (such as, for example, bleomycin), a topoisomerase II inhibitor (such as, for example, amsacrine, dactinomycin, daunorubicin, idarubicin, mitoxantrone, doxorubicin, etoposide, and teniposide), a DNA minor groove binding agent (such as, for example, plicamydin), a tubulin interactive agent (such as, for example, vincristine, vinblastine, and paclitaxel), a hormonal agent (such as, for example, estrogens, conjugated estrogens, ethinyl estradiol,

d i ethyl sti I besterol , chlortrianisen, idenestrol, progestins (such as hydroxyprogesterone caproate, medroxyprogesterone, and megestrol), and androgens (such as testosterone, testosterone propionate, fluoxymesterone, and methyltestosterone)), an adrenal

corticosteroid (such as, for example, prednisone, dexamethasone, methylprednisolone, and prednisolone), a leutinizing hormone releasing agent or gonadotropin-releasing hormone antagonist (such as, for example, leuprolide acetate and goserelin acetate), an antihormonal agent (such as, for example, tamoxifen), an antiandrogen agent (such as flutamide), an antiadrenal agent (such as mitotane and aminoglutethimide), and a natural product or derivative thereof (such as, for example, vinca alkaloids, antibiotics, enzymaes and epipodophyllotoxins, including, for example vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-C, paclitaxel,

mithramycin, deoxyco-formycin, mitomycin-C, L-asparaginase, and teniposide.

[00109] In some embodiments, a PTEN inhibitor is administered to a subject in combination with temozolomide (TEMODAR®) (e.g., for treating primary malignant brain tumors); or with docetaxel (TAXOTERE®) (e.g., for treating metastatic breast cancer).

[00110] In one embodiment, a PTEN inhibitor is administered to a subject in combination with cyclophosphamide. Cyclophosphamide (CTX, CYTOXAN®, or NEOSAR®) is an oxazahosphorine drug and analogs include ifosfamide (IFO, Ifex), perfosfamide, trophosphamide (trofosfamide; Ixoten), and pharmaceutically acceptable salts, solvates, prodrugs and metabolites thereof (U.S. 2007/0202077, which is specifically incorporated herein by reference in its entirety). Ifosfamide (MITOXANA®) is a structural analog of cyclophosphamide and its mechanism of action is considered to be identical or substantially similar to that of cyclophosphamide. Perfosfamide (4-hydroperoxycyclophosphamide) and trophosphamide are also alkylating agents, which are structurally related to

cyclophosphamide. For example, perfosfamide alkylates DNA, thereby inhibiting DNA replication and RNA and protein synthesis. New oxazaphosphorines derivatives have been designed and evaluated with an attempt to improve the selectivity and response with reduced host toxicity (Liang et al., Curr Pharm Des. 2007; 13(9):963-78). These include mafosfamide (NSC 345842), glufosfamide (D19575, beta-D-glucosylisophosphoramide mustard), S-(-)-bromofosfamide (CBM-11), NSC 612567 (aldophosphamide

perhydrothiazine) and NSC 613060 (aldophosphamide thiazolidine). Mafosfamide is an oxazaphosphorine analog that is a chemically stable 4-thioethane sulfonic acid salt of 4- hydroxy-CPA. Glufosfamide is IFO derivative in which the isophosphoramide mustard, the alkylating metabolite of IFO, is glycosidically linked to a beta-D-glucose molecule. Additional cyclophosphamide analogs are described in U.S. 5,190,929, which is specifically

incorporated herein by reference in its entirety.

[001 11] In human clinical trials where CTX has been used as an immunopotentiating agent, a dose of 300 mg/m² has usually been used. For an average male (6 ft, 170 pound (78 kg) with a body surface area of 1.98 m²), 300 mg/m² is 8 mg/kg, or 624 mg of total drug. In mouse models of cancer, efficacy has been seen at doses ranging from 15 - 150 mg/kg, which relates to 0.45 - 4.5 mg of total drug in a 30g mouse (Machiels et al. Cancer Res. 61 :3689-3697 (2001), Hengst et al Cancer Res. 41 :2163-2167 (1981), Hengst Cancer Res. 40:2135-2141 (1980)). The methods of the invention would allow a lower dose of CTX to be used in combination with a PTEN inhibitor without reducing its anti-cancer or anti-tumor efficacy. For example, CTX may be administered at a dose reduced at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% compared to the 8 mg/kg dose for an average male or compared to a 15-150 mg/kg dose for a mouse, when administered in combination with a PTEN inhibitor.

[001 12] For larger mammals, such as a primate, preferably human, patient, such mg/m2 doses may be used but unit doses administered over a finite time interval may be preferred. Such unit doses may be administered on a daily basis for a finite time period, such as up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days or up to 20 days or up to 25 days, are all specifically contemplated by the invention. The same regimen may be applied for the other potentiating agents recited herein.

[001 13] In other embodiments, the chemotherapeutic agent may be Sunitinib

(SUTENT®), 8ηίί-ΤΘΡβ, Imatinib (GLEEVAC®), a mitosis inhibitor (such as paclitaxel), an aromatase inhibitor (e.g. Letrozole) or an angiogenesis inhibitor (VEGF inhibitors e.g.

Avastin, VEGF-Trap) (see, for example, Li et al., Clin Cancer Res. 2006 Nov

15; 12(22):6808-16), anthracycline, oxaliplatin, doxorubicin, a TLR4 antagonist or an IL-18 antagonist.

[00114] Chemotherapeutic agents that kill tumor cells may be administered in combination with a PTEN inhibitor according to the methods of the invention. Non-limiting examples of such agent include imatinib, sunitinib, trastuzumab, cetuximab, gefitinib, erlotinib, panituzumab, bevacizumab, NEXAVAR^® (sorafenib), venurafinib, bortezomib, carfilzomib, lenolidomide and rituximab.

[00115] For example, one standard dose of sorafenib for treating cancer is 400 mg (2 x 200 mg tablets) taken twice daily. The methods of the invention would allow a lower dose of sorafenib to be used in combination with a PTEN inhibitor without reducing its anti-cancer or anti-tumor efficacy. For example, sorafenib may be administered at a dose reduced at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% compared to the 400 mg twice daily dose, when administered in combination with a PTEN inhibitor.

[00116] Furthermore, the methods of the invention would allow a lower dose of any one of adriamycin, cisplatin, carboplatin, oxaliplatin, cyclophosphamide, ifosfamide, temozolomide, gemcitabine, pactilaxel, docetaxel or etoposide to be used in combination with a PTEN inhibitor without reducing the chemotherapeutic agent's anti-cancer or antitumor efficacy. For example, adriamycin, cisplatin, carboplatin, oxaliplatin,

cyclophosphamide, ifosfamide, temozolomide, gemcitabine, pactilaxel, docetaxel or etoposide may be administered at a dose reduced at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% compared to the daily or aggregate standard dose of the corresponding chemotherapeutic agent shown in Table 1 , when administered in combination with a PTEN inhibitor.

Table 1. Examples of standard doses of selected chemotherapeutic agents

carboplatin

400 1 ,2 21

(sarcoma)

oxaliplatin 85-130 1 21

cyclophosphamide 300-600* first 2-5 days 21-28

ifosfamide 1800-2400** first 3-5 days 21-28

temozolomide 200 1 ,2,3,4,5 28

gemcitabine 1000-1250 1 ,8 21

gemcitabine 1000-1250 1 ,8, 15 28

pactilaxel 135 1 21

docetaxel 100 1 21

etoposide 50 1 ,2,3,4,5 21-28

etoposide 100 1 ,2,3 21-28

*aggregate dose 1500-1800 mg/m^A2/cycle

**aggregate dose 1500-1800 mg/m^A2/cycle

GCT = germ cell tumor / testicular cancer

[00117] In some embodiments, the disclosed methods involve co-administration with a PD-1 antagonist. PD-1 antagonists, also referred to herein as inhibitors of the PD-1/PD-L pathway, include, but are not limited to, antibodies, peptides, nucleic acid molecules

(including, for example, an antisense molecule, a PNA, or an RNAi), peptidomimetics, small molecules, a soluble PD-1 ligand polypeptide, or a chimeric polypeptide (for example, a chimeric PD-1 ligand/lmmunoglobulin molecule). An antibody may be an intact antibody, an antibody binding fragment, or a chimeric antibody. A chimeric antibody may include both human and non-human portions. An antibody may be a polyclonal or a moncoclonal antibody. An antibody may be a derived from a wide variety of species, including, but not limited to mouse and human. An antibody may be a humanized antibody. An antibody may be linked to another functional molecule, for example, another peptide or protein, a toxin, a radioisotype, a cytotoxic agent, cytostatic agent, a polymer, such as, for example, polyethylene glycol, polypropylene glycol or polyoxyalkenes.

[00118] Programmed Death-1 (PD-1) is a member of the CD28 family of receptors that delivers a negative immune response when induced on T-cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that decreases T-cell multiplication and/or the strength and/or duration of a T-cell response. Suitable PD-1 antagonists are described in U.S. 8, 114,845, U.S. 8,609,089, and U.S. 8,709,416, each of which is specifically incorporated by reference herein in its entirety, and include compounds or agents that either bind to and block a ligand of PD-1 to interfere with or inhibit the binding of the ligand to the PD-1 receptor, or bind directly to and block the PD-1 receptor without inducing inhibitory signal transduction through the PD-1 receptor.

[00119] In some embodiments, the PD-1 receptor antagonist binds directly to the PD- 1 receptor without triggering inhibitory signal transduction and also binds to a ligand of the PD-1 receptor to reduce or inhibit the ligand from triggering signal transduction through the PD-1 receptor. By reducing the number and/or amount of ligands that bind to PD-1 receptor and trigger the transduction of an inhibitory signal, fewer cells are attenuated by the negative signal delivered by PD-1 signal transduction and a more robust immune response can be achieved.

[00120] It is believed that PD-1 signaling is driven by binding to a PD-1 ligand (such as B7-H1 or B7-DC) in close proximity to a peptide antigen presented by major

histocompatibility complex (MHC) (see, for example, Freeman, Proc. Natl. Acad. Sci. U. S. A, 105: 10275-10276 (2008)). Therefore, proteins, antibodies or small molecules that prevent co-ligation of PD-1 and TCR on the T-cell membrane are also useful PD-1 antagonists.

[00121] In preferred embodiments, the PD-1 receptor antagonists are small molecule antagonists or antibodies that reduce or interfere with PD-1 receptor signal transduction by binding to ligands of PD-1 or to PD-1 itself, especially where co-ligation of PD-1 with TCR does not follow such binding, thereby not triggering inhibitory signal transduction through the PD-1 receptor.

[00122] Other PD-1 antagonists contemplated by the methods of this invention include antibodies that bind to PD-1 or ligands of PD-1 , and other antibodies. Suitable anti- PD-1 antibodies include, but are not limited to, those described in the following publications: WO 2003/099196; WO 2006/121168; WO 2009/014708; WO 2004/004771 ; WO

2004/072286; WO 2004/056875; WO 2008/083174; WO 2007/005874; WO 2009/073533; and Berger et al., Clin. Cancer Res., 14:30443051 (2008); each of which is specifically incorporated by reference herein in its entirety.

[00123] A specific example of an anti-PD-1 antibody is an antibody described in US 2007/0166281 at par. 42, a human anti-PD-1 antibody, preferably administered at a dose of 3 mg/kg.

[00124] Exemplary anti-B7-H1 antibodies include, but are not limited to, those described in the following publications: WO 2006/133396; WO 2008/083174; and US 2006/01 10383; each of which is specifically incorporated by reference herein in its entirety. [00125] A specific example of an anti-B7-H1 antibody is an antibody described in WO/2007/005874, a human anti-B7-H1 antibody.

[00126] Additional anti-PD-1 and anti-B7-H1 antibodies are disclosed in U.S.

2014/0044738, which is specifically incorporated by reference herein in its entirety.

[00127] For anti-B7-DC antibodies see U.S. 7,411 ,051 , U.S. 7,052,694, U.S.

7,390,888, and U.S. 2006/0099203, each of which is specifically incorporated by reference herein in its entirety.

[00128] Other exemplary PD-1 receptor antagonists include, but are not limited to B7- DC polypeptides, including homologs and variants of these, as well as active fragments of any of the foregoing, and fusion proteins that incorporate any of these. In one embodiment, the fusion protein includes the soluble portion of B7-DC coupled to the Fc portion of an antibody, such as human IgG, and does not incorporate all or part of the transmembrane portion of human B7-DC.

[00129] The PD-1 antagonist can also be a fragment of a mammalian B7-H1 , preferably from mouse or primate, preferably human, wherein the fragment binds to and blocks PD-1 but does not result in inhibitory signal transduction through PD-1. The fragments can also be part of a fusion protein, for example an Ig fusion protein.

[00130] Other useful polypeptides PD-1 antagonists include those that bind to the ligands of the PD-1 receptor. These include the PD-1 receptor protein, or soluble fragments thereof, which can bind to the PD-1 ligands, such as B7-H1 or B7-DC, and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction. B7-H1 has also been shown to bind the protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 1 11-122, (2007)). Such fragments also include the soluble ECD portion of the PD-1 protein that includes mutations, such as the A99L mutation, that increases binding to the natural ligands (Molnar et al., PNAS, 105:10483-10488 (2008)). B7-1 or soluble fragments thereof, which can bind to the B7-H1 ligand and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction, are also useful.

[00131] PD-1 and B7-H1 anti-sense nucleic acids, both DNA and RNA, as well as siRNA molecules can also be PD-1 antagonists. Such anti-sense molecules prevent expression of PD-1 on T-cells as well as production of T-cell ligands, such as B7-H1 , PD-L1 and/or PD-L2. For example, siRNA (for example, of about 21 nucleotides in length, which is specific for the gene encoding PD-1 , or encoding a PD-1 ligand, and which oligonucleotides can be readily purchased commercially) complexed with carriers, such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 1 19(8): 2231-2244 (2009), are readily taken up by cells that express PD-1 as well as ligands of PD-1 and reduce expression of these receptors and ligands to achieve a decrease in inhibitory signal transduction in T-cells, thereby activating T-cells.

[00132] One or more PD-1 antagonists may include a combination of PD-1 antagonists. For example, one or more inhibitors of PD-1 , one or more inhibitors of PD-L1 , and/or one or more inhibitors of PD-L2 may be administered. One or more of such inhibitors may be an antibody. For example, to inhibit the PD-1/PD-L pathway a mixture of inhibitors of PD-1 , PD-L1 , and/or PD-L2 may be used in combination. In some embodiments, one or more inhibitors of PD-1 and one or more inhibitors of PD-L1 may be administered. In some embodiments, one or more inhibitors of PD-1 and one or more inhibitors of PD-L2 may be administered. In some embodiments, one or more inhibitors of PD-1 , one or more inhibitors of PD-L1 , and one or more inhibitors of PD-2 may be administered. A mixture or cocktail of inhibitors of the PD-1/PD-L pathway may be administered. For example, a cocktail of antibodies to PD-1 , PD-L1 , and/or PD-L2 may be administered.

[00133] Examples of suitable PD-1 pathway antagonists are provided in U.S.

2011/0223188, U.S. 201 1/0195068, U.S. 201 1/0159023, U.S. 2012/0114649, and U.S. 2013/0017199, each of which is specifically incorporated by reference herein in its entirety.

[00134] Other molecules useful in mediating the effects of T-cells in an immune response are also contemplated as additional therapeutic agents used in the methods and compositions of the invention. In one embodiment, the molecule is an antagonist of CTLA4, for example an antagonistic anti-CTLA4 antibody. CTLA4 (Cytotoxic T-Lymphocyte Antigen 4) is a CD28-family receptor expressed on CD4+ T-cells. It binds the same ligands as CD28 (CD80 and CD86 on B cells and dendritic cells), but with higher affinity than CD28. In contrast to CD28, which enhances cell function when bound at the same time as the T-cell receptor, CTLA4 inhibits T-cell functioning. CTLA4 blockade releases inhibitory controls on T-cell activation and proliferation, inducing antitumor immunity in both preclinical and early clinical trials (Quezada et al., 2006, J Clin Invest; 1 16: 1935-1945, U.S. Pat. No. 7,229,628).

[00135] Blockade of CTLA4 with anti-CTLA4 antibodies can induce rejection of several types of established transplantable tumors in mice, including colon carcinoma, fibrosarcoma, prostatic carcinoma, lymphoma, and renal carcinoma (Leach et al., 1996, Science; 271 : 1734-1736; Kwon et al., 1997, Proc Natl Acad Sci USA; 94:8099-8103; Yang et al., 1997, Cancer Res; 57:4036-4041 ; Shrikant et al., 1999, Immunity; 1 1 :483-493; and Sotomayor et al., 1999, Proc Natl Acad Sci USA; 96: 11476-1 1481). Fully human anti- CTLA4 are being used in clinical trials with patients with melanoma or ovarian cancer (Hodi et al., 2003, Proc Natl Acad Sci USA; 100:4712-471717; Ribas et al., 2004, J Immunother; 27:354-367; and Phan et al., 2003, Proc Natl Acad Sci USA 100:8372-8377). [00136] Antibodies to block CTLA4 (such as Medarex MDX0101) have been the subject of clinical trials (see, for example, Peggs et al., 2006, Curr Opin Immunol; 18:206- 213). Other suitable anti-CTLA4 antibodies include those described in WO 2007/056539. Despite great potential, some anti-CTLA4 antibodies only show anti-tumor efficacy at doses that are toxic, due to development of nonspecific autoimmunity. In some embodiments, immunomodulators such as anti-CTLA4 can be administered at a lower, less toxic dosage when co-administered with the disclosed PTEN inhibitor compositions.

[00137] Dosages for anti-PD-1 , anti-B7-H1 , and anti-CTLA4 antibody, are known in the art and can be in the range of 0.1 to 100 mg/kg, with shorter ranges of 1 to 50 mg/kg preferred and ranges of 10 to 20 mg/kg being more preferred. An appropriate dose for a human subject is between 5 and 15 mg/kg, with 10 mg/kg of antibody (for example, human anti-PD-1 antibody) most preferred.

[00138] Specific examples of an anti-CTLA4 antibody useful in the methods of the invention are Ipilimumab, a human anti-CTLA4 antibody, preferably administered at a dose of about 10 mg/kg, and Tremelimumab a human anti-CTLA4 antibody, preferably administered at a dose of about 15 mg/kg. See also Sammartino, et al., Clinical Kidney Journal, 3(2): 135-137 (2010), published online December 2009.

[00139] In other embodiments, the antagonist is a small molecule. A series of small organic compounds have been shown to bind to the B7-1 ligand to prevent binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368 (2002). Such small organics could be administered alone or together with an anti-CTLA4 antibody to reduce inhibitory signal transduction of T-cells.

[00140] In a specific combination, a PTEN inhibitor is administered to a subject in combination with ipilimumab (YERVOY^®) (e.g., to treat melanoma).

[00141] The invention provides compositions comprising a PTEN inhibitor and a chemotherapeutic (e.g., antineoplastic) agent for improving the efficacy of anti-tumor immunotherapy are provided. The compositions are typically pharmaceutical compositions including an effective amount of a PTEN-inhibitor drug. The compositions may additionally comprise a vaccine directed against a tumor or a cancer antigen.

[00142] Pharmaceutical compositions can be supplied as a kit comprising a container that comprises the pharmaceutical composition as described herein. A pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection.

Alternatively, such a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a pharmaceutical composition. Such a kit can further comprise written information on indications and usage of the pharmaceutical composition.

[00143] The disclosure will be further clarified by the following examples, which are intended to be purely exemplary of the disclosure and in no way limiting.

EXAMPLES

Example 1 : General methods

[00144] The B16F10, LLC, EL4 and E.G7 cell lines were obtained from the American

Type Culture Collection (Manassas, VA). B16-OVA is the B16F10 cell line transfected with full-length chicken ovalbumin, clone M04 (see, Falo et al., Nat. Med. 1 , 649-653 (1995)).

Tumor implantation was performed as described previously (Sharma et al., J. Clin. Invest.

117, 2570-2582 (2007)), using 1 x10⁵ cells for B16F10 and 1 x10⁶ cells for other cell lines

(large inocula were used to ensure rapid tumor engraftment and immune suppression).

Tumor volume was calculated from orthogonal diameters using the formula

Mice received approved euthanasia when tumors reached a size of 300 mm²; death was not used a planned endpoint in any study.

[00145] OT-I mice (CD8⁺, recognizing the SIINFEKL (SEQ ID NO: 1) peptide of ovalbumin (OVA) on H2K (Hogquist et al., Cell 76, 17-27 (1994).)); and pmel-1 mice, B6.Cg-T/7y7^a/CyTg(TcraTcrb)8Rest/J, recognizing a peptide from human gp100 (Overwijk et al., J. Exp. Med. 198, 569-580 (2003)), were obtained from Jackson Laboratory, Bar Harbor, ME. CD8⁺ effector cells were FACS-sorted from spleens of OT-I or pmel-1 mice.

[00146] CpG-1826 (phosphorothioate oligo 5'-TCCATGACGTTCCTGAGCTT-3' (SEQ ID NO:2)) was synthesized from the published sequence (Chu et al., J. Exp. Med. 186, 1623-1631 (1997)). Whole OVA protein was obtained from Sigma (#A-5503). Human gp100₂₅-33 (KVPRNQDWL (SEQ ID NO:3)) was synthesized from the published sequence (Overwijk et al., J. Exp. Med. 198, 569-580 (2003)). Vaccines were prepared with 100 μg of OVA protein or 25 μg peptide, with 50 μg CpG-1826 in incomplete Freund's adjuvant (I FA, Sigma F-5506) and administered in the hind-limb footpad. Popliteal lymph nodes (LNs) were harvested 4 days later.

[00147] To assess responses to the vaccines, mice received a carboxyfluorescein succinimidyl ester (CFSE)-labeled cohort of resting CD8⁺ pmel-1 transgenic T-cells

(recognizing a peptide from the human gp100) or CFSE-labeled OT-I cells (recognizing a peptide of OVA). To prevent nonspecific activation, responder cells were always purified by negative selection. For all adoptive transfers, OT-I or pmel-1 spleen cells were enriched by negative selection using magnetic beads (mouse CD8 isolation kit II, #130-095-236, Miltenyi Biotech). Staining for bead isolation was performed on ice, with short incubation times. Mice received 2* 10⁶ enriched CD8⁺ cells via tail-vein. The labeled vaccine-specific resting T-cells were used to determine whether tumor-bearing hosts contained a new, systemic population of activated Tregs in response to vaccine treatment. After vaccine treatment, the transferred T-cells were stained for the differentiation marker granzyme B (GzmB) and CFSE dye dilution and analyzed by FACS. CFSE dye dilution allows tracing of multiple

generations of cells. In mice without B16F10 tumors, immunization with cognate antigen generated a robust response in vaccine-draining lymph nodes (VDLNs), driving proliferation of pmel-1 T-cells and up-regulation of the differentiation marker granzyme B. However, the presence of a growing B16F10 tumor, even at a remote site, caused progressive loss of response to vaccine. A second tumor type, E.G7 lymphoma, also showed tumor-induced inhibition of vaccine response at distant sites.

[00148] VO-OHpic (Biovision #1801-5) was used at 1 μΜ in vitro unless otherwise specified and at 10 mg/kg/d in vivo, administered in 10% DMSO.

[00149] For FACS staining, lymph nodes were prepared by rapidly passing through a 40 μηι mesh, then stained using short incubation times (10 min on ice), as described (Sharma et al., Immunity 38, 998-1012 (2013)). Tumors were disaggregated by treating for 1 hr with 1 mg/mL collagenase (C5138, Sigma), 0.1 mg/mL DNAse (D5025, Sigma), and 0.1 mg/mL hyaluronidase (H3884, Sigma) in RPMI 1640 medium.

[00150] The following conjugated mAbs were obtained from BD-Pharmingen against: CD86 (clone GL1); CD1 1c (clone HL3); Ly6c (clone AL-21). Conjugated antibodies obtained from eBioscience were against: Foxp3 (clone FJK-16s); granzyme B (clone NGZB); PD-L1 (clone MIH5); CD103 (Ber-ACT8) and Ly6c (clone HK1.4).

[00151] Intracellular antigens were detected using fixation-permeabilization reagent and matching perm-wash buffer from eBioscience (Cat. #00-5521), with blocking using 5% normal donkey serum, then acquired immediately after staining. Unconjugated anti-Fox03a (rabbit mAb, clone 75D8, Cell Signaling Technology) was used at 4 μg/ml in perm-wash buffer, and was detected with donkey-anti-rabbit-PE (Jackson ImmunoResearch #711-116- 152) 1 : 100 dilution. All washes were in perm-wash buffer in the cold.

[00152] Multiple treatment groups were compared by ANOVA with Tukey's HSD correction. Where given, error bars show standard deviation.

Example 2: Rapid reconfiguration of the suppressive tumor milieu when PTEN is inhibited

[00153] The effect of pharmacologic inhibition of PTEN on an existing,

immunosuppressive tumor microenvironment was tested. B16F10 tumors were grown in WT (wild-type) hosts, then treated with pmel-1 T-cells and hgp100 vaccine, with or without PTEN inhibitor VO-OHpic (10 mg/kg) (Fig. 1A). In the absence of VO-OHpic, the pmel-1/vaccine treatment had little effect on these large established tumors. However, if PTEN was blocked then pmel-1/vaccine became able to drive rapid tumor involution. In the case of E.G7 tumors (Fig. 1 B), which bear a strong OVA (ovalbumin) xenoantigen, treatment with high- affinity OT-I cells plus OVA vaccine was sufficient to slow tumor growth. However, in order to shrink the size of a large established tumor, addition of VO-OHpic (10 mg/kg) was required.

[00154] In both models, the enhanced anti-tumor response was accompanied by widespread reconfiguration of the intratumoral milieu (Fig. 1C). B16F10 tumors were treated as in Fig. 1A, then disaggregated on day 4 after vaccine and stained as shown. The results are representative of 5 experiments using both B16F10 and E.G7 tumors. This included loss of Fox03a expression in Tregs; proliferation of CFSE-labeled effector cells within the tumor; and emergence of activated Ly6c⁺CD1 1 b⁺ myeloid DCs (dendritic cells). Sorting studies on these DCs showed that they were able to robustly cross-present endogenous tumor antigen acquired in vivo (Fig. 1 D). B16-OVA tumors bearing a nominal ovalbumin transgene were treated with pmel-1/vaccine, with or without VO-OHpic. The Ly6c^NEG or Ly6c⁺ fraction of DCs was sorted as shown, and tested for ability to present endogenous OVA antigen to OT-I responder cells in vitro. In contrast, when control Ly6c^NEG DCs were isolated from tumors receiving the same pmel-1 /vaccine treatment but without VO-OHpic, these DCs were suppressive and did not cross-present endogenous tumor antigens effectively. Thus, taken together, these data showed that it was possible to reconfigure an established, suppressive tumor microenvironment into an immunogenic milieu when PTEN was blocked.

Example 3: Inhibition of PTEN fundamentally alters the response to chemotherapy

[00155] If PTEN was blocked at the time of chemotherapy, even a modest dose of chemotherapy became able to trigger rapid and prolonged regression of established tumors

(Fig. 2A). Mice with established B16F10 tumors were treated with a single dose of cyclophosphamide (CTX, 150 mg/kg) or VO-OHpic (10 mg/kg/d) or both, as shown.

Chemotherapy by itself was not effective, and VO-OHpic alone had no effect; but together the two agents displayed striking synergy. Consistent with the need to "de-activate" an existing pool of pre-activated, PTEN-dependent Tregs, the initial tumor regression was influenced primarily by the PTEN pathway.

[00156] Analysis of the tumor microenvironment showed that treatment with chemotherapy alone had little effect on the suppressive milieu, which remained dominated by Fox03a⁺ Tregs and PD-L1⁺ DCs (Fig. 2B). However, addition of PTEN inhibitor abrogated Fox03a expression in Tregs, down-regulated PD-L1 , and increased the number of inflammatory Ly6c⁺CD11 b⁺ CD1 1c⁺ DCs. Of note, this new population of Ly6c⁺CD11 b⁺ DCs also expressed the integrin marker CD103. A similar CD103⁺ phenotype has recently been associated with pro-inflammatory and cross-presenting DCs in the tumor

microenvironment (Broz et al., Cancer Cell 26, 638-652 (2014); Ruffell et al., Cancer Cell 26, 623-637 (2014)). The contribution of the adaptive immune response was required for tumor regression (Fig. 2C). In the absence of an adaptive immune system (Rag1-KO mice), the anti-tumor effect of CTX+VO-OHpic was lost.

Example 4: Pre-vaccination and PTEN inhibition improve effect of chemotherapy

[00157] Combining pre-vaccination with PTEN inhibition and chemotherapy reduced volume of Lewis Lung Carcinoma (LLC) tumors transfected with the nominal antigen (LLC- gplOO tumors) to a greater extent than the combination of any two of these treatments (Fig. 3). As shown in Fig. 3, groups of mice with established LLC-gp100 tumore were treated with various combinations of vaccinattion with hgp100/CpG/IFA (incomplete Freund's adjuvant), CTX (cyclophosphamide; 150 mg/kg) and VO-OHpic. The group treated with vaccine, VO- OHpic and CTX showed a slower tumor growth than the groups treated with vaccine and CTX; vaccine and VO-OHpic; or VO-OHpic and CTX.

Example 5: Inhibition of PTEN allows reduction in chemotherapeutic agent dose

[00158] PTEN inhibition allowed the use of lower doses of chemotherapy in mice with established B16F10 tumors (Figs. 4A and 4B). The combination of a PTEN inhibitor (VO- OHpic) and a chemotherapeutic agent (cyclophosphamide (CTX)) created a greater anti- tumor effect than could be achieved even by a larger dose of chemotherapy alone. Mice with B16F10 tumors were treated with 0 or 10 mg/kg VO-OHpic at days 9, 10, 1 1 , 12, and 13 and with 0, 25, 50, or 150 mg/kg CTX at day 10, as shown in the table in Fig. 4B.

Surprisingly, tumor regression was seen with only 50 mg/kg of CTX (when PTEN-inhibitor was given). This result is advantageous in chemotherapeutic treatment because 50 mg/kg of CTX is a minimally-toxic dose and much lower than the maximum tolerated dose (MTD) in mice. The PTEN inhibitor increased the efficacy of low-dose CTX into the type usually seen with high-dose CTX as a single agent (e.g., 3- to 10-fold higher).

Example 6: Absence of PTEN-Tregs contributes to failure to creat a suppressive tumor microenvironment Treg

[00159] The effect of PTEN -KO hosts on tumor growth was tested. Mice with a BAC-transgenic GFP-Cre fusion protein under the Foxp3 promoter (Foxp3^GFP'CRE) were obtained from Jackson Laboratories (NOD/ShiLt- Tg(Foxp3-EGFP/cre)1Jbs/J) (Zhou et al., J. Exp. Med. 205, 1983-1991 (2008); Zhou et al., Nat. Immunol. 10, 1000-1007 (2009)) and back-crossed onto the B6 background. These were used for intercrosses with floxed alleles

Treg „_FP„

to create PTEN -KO mice, as follows. Foxp3 mice were crossed with mice bearing loxP sites flanking exon 5 of the PTEN gene (Lesche et al., Genesis 32, 148-149 (2002)) (B6.129S4-Pten ^im1Hw J, Jackson Laboratories). The resulting strain was maintained as hemizygous for the GFP-Cre and homozygous for pten^loxP/loxP.

Treg

[00160] Aggressive melanoma tumors implanted in PTEN -KO hosts grew much slower than the same tumors implanted in wild-type (WT) parental strains (Fig. 5A). Slower growth was also seen with E.G7 (EL4-OVA) and LLC tumors. Analysis of immune cells

+

infiltrating the tumors (Fig. 5B) showed that WT hosts contained many PTEN Tregs that also co-expressed Fox03a and PD-1 , consistent with a suppressive phenotype. In contrast, the

Treg

Tregs in PTEN -KO tumors did not express Fox03a or PD-1 ; instead, they appeared unstable, with many expressing pro-inflammatory markers such as IL-2, CD40L and IL-17 (Fig. 5B, lower panels). All of these "re-programmed" Tregs continued to express residual Foxp3 (Fig. 5B, bottom graph) thus showing that they derived from former Tregs.

Treg

[00161] The tumor-infiltrating CD8 T cells in PTEN -KO hosts appeared more activated (Fig. 5C). Fewer CD8⁺ cells were PD-1⁺ (implying an "exhausted" phenotype) and more had an activated phenotype expressing CD103⁺, CD69⁺ and IFNv. Dendritic cells

Treg

(DCs) in tumors from PTEN -KO mice also showed a more activated phenotype (Fig. 5D). More DCs expressed an activated myeloid DC phenotype of Ly6c⁺CD1 1 b⁺CD103⁺, which has been associated with anti-tumor immune surveillance. Many of these CD103⁺ DCs produced IL-6 (bottom panels). This was significant because IL-6 is a key driver of Treg reprogramming. Similar changes were seen when E.G7 lymphoma tumors were grown in

PTEN^Tre9-KO hosts (Figs. 6A, 6B, and 6C).

Claims

CLAIMS:

1. A method of reducing tumor volume in a subject in need thereof, the method comprising administering a phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase (PTEN) inhibitor, an antineoplastic chemotherapeutic agent and a vaccine to the subject, wherein the vaccine targets or comprises a tumor antigen.

2. A method of treating cancer in a subject in need thereof, the method comprising administering a phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase (PTEN) inhibitor, an antineoplastic chemotherapeutic agent and a vaccine to the subject, wherein the vaccine targets or comprises a tumor antigen.

3. The method of claim 1 or 2, wherein the method comprises:

(i) administering the vaccine to the subject; and

(ii) after step (i), administering the PTEN inhibitor and the antineoplastic

chemotherapeutic agent to the subject.

4. The method of claim 1 or 2, wherein the method comprises:

(i) administering the PTEN inhibitor and the vaccine to the subject; and

(ii) after step (i), administering the antineoplastic chemotherapeutic agent to the subject.

5. The method of claim 3 or 4, wherein step (ii) is performed at least about one week, at least about two weeks, at least about three weeks or at least about four weeks after step

6. The method of claim 3 or 5, wherein the PTEN inhibitor and the chemotherapeutic agent are administered to the subject concurrently or sequentially.

7. The method of claim 4 or 5, wherein the PTEN inhibitor and the vaccine are administered to the subject concurrently or sequentially.

8. The method of any one of claims 3-7, wherein the method comprises:

(i) administering the PTEN inhibitor and the vaccine to the subject; and

(ii) after step (i), administering the PTEN inhibitor and the antineoplastic

chemotherapeutic agent to the subject.

9. The method of claim 8, wherein the same PTEN inhibitor is administered to the subject in steps (i) and (ii).

10. The method of any one of claims 1-9, wherein the vaccine targets or comprises an antigen from a lung tumor, a breast tumor, an ovarian tumor, a brain tumor, a pancreatic tumor, a colon tumor or a melanoma tumor.

11. The method of claim 10, wherein the vaccine targets or comprises human gp100, NY-ESO-1 , Mud or EGFR-vlll.

12. The method of any one of claims 1-11 , wherein the PTEN inhibitor is a small molecule, a nucleic acid or a protein.

13. The method of any one of claims 1-12, wherein the PTEN inhibitor is N-(9, 10- Dioxo-9, 10-dihydrophenanthren-2-yl)-2,2-dimethylpropionamide; or 3,4-Dephostatin, ethyl-.

14. The method of any one of claims 1-12, wherein the PTEN inhibitor is a vanadium complex.

15. The method of claim 14, wherein the PTEN inhibitor is VO-OHpic.

16. The method of any one of claims 1 and 3-15, wherein the tumor is a refractory tumor.

17. The method of any one of claims 1 and 3-16, wherein the tumor is a lung tumor, a breast tumor, an ovarian tumor, a brain tumor, a pancreatic tumor, a colon tumor or a melanoma tumor.

18. The method of any one of claims 2-15, wherein the cancer is lung cancer, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, colon cancer or melanoma.

19. The method of any one of claims 1-18, wherein the method further comprises

(iii) at least about one week, at least about two weeks, at least about three weeks or at least about four weeks after step (ii), administering the vaccine and, optionally, the PTEN inhibitor to the subject; and

(iv) after step (iii), administering the chemotherapeutic agent and, optionally, the PTEN inhibitor to the subject.

20. The method of claim 19, wherein step (iv) is performed at least about one week, at least about two weeks, at least about three weeks or at least about four weeks after step (iii).

21. The method of claim 19 or 20, wherein the PTEN inhibitor and the

chemotherapeutic agent of step (iii) are administered to the subject concurrently or sequentially.

22. The method of claim 19 or 20, wherein the PTEN inhibitor and the chemotherapeutic agent of step (iv) are administered to the subject concurrently or sequentially.

23. The method of claim 19, wherein the same PTEN inhibitor is administered to the subject in steps (iii) and (iv).

24. A method of reducing tumor volume in a subject in need thereof, the method comprising administering a phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase (PTEN) inhibitor and a chemotherapeutic agent to the subject, wherein the chemotherapeutic agent is administered at a reduced dose compared to the standard dose used to reduce tumor volume when the chemotherapeutic agent is administered without any PTEN inhibitor.

25. The method of claim 24, wherein the PTEN inhibitor and the chemotherapeutic agent are administered to the subject concurrently or sequentially.

26. The method of claim 24 or 25, wherein the chemotherapeutic agent is administered at a dose reduced at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% compared to the standard dose used to reduce tumor volume when the chemotherapeutic agent is administered without any PTEN inhibitor.

27. A method of treating cancer in a subject in need thereof, the method comprising administering a phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase (PTEN) inhibitor and a chemotherapeutic agent to the subject, wherein the chemotherapeutic agent is administered at a reduced dose compared to the standard dose used to treat cancer when the chemotherapeutic agent is administered without any PTEN inhibitor.

28. The method of claim 27, wherein the PTEN inhibitor and the chemotherapeutic agent are administered to the subject concurrently or sequentially.

29. The method of claim 27 or 28, wherein the chemotherapeutic agent is administered at a dose reduced at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% compared to the standard dose used to treat cancer when the chemotherapeutic agent is administered without any PTEN inhibitor.

30. The method of any one of claims 24-29, wherein the PTEN inhibitor and the chemotherapeutic agent are administered to the subject in multiple cycles.

31. The method of claim of claim 30, wherein the PTEN inhibitor and the

chemotherapeutic agent are administered to the subject about every two weeks, about every ten days, about every one week, about every six days, about every five days, about every four days, about every three days, about every two days or about every one day.