METHODS FOR ENHANCING THE EFFICACY OF A TUMOR-DIRECTED IMMUNE RESPONSE

Abstract

As described below, the present invention features methods for enhancing the efficacy of a tumor antigen in inducing an anti-cancer immune response in a subject by administering an OX40 agonist and an Indoleamine 2,3-dioxygenase (IDO) inhibitor with the tumor antigen.

Claims

1. A method for enhancing an immune response against a tumor antigen in a subject, the method comprising administering to the subject an OX40 agonist, an Indoleamine 2,3-dioxygenase (IDO) inhibitor, and an immunogenic composition comprising a tumor antigen, thereby enhancing the subject's immune response against the tumor antigen relative to administration of the immunogenic composition alone.

2. A method for delaying or reducing tumor growth in a subject, the method comprising administering to the subject an OX40 agonist, an Indoleamine 2,3-dioxygenase (IDO) inhibitor, and an immunogenic composition comprising a tumor antigen, thereby delaying or reducing tumor growth in the subject relative to an untreated control subject.

3. A method for enhancing an immune response against a tumor antigen in a subject, the method comprising administering to the subject radiation or a chemotherapeutic sufficient to induce tumor cell apoptosis, an OX40 agonist, and an Indoleamine 2,3-dioxygenase (IDO) inhibitor, thereby enhancing the subject's immune response against the tumor antigen relative to administration of the radiation or a chemotherapeutic alone.

4. A method for delaying or reducing tumor growth in a subject, the method comprising administering to the subject radiation or a chemotherapeutic sufficient to induce tumor cell apoptosis, an OX40 agonist, and an Indoleamine 2,3-dioxygenase (IDO) inhibitor, thereby delaying or reducing tumor growth in the subject relative to an untreated control subject.

5. The method of any of claims 1-4, wherein the method increases the ratio of CD8.sup.+ T cells to regulatory T cells within a tumor of the subject.

6. The method of any of claims 1-4, wherein the amount of OX40 agonist administered is sufficient to increase the CD8.sup.+ T cells to regulatory T cell ratio within a tumor of the subject.

7. The method of any of claims 1-4, wherein the amount of OX40 agonist administered is sufficiently low so as not to increase tumor-infiltrating regulatory T cells in the subject.

8. A method for increasing the CD8.sup.+ T cells to regulatory T cell ratio within a tumor in a subject, the method comprising administering to the subject an effective amount of an OX40 agonist, an Indoleamine 2,3-dioxygenase (IDO) inhibitor, and an immunogenic composition comprising a tumor antigen, thereby increasing the CD8/Treg ratio with the tumor.

9. A method for increasing the CD8.sup.+ T cells to regulatory T cell ratio within a tumor in a subject, the method comprising administering to the subject radiation or a chemotherapeutic sufficient to induce tumor cell apoptosis, an effective amount of an OX40 agonist, and an Indoleamine 2,3-dioxygenase (IDO) inhibitor, thereby increasing the CD8/Treg ratio with the tumor.

10. The method of any of claims 1-9, wherein the effective amount of OX40 agonist administered is sufficiently low so as not to increase tumor-infiltrating Treg cells in the subject.

11. The method of any of claims 1-9, wherein the administration increases the subject's anti-tumor immune response relative to the administration of the immunogenic composition alone.

12. The method of any of claims 1-11, wherein the Indoleamine 2,3-dioxygenase (IDO) inhibitor is 1-methyltryptophan (1-MT), the D isomer of 1-methyl-tryptophan, or NLG919.

13. The method of claim 12, wherein the IDO inhibitor is 1-MT.

14. The method of any of claims 1-13, wherein the OX40 agonist specifically binds OX40.

15. The method of any of claims 1-14, wherein the OX40 agonist is an antibody that specifically binds OX40 or an antigen-binding fragment thereof.

16. The method of claim 15, wherein the antibody or antigen binding fragment thereof is a monoclonal antibody.

17. The method of claim 16, wherein the antibody or antigen binding fragment thereof is a chimeric antibody.

18. The method of claim 16, wherein the antibody or antigen binding fragment thereof is a humanized antibody.

19. The method of claim 16, wherein the antibody or antigen-binding fragment thereof binds to the same OX40 epitope as mAb 9B12.

20. The method of any of claims 1-16, wherein the tumor antigen is selected from the group consisting of alpha fetoprotein, carcinoembryonic antigen, cdk4, beta-catenin, CA125, caspase-8, epithelial tumor antigen, an HPV antigen, HPV16 antigen, CTL epitope from HPV16 E7 antigen, melanoma associated antigen (MAGE)-1, MAGE-3, tyrosinase, surface Ig idiotype, Her-2/neu, MUC-1, prostate specific antigen (PSA), sialyl Tn (STn), heat shock proteins, gp96, ganglioside molecules GM2, GD2, GD3, carcinoembryonic antigen (CEA) and MART-1.

21. The method of claim 20, wherein the amount of tumor antigen is sufficient to induce an anti-cancer immune response in the subject.

22. The method of claim 20, wherein the method stimulates T-lymphocyte activity in the subject.

23. The method of any of claims 1-22, wherein the immunogenic composition further comprises an adjuvant.

24. The method of any of claims 1-23, wherein the subject has a cancer selected from the group consisting of an HPV-associated cancer, cervical cancer, penile cancer, anal cancer, squamous cell carcinoma of the head and neck and cancer of the vulvar.

25. The method of any of claims 1-24, wherein the method increases subject survival by at least 10%, 20%, or 30% relative to a subject that received administration of the immunogenic composition only.

26. The method of any of claims 1-25, wherein the method reduces tumor growth by at least about 20% relative to tumor growth in an untreated control subject or induces tumor regression.

27. The method of any of claims 1-25, wherein the subject is a human patient.

28. The method of any of claims 1-25, wherein the immunogenic composition is a cancer vaccine.

29. The method of any of claims 1-28, wherein the chemotherapeutic is selected from the group consisting of anthracyclines or oxaliplatin.

30. The method of any of claims 1-29, wherein the anthracycline is daunorubicin, doxorubicin, epirubicin, idarubicin, or valrubicin.

31. A method for enhancing an immune response against an HPV tumor antigen in a subject, the method comprising administering to the subject an agonist OX40 antibody that binds the same OX40 epitope as mAb 9B12, 1-MT, and an immunogenic composition comprising an HPV16 antigen and an adjuvant, thereby enhancing the subject's immune response against the tumor antigen relative to administration of the immunogenic composition alone.

32. A method for treating an HPV-related cancer in a subject, the method comprising administering to the subject an agonist OX40 antibody that binds the same OX40 epitope as mAb 9B12, 1-MT, and an immunogenic composition comprising an HPV16 antigen and an adjuvant, treating an HPV-related cancer in the subject.

33. The method of claim 31 or 32, wherein the method increases the ratio of CD8.sup.+ T cells to regulatory T cell ratio within a tumor of the subject.

34. The method of claim 31 or 32, wherein the amount of OX40 agonist administered is sufficient to increase the CD8.sup.+ T cells to regulatory T cell ratio within a tumor of the subject.

35. The method of claim 31 or 32, wherein the amount of OX40 agonist administered is sufficiently low so as not to increase tumor-infiltrating Treg cells in the subject.

36. The method of claim 31 or 32, wherein the administration increases the subject's anti-tumor immune response relative to the administration of the immunogenic composition alone.

37. The method of any of claims 1-36, wherein the administration is by intravenous infusion.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIGS. 1A and 1B show that administration of a cancer vaccine in combination with an anti-OX40 agonist antibody increased survival of mice in a tumorigenic mouse model. FIG. 1A depicts the design of a study of the effect of a cancer vaccine (CTL epitope from HPV16 E7 antigen, PADRE Thelper epitope and QuilA adjuvant) and anti-OX40 by measuring survival of mice in a mouse model of tumorigenesis. FIG. 1B are graphs showing that administering cancer vaccine and anti-OX40 agonist antibody at 1 mg/kg enhanced survival of mice in a tumorigenic mouse model (top right panel), compared to administering cancer vaccine alone or anti-OX40 alone, or administering cancer vaccine and anti-OX40 at lower (0.5 mg/kg, top left panel) or higher (2.5 mg/kg, bottom panel) doses. Of the mice receiving cancer vaccine and anti-OX40 agonist antibody at 1 mg/kg, 20% were alive at up to about 62 days after injection of TC-1 tumor cells.

[0049] FIGS. 2A and 2B show that administration of a cancer vaccine in combination with an anti-OX40 agonist antibody inhibited tumor growth in mice in a tumorigenic mouse model. FIG. 2A depicts the design of a study of the effect of a cancer vaccine (CTL epitope from HPV16 E7 antigen, PADRE Thelper epitope and QuilA adjuvant) and anti-OX40 by measuring tumor growth in mice in a mouse model of tumorigenesis. FIG. 2B are graphs showing that administering cancer vaccine and anti-OX40 agonist antibody at 1 mg/kg inhibited tumor growth in a tumorigenic mouse model (bottom, middle panel), compared to administering cancer vaccine alone (bottom, left panel) or anti-OX40 alone (1 mg/kg, top middle panel; 2.5 mg/kg, top right panel), or administering cancer vaccine and anti-OX40 at higher (2.5 mg/kg; bottom right panel) doses. Of the mice receiving cancer vaccine and anti-OX40 agonist antibody at 1 mg/kg, mice showed reduced tumor volume and/or delays in tumor growth compared to the other study groups.

[0050] FIGS. 3A-3C show that administration of a cancer vaccine in combination with an anti-OX40 agonist antibody stimulated an antigen-specific immune response in mice in a tumorigenic mouse model. FIG. 3A depicts the design of a study of the effect of a cancer vaccine (CTL epitope from HPV16 E7 antigen, PADRE Thelper epitope and QuilA adjuvant) and anti-OX40 by evaluating antigen-specific immune response and tumor-infiltrating T cell profiles in a mouse model of tumorigenesis. FIG. 3B are graphs showing that mice receiving the cancer vaccine and anti-OX40 agonist antibody showed increases in antigen (E7) specific CD8 T cells in a tumorigenic mouse model, compared to mice receiving cancer vaccine alone or anti-OX40 alone. Mice receiving cancer vaccine and anti-OX40 agonist antibody at 1 mg/kg showed the highest ratio of E7-specific CD8 T cells:total CD8 T cells compared to the other groups receiving the cancer vaccine and anti-OX40. FIG. 3C are graphs showing that mice receiving the cancer vaccine and anti-OX40 agonist antibody showed increases in antigen (E7) specific CD8 T cells in a tumorigenic mouse model, compared to mice receiving cancer vaccine alone or anti-OX40 alone. Mice receiving cancer vaccine and anti-OX40 agonist antibody at 1 mg/kg showed the highest ratio of E7-specific CD8 T cells:regulatory T cells (Treg) compared to the other groups receiving the cancer vaccine and anti-OX40 at lower (0.5 mg/kg) or higher (2.5 mg/kg) doses.

[0051] FIGS. 4A-4C show that administration of a cancer vaccine in combination with an anti-OX40 agonist antibody increased tumor infiltration by regulatory T cells (Treg) in a tumorigenic mouse model. FIG. 4A depicts the design of a study of the effect of anti-OX40 antibody based treatment by evaluating splenic and tumor-infiltrating T cell profiles in a mouse model of tumorigenesis. FIG. 4B are graphs showing that mice receiving anti-OX40 agonist antibody (1 mg/kg; 1.75 mg/kg; 2.5 mg/kg) showed a significant increase in non-Treg CD4 T cells (CD4.sup.+ FoxP3.sup.) in spleen, compared to mice receiving no treatment. Mice receiving anti-OX40 agonist antibody (1 mg/kg; 1.75 mg/kg; 2.5 mg/kg) had similar levels of CD8.sup.+ cells (top left panel) and Treg cells (CD4.sup.+ Foxp3.sup.+; bottom panel) in spleen, compared to mice receiving no treatment. FIG. 4C are graphs showing that mice receiving anti-OX40 showed increased levels of tumor infiltrating Treg cells with increasing dosage of anti-OX40 (bottom panel).

[0052] FIGS. 5A-5D show that administration of a cancer vaccine in combination with an anti-OX40 agonist antibody and an indoleamine 2,3-dioxygenase (IDO) inhibitor inhibited tumor growth and increased survival in a tumorigenic mouse model. FIG. 5A depicts the design of a study of the effect of a cancer vaccine (CTL epitope from HPV16 E7 antigen, PADRE Thelper epitope and QuilA adjuvant), anti-OX40, and 1-methyltryptophan (1-MT) by measuring tumor growth and survival of mice in a mouse model of tumorigenesis. FIG. 5B are graphs showing that mice receiving the cancer vaccine, anti-OX40 agonist antibody (1 mg/kg), and 1-MT significantly inhibited and/or delayed tumor growth in a tumorigenic mouse model (panel h), compared to other groups of mice, including mice receiving cancer vaccine and anti-OX40 (panel 0 or cancer vaccine and 1-MT (panel g). Of the mice receiving cancer vaccine, anti-OX40 agonist antibody, and 1-MT, two mice showed a complete reduction in tumor volume 35 days after injection of TC-1 tumor cells. FIG. 5C is a graph showing increased survival of mice receiving the cancer vaccine, anti-OX40 agonist antibody (1 mg/kg), and 1-MT, compared to other groups of mice, including mice receiving cancer vaccine and anti-OX40 (blue) or cancer vaccine and 1-MT (green). Mice receiving cancer vaccine, anti-OX40 agonist antibody, and 1-MT showed reduced tumor volume and/or delays in tumor growth compared to the other study groups. Up to 80% survival was observed in these mice at up to about 55 days after injection of TC-1 tumor cells. FIG. 5D are graphs showing that inhibition of tumor growth and survival of mice were correlated. The tumor volumes and days survival were plotted for individual mice in the study.

DETAILED DESCRIPTION OF THE INVENTION

[0053] The invention features methods that are useful for enhancing the efficacy of a cancer vaccine.

[0054] The invention is based, at least in part, on the discovery that targeting the effector arm of the immune system with an agonist anti-OX40 antibody and the suppressor arm of the immune system with an IDO inhibitor enhanced the efficacy of a cancer vaccine. The OX40 molecule is a co-stimulatory receptor expressed on T cells that can lead to the proliferation and enhancement of T cell effector function when targeted with an agonist antibody. However, the effector T cells are suppressed by Indoleamine 2,3-dioxygenase (IDO) inhibitor, which is secreted by tumors as a protective mechanism against the tumor's destruction. As reported in detail below, treatment with anti-OX40 agonist antibody in combination with a cancer vaccine leads to the enhancement of antigen-specific T cell responses. The dose of 1 mg/kg anti-OX40 antibody stimulates the effector arm of T cells, which ultimately leads to a significant increase of CD8.sup.+/regulatory T cell (Treg) ratio within tumors. Further, this combination of vaccine and anti-OX40 antibody treatment lead to a significant inhibition of tumor growth and prolonged mouse survival compared to untreated tumor (TC-1) bearing mice. A complete tumor regression was observed in 20% of treated mice. This effect was significantly enhanced, when the vaccine and anti-OX40 antibody treatment was combined with 1-MT, an indoleamine-(2,3)-dioxygenase (IDO) activity inhibitor. IDO has been shown to be secreted by tumor cells, suppressive dendritic cells and macrophages in tumor environment, and is known to be responsible for suppressing effector cells and inducing regulatory T cells. These data demonstrate that the combination of vaccine and anti-OX40 antibody with 1-methyltryptophan (1-MT, IDO inhibitor) lead to a more profound inhibition of tumor growth and complete regression of established tumors in 60% of mice. In conclusion, these findings indicate that simultaneous targeting of the effector arm of immunity with an anti-OX40 antibody and the suppressor arm of immunity with 1-MT, has a synergistic effect resulting in tumor eradication and is a promising strategy that can enhance the overall efficacy of cancer treatment in patients.

OX40

[0055] OX40 is a TNF-receptor family member that is expressed primarily on activated CD4.sup.+ and CD8.sup.+ T cells and regulatory T cells. OX40 agonists have potent anti-tumor activity against multiple tumor types, which is dependent on CD4.sup.+ and CD8.sup.+ T cells (Kjaergaard, J., et al. Cancer Res 60, 5514-5521 (2000); Weinberg, A. D., et al. J Immunol 164, 2160-2169 (2000); Gough, M. J., et al. Cancer Res 68, 5206-5215 (2008); Piconese, S., Valzasina, B. & Colombo, M. P. J Exp Med 205, 825-839 (2008)). OX40 agonists enhanced T cell proliferation, effector cytokine production, cytotoxicity, and decreased activation-induced cell death and increased the generation of memory T cells in non-human model systems (Gramaglia, I., et al. J Immunol 165, 3043-3050. (2000); Maxwell, J. R., et al. J Immunol 164, 107-112 (2000); Lee, S. W., et al. J Immunol 177, 4464-4472 (2006); Ruby, C. E. & Weinberg, A. D. Cancer Immunol Immunother 58, 1941-1947 (2009)).

OX40 Agonists

[0056] OX40 agonists interact with the OX40 receptor on CD4.sup.+ T-cells during, or shortly after, priming by an antigen resulting in an increased response of the CD4.sup.+ T-cells to the antigen. An OX40 agonist interacting with the OX40 receptor on antigen specific CD4.sup.+ T-cells can increase T cell proliferation as compared to the response to antigen alone. The elevated response to the antigen can be maintained for a period of time substantially longer than in the absence of an OX40 agonist. Thus, stimulation via an OX40 agonist enhances the antigen specific immune response by boosting T-cell recognition of antigens, e.g., tumor cells. OX40 agonists are described, for example, in U.S. Pat. Nos. 6,312,700, 7,504,101, 7,622,444, and 7,959,925, which are incorporated herein by reference in their entireties. Methods of using such agonists in cancer treatment are described, for example, in WO/2013/119202 and in WO/2013/130102, each of which are incorporated herein by reference in its entirety.

[0057] OX40 agonists include, but are not limited to OX40 binding molecules, e.g., binding polypeptides, e.g., OX40 ligand (OX40L) or an OX40-binding fragment, variant, or derivative thereof, such as soluble extracellular ligand domains and OX40L fusion proteins, and anti-OX40 antibodies (for example, monoclonal antibodies such as humanized monoclonal antibodies), or an antigen-binding fragment, variant or derivative thereof. Examples of anti-OX40 monoclonal antibodies are described, for example, in WO 95/12673 and WO/95/21915, the disclosures of which are incorporated herein by reference in their entireties. In certain embodiments, the anti-OX40 monoclonal antibody is 9B12, or an antigen-binding fragment, variant, or derivative thereof, as described in Weinberg, A. D., et al. J Immunother 29, 575-585 (2006), which is incorporated herein by reference in its entirety.

[0058] 9B12 is a murine IgG1, anti-OX40 mAb directed against the extracellular domain of human OX40 (CD134) (Weinberg, A. D., et al. J Immunother 29, 575-585 (2006)). It was selected from a panel of anti-OX40 monoclonal antibodies because of its ability to elicit an agonist response for OX40 signaling, stability, and for its high level of production by the hybridoma. For use in clinical applications, 9B12 mAb is equilibrated with phosphate buffered saline, pH 7.0, and its concentration is adjusted to 5.0 mg/ml by diafiltration.

[0059] OX40 agonists include a fusion protein in which one or more domains of OX40L is covalently linked to one or more additional protein domains. Exemplary OX40L fusion proteins that can be used as OX40 agonists are described in U.S. Pat. No. 6,312,700, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, an OX40 agonist includes an OX40L fusion polypeptide that self-assembles into a multimeric (e.g., trimeric or hexameric) OX40L fusion protein. Such fusion proteins are described, e.g., in U.S. Pat. No. 7,959,925, which is incorporated by reference herein in its entirety. The multimeric OX40L fusion protein exhibits increased efficacy in enhancing antigen specific immune response in a subject, particularly a human subject, due to its ability to spontaneously assemble into highly stable trimers and hexamers.

[0060] In another embodiment, an OX40 agonist capable of assembling into a multimeric form includes a fusion polypeptide comprising in an N-terminal to C-terminal direction: an immunoglobulin domain, wherein the immunoglobulin domain includes an Fc domain, a trimerization domain, wherein the trimerization domain includes a coiled coil trimerization domain, and a receptor binding domain, wherein the receptor binding domain is an OX40 receptor binding domain, e.g., an OX40L or an OX40-binding fragment, variant, or derivative thereof, where the fusion polypeptide can self-assemble into a trimeric fusion protein. In one aspect, an OX40 agonist capable of assembling into a multimeric form is capable of binding to the OX40 receptor and stimulating at least one OX40 mediated activity. In certain aspects, the OX40 agonist includes an extracellular domain of OX40 ligand.

[0061] The trimerization domain of an OX40 agonist capable of assembling into a multimeric form serves to promote self-assembly of individual OX40L fusion polypeptide molecules into a trimeric protein. Thus, an OX40L fusion polypeptide with a trimerization domain self-assembles into a trimeric OX40L fusion protein. In one aspect, the trimerization domain is an isoleucine zipper domain or other coiled coli polypeptide structure. Exemplary coiled coil trimerization domains include: TRAF2 (GENBANK Accession No. Q12933, amino acids 299-348; Thrombospondin 1 (Accession No. P07996, amino acids 291-314; Matrilin-4 (Accession No. O95460, amino acids 594-618; CMP (matrilin-1) (Accession No. NP-002370, amino acids 463-496; HSF1 (Accession No. AAX42211, amino acids 165-191; and Cubilin (Accession No. NP-001072, amino acids 104-138. In certain specific aspects, the trimerization domain includes a TRAF2 trimerization domain, a Matrilin-4 trimerization domain, or a combination thereof. In particular embodiments, an OX40 agonist is modified to increase its serum half-life. For example, the serum half-life of an OX40 agonist can be increased by conjugation to a heterologous molecule such as serum albumin, an antibody Fc region, or PEG. In addition, in certain embodiments mutations such as deletion, addition, or substitution mutations may be made to the antibodies or functional parts to improve their half-life. In one embodiment, the Fc region may be mutated to include one, two, or all three of the following substitutions M252Y, S254T, and T256E, wherein the numbering corresponds to the EU index in Kabat. In one embodiment, the Fc region may be mutated to include all of the following substitutions M252Y, S254T, and T256E, wherein the numbering corresponds to the EU index in Kabat. Dall'Acqua et al., Properties of Human IgG1s Engineered for Enhanced Binding to the Neonatal Fc Receptor (FcRn), J Biol Chem 281(33):23514-23524 (2006). The embodiment with all three substitutions is denoted as the YTE variant. Expressed differently, in one embodiment, the antibody or functional part has an Fc region having Y at position 252Y, T at position 254T, and E at position 256, wherein the numbering corresponds to the EU index in Kabat.

[0062] In certain embodiments, OX40 agonists can be conjugated to other therapeutic agents or toxins to form immunoconjugates and/or fusion proteins.

[0063] In certain aspects, an OX40 agonist can be formulated so as to facilitate administration and promote stability of the active agent. In certain aspects, pharmaceutical compositions in accordance with the present disclosure comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. Suitable formulations for use in the treatment methods disclosed herein are described, e.g., in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).

[0064] Desirably, administration of an OX40 agonist results in an enhanced T-lymphocyte response to antigens on a variety of cancer cells, because the activation of OX40, while functioning in concert with antigenic stimulation of T-lymphocytes, is not antigen or cell-specific itself. Thus, administration of the OX40 agonist can be used to enhance an immune response against virtually any tumor antigen.

[0065] An effective amount of an OX40 agonist to be administered can be determined by a person of ordinary skill in the art by well-known methods.

[0066] Clinical response to administration of an OX40 agonist can be assessed using diagnostic techniques known to clinicians, including but not limited to magnetic resonance imaging (MRI) scan, x-radiographic imaging, computed tomographic (CT) scan, flow cytometry or fluorescence-activated cell sorter (FACS) analysis, histology, gross pathology, and blood chemistry, including but not limited to changes detectable by ELISA, RIA, and chromatography. In addition, the subject undergoing therapy with an OX40 agonist may experience the beneficial effect of an improvement in the symptoms associated with the disease.

[0067] Administration of the OX40 agonist can be via any usable route, as determined by the nature of the formulation and the needs of the patient. In certain embodiments, the OX40 agonist is administered by IV infusion.

[0068] Given that immune stimulation with OX40 agonists is not antigen-specific, a variety of cancers can be treated by the methods provided herein, for example in certain aspects, the cancer is a solid tumor, or a metastasis thereof. Types of cancers include, but are not limited to melanoma, gastrointestinal cancer, renal cell carcinoma, prostate cancer, lung cancer, or any combination thereof. The site of metastasis is not limiting and can include, for example metastases in the lymph node, lung, liver, bone, or any combination thereof.

[0069] The cancer treatment methods provided herein include conventional or non-conventional cancer treatments in addition to the administration of a cancer vaccine, an OX40 agonist, and the IDO inhibitor. By non-limiting example, administration of a cancer vaccine, an OX40 agonist, and the IDO inhibitor can be combined with surgery, radiation, chemotherapy, immunotherapy, targeting anti-cancer therapy, hormone therapy, or any combination thereof.

[0070] Effective treatment with an OX40 agonist includes, for example, reducing the rate of progression of the cancer, retardation or stabilization of tumor or metastatic growth, tumor shrinkage, and/or tumor regression, either at the site of a primary tumor, or in one or more metastases.

[0071] As reported herein below, administration of the OX40 agonist and the IDO inhibitor unexpectedly enhances the efficacy of the immunogenic composition comprising a tumor antigen.

Indoleamine 2,3-Dioxygenase Inhibitor

[0072] Tryptophan (Trp) is an essential amino acid required for the biosynthesis of proteins, niacin and the neurotransmitter 5-hydroxytryptamine (serotonin). The enzyme indoleamine 2,3-dioxygenase (also known as INDO or IDO) catalyzes the first and rate limiting step in the degradation of L-tryptophan to N-formyl-kynurenine. In human cells, IFN- stimulation induces activation of IDO, which leads to a depletion of Trp, thereby arresting the growth of Trp-dependent intracellular pathogens, such as Toxoplasma gondii and Chlamydia trachomatis. IDO activity also has an antiproliferative effect on many tumor cells, and IDO induction has been observed in vivo during rejection of allogeneic tumors, indicating a possible role for this enzyme in the tumor rejection process.

[0073] It has been observed that HeLa cells co-cultured with peripheral blood lymphocytes (PBLs) acquire an immunoinhibitory phenotype through up-regulation of IDO activity. A reduction in PBL proliferation upon treatment with interleukin-2 (IL-2) was believed to result from IDO released by the tumor cells in response to IFN- secretion by the PBLs. This effect was reversed by treatment with 1-methyl-tryptophan (1-MT), a specific IDO inhibitor. It was proposed that IDO activity in tumor cells may serve to impair antitumor responses (Logan, et al., 2002, Immunology, 105: 478-87).

[0074] Small molecule inhibitors of IDO useful in the methods of the invention are described, for example, in PCT Publication WO 99/29310, which reports methods for altering T cell-mediated immunity comprising altering local extracellular concentrations of tryptophan and tryptophan metabolites, using an inhibitor of IDO such as 1-methyl-DL-tryptophan, p-(3-benzofuranyl)-DL-alanine, p-[3-benzo(b)thienyl]-DL-alanine, and 6-nitro-L-tryptophan) (Munn, 1999). Compounds having indoleamine-2,3-dioxygenase (IDO) inhibitory activity are further reported in WO 2004/094409; and U.S. Patent Application Publication No. 2004/0234623 is directed to methods of treating a subject with a cancer or an infection by the administration of an inhibitor of indoleamine-2,3-dioxygenase in combination with other therapeutic modalities. IDO inhibitors, including Indoximod, the D isomer of 1-methyl-tryptophan, and NLG919, are known in the art and are commercially available, for example, from NewLink Genetics (Ames, Iowa). Other IDO inhibitors are described, for example, in US Patent Publication Nos. 20130289083, which is incorporated herein by reference in its entirety.

Generating an Anti-Cancer Immune Response

[0075] Cancer vaccines are potentially useful as therapeutics for the treatment of specific types of cancers. Advantageously, these vaccines may be tailored to treat the cancers of particular individuals, by generating immunogenic compositions that target specific tumor antigens expressed on a tumor in a subject. Cancer vaccines typically contain inactivated tumor cells or tumor antigens that stimulate a patient's immune system. The immune system responds to this stimulation by generating immunoresponsive cells that target the cancer. Unlike vaccines for other disease that prevent the occurrence of the disease, cancer vaccines are typically administered after a subject has been identified as having a neoplasia.

[0076] Antigen vaccines use tumor-specific antigensproteins displayed on a tumor cellto stimulate the immune system. By injecting these antigens into the cancerous area of the patient, the immune system produces antibodies or cytotoxic T lymphocytes to attack cancer cells that carry that specific antigen. Multiple antigens can be used in this type of vaccine to vary the immune system response.

[0077] Suitably, the tumor antigen is a tumor specific antigen (TSA) or a tumor associated antigen (TAA). Several tumor antigens and their expression patterns are known in the art and can be selected based on the tumor type to be treated. Non-limiting examples of tumor antigens include alpha fetoprotein (hepatocellular carcinoma), carcinoembryonic antigen (bowel cancer), cdk4 (melanoma), beta-catenin (melanoma), BING-4, CA125 (ovarian cancer), calcium-activated chloride channel 2, carcinoembryonic antigen, caspase-8 (squamous cell carcinoma), CDK4, CML66, cyclin-B1, Ep-Cam, epithelial tumor antigen (breast cancer), EphA3, fibronectin, an HPV antigen, HPV16 antigen, HPV 36, 37, CTL epitope from HPV16 E7 antigen, ART-2, melanoma associated antigen (MAGE)-1 and MAGE-3 (melanoma, breast, glioma), mesothelin, SAP-1, surviving, telomerase, tyrosinase (melanoma), surface Ig idiotype (e.g., BCR) (lymphoma), Her-2/neu (breast, ovarian), MUC-1 (breast, pancreatic), TAG-72, tyrosinase (melanoma), and HPV E6 and E7 (cervical carcinoma). Additional suitable tumor antigens include prostate specific antigen (PSA), RAS, sialyl Tn (STn), heat shock proteins and associated tumor peptides (e.g., gp96), ganglioside molecules (e.g., GM2, GD2, and GD3), carcinoembryonic antigen (CEA) and MART-1.

[0078] Typically immunogenic compositions comprising a tumor antigen are prepared in an injectable form, either as a liquid solution or as a suspension. Solid forms suitable for injection may also be prepared as emulsions, or with the polypeptides encapsulated in liposomes. The tumor antigen(s) are injected in any suitable carrier known in the art. Suitable carriers typically comprise large macromolecules that are slowly metabolized, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and inactive virus particles. Such carriers are well known to those skilled in the art. These carriers may also function as adjuvants.

[0079] Adjuvants are immunostimulating agents that enhance vaccine effectiveness. Effective adjuvants include, but are not limited to, aluminum salts such as aluminum hydroxide and aluminum phosphate, muramyl peptides, bacterial cell wall components, saponin adjuvants, and other substances that act as immunostimulating agents to enhance the effectiveness of the composition.

[0080] Immunogenic compositions (e.g., cancer vaccines) are administered in a manner compatible with the dose formulation. By an effective amount is meant a single dose, or a vaccine administered in a multiple dose schedule, that is effective for the treatment or prevention of a disease or disorder. Preferably, the dose is effective to inhibit the growth of a neoplasm. The dose administered will vary, depending on the subject to be treated, the subject's health and physical condition, the capacity of the subject's immune system to produce antibodies, the degree of protection desired, and other relevant factors. Precise amounts of the active ingredient required will depend on the judgment of the practitioner.

[0081] As reported herein below, administration of the IDO inhibitor (e.g., 1-MT) and the OX40 agonist (e.g., OX40 antibody agonist) synergistically enhances the efficacy of the cancer vaccine. Preferably, administration of the immunogenic composition comprising a tumor antigen, the OX40 antibody agonist, and the IDO inhibitor reduces or delays tumor growth, induces tumor regression, or increases patient survival relative to administration of such agents alone.

[0082] In addition to the use of cancer vaccines, therapies that induce tumor cell apoptosis release tumor antigens into the body that are capable of inducing an anti-cancer immune response. In one embodiment, radiation may be used to induce tumor cell apoptosis. Accordingly, the invention provides methods for enhancing the efficacy of a tumor antigen in inducing an anti-cancer immune response by administering an OX40 agonist and an Indoleamine 2,3-dioxygenase (IDO) inhibitor in combination with radiation therapy. In another embodiment, chemotherapy that induces tumor cell apoptosis (e.g., anthracyclines, oxaliplatin) can be administered in combination with an OX40 agonist and an Indoleamine 2,3-dioxygenase (IDO) inhibitor. Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, and valrubicin.

[0083] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, 1989); Oligonucleotide Synthesis (Gait, 1984); Animal Cell Culture (Freshney, 1987); Methods in Enzymology Handbook of Experimental Immunology (Weir, 1996); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, 1987); Current Protocols in Molecular Biology (Ausubel, 1987); PCR: The Polymerase Chain Reaction, (Mullis, 1994); Current Protocols in Immunology (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0084] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1. Treatment with a Cancer Vaccine and an Anti-OX40 Agonist Antibody Increased Survival in a Tumorigenic Mouse Model

[0085] A study was performed in a tumorigenic mouse model to determine the effect of treatment with a cancer vaccine (CTL epitope from HPV16 E7 antigen, PADRE T-helper epitope and QuilA adjuvant) and OX40 agonist on survival (FIG. 1A). Mice (C57BL6; female, 6-8 weeks old) were injected with TC-1 tumor cells (710.sup.4 s.c.). Administration of anti-OX40 antibody (clone OX86) was started at day 4 or day 10 after injection with TC-1 cells. The first day of tumor appearance was around day 10. Anti-OX40 antibody was administered twice a week at 0.5, 1.0, and 2.5 mg/kg doses, i.p. In all, 14 groups of mice (n=5/group) were studied, including mice receiving no treatment, vaccine alone, anti-OX40 alone at day 4 (0.5, 1.0, and 2.5 mg/kg), anti-OX40 alone at day 10 (0.5, 1.0, and 2.5 mg/kg), anti-OX40 at day 4 (0.5, 1.0, and 2.5 mg/kg) and cancer vaccine, and anti-OX40 at day 10 (0.5, 1.0, and 2.5 mg/kg) and cancer vaccine. The study was repeated twice.

[0086] Mice administered cancer vaccine and anti-OX40 agonist antibody at 1 mg/kg showed enhanced survival, compared to mice administered cancer vaccine alone or anti-OX40 alone (FIG. 1B). In particular, mice administered cancer vaccine and anti-OX40 at lower (0.5 mg/kg, top left panel) or higher (2.5 mg/kg, bottom panel) doses did not show enhanced survival. Of the mice receiving cancer vaccine and anti-OX40 agonist antibody at 1 mg/kg, 20% were alive at up to about 62 days after injection of TC-1 tumor cells. No significant difference in survival was provided when anti-OX40 antibody was given prior to tumor appearance (day 4 after tumor implantation) or on the first day of tumor appearance (day 10 after tumor implantation).

Example 2. Treatment with a Cancer Vaccine and an Anti-OX40 Agonist Antibody Decreased Tumor Growth in a Tumorigenic Mouse Model

[0087] A study was performed in a tumorigenic mouse model to determine the effect of treatment with a cancer vaccine (CTL epitope from HPV16 E7 antigen, PADRE Thelper epitope and QuilA adjuvant) and OX40 agonist on tumor growth (FIG. 2A). Mice (C57BL6; female, 6-8 weeks old) were injected with TC-1 tumor cells (710.sup.4 s.c.). Administration of anti-OX40 antibody (clone OX86) was started at day 4 or day 10 after injection with TC-1 cells. Anti-OX40 antibody was administered twice a week at 1.0, and 2.5 mg/kg doses, i.p. In all, 6 groups of mice (n=5/group) were studied, including mice receiving no treatment, vaccine alone, anti-OX40 alone (1.0, and 2.5 mg/kg), and anti-OX40 (1.0 and 2.5 mg/kg) and cancer vaccine. The study was repeated twice.

[0088] Mice administered cancer vaccine and anti-OX40 agonist antibody at 1 mg/kg showed reduced tumor volume and/or delays in tumor growth (FIG. 2B: bottom, middle panel), compared to mice administered cancer vaccine alone (FIG. 2B: bottom, left panel) or anti-OX40 alone (FIG. 2B: top middle panel, top right panel). Consistent with other results (see, e.g., FIG. 1B), mice administered cancer vaccine and anti-OX40 at a higher dose (2.5 mg/kg) did not show the same effect on tumor growth as mice receiving cancer vaccine and anti-Ox40 agonist antibody at 1 mg/kg (FIG. 2B: bottom right panel). Thus, treatment with a cancer vaccine and anti-OX40 agonist antibody at 1 mg/kg inhibited tumor growth in mice.

Example 3. Treatment with Cancer Vaccine and an Anti-OX40 Agonist Antibody Increased CD8/Treg Ratio within a Tumor in a Tumorigenic Mouse Model

[0089] A study was performed in a tumorigenic mouse model to evaluate the effect of treatment with a cancer vaccine (CTL epitope from HPV16 E7 antigen, PADRE Thelper epitope and QuilA adjuvant) and OX40 agonist on immune response (FIG. 3A). Mice (C57BL6; female, 6-8 weeks old) were injected with TC-1 tumor cells (710.sup.4 s.c.). Administration of anti-OX40 antibody (clone OX86) was started at day 10 after injection with TC-1 cells. Anti-OX40 antibody was administered twice a week at 0.5, 1.0, or 2.5 mg/kg doses, i.p. In all, 8 groups of mice (n=5/group) were studied, including mice receiving no treatment, vaccine alone, anti-OX40 alone (0.5 and 1.0 mg/kg), and anti-OX40 (0.5, 1.0, and 2.5 mg/kg) and cancer vaccine. The study was repeated twice. FIGS. 3B and 3C show the effect of various doses of Ox40 alone, or in combination with vaccine on CD8 T cells, E7-specific CD8 T cells, T reg cells, and the CD8/Treg ratio.

Example 4. Treatment with Cancer Vaccine and an Anti-OX40 Agonist Antibody Stimulated an Antigen-Specific Immune Response in a Tumorigenic Mouse Model

[0090] A study was performed in a tumorigenic mouse model to evaluate the effect of treatment with a cancer vaccine (CTL epitope from HPV16 E7 antigen, PADRE Thelper epitope and QuilA adjuvant) and OX40 agonist on immune response (FIG. 4A). Mice (C57BL6; female, 6-8 weeks old) were injected with TC-1 tumor cells (710.sup.4 s.c.). Administration of anti-OX40 antibody (clone OX86) was started at day 10 after injection with TC-1 cells. Anti-OX40 antibody was administered twice a week at 1.0, 1.75, or 2.5 mg/kg doses, i.p. In all, 4 groups of mice (n=5/group) were studied, including mice receiving no treatment and anti-OX40 alone (1.0, 1.75, and 2.5 mg/kg). The study was repeated twice.

[0091] No effect on percentage or number of CD8.sup.+ T-cells in spleen (FIG. 4B: top left panel) or tumor (FIG. 4C: top right panel) was detected after treatment with anti-OX40 antibody for all doses. All three doses significantly increased non-Treg CD4 T cells (CD4.sup.+ FoxP3.sup.) in the spleen (FIG. 4B, top right panel) and within the tumor (FIG. 4C, top right panel). Mice receiving anti-OX40 agonist antibody had similar levels of Treg cells (CD4.sup.+ Foxp3.sup.+; FIG. 4B: bottom panel), compared to mice receiving no treatment. However, mice receiving anti-OX40 showed increased levels of tumor infiltrating Treg cells with increasing dosage of anti-OX40 (FIG. 4C: bottom panel). Without being bound to a particular theory, the increase in tumor infiltrating Treg cells might explain a lack of therapeutic effect when 2.5 mg/kg of anti-OX40 Ab was used with the cancer vaccine.

Example 5. Treatment with Cancer Vaccine, Anti-OX40 Agonist Antibody, and Indoleamine 2,3-Dioxygenase (IDO) Inhibitor Decreased Tumor Growth and Increased Survival in a Tumorigenic Mouse Model

[0092] Mouse tumor TC-1, which expresses the E7 oncoprotein from HPV-16, is used as a surrogate for human tumors infected with HPV-16. Mice (C57BL6; female, 6-8 weeks old) were injected with TC-1 tumor cells (7104 s.c.) at Day 0. Administration of anti-OX40 antibody (clone OX86) was started at day 10 after injection with TC-1 cells. The anti-OX40 antibody was administered twice a week at 1.0 mg/kg, i.p. Starting at day 10, 1-methyl-tryptophan (1-MT) was administered to the mice by adding 1-MT (2 mg/mL) to their drinking water. In all, 8 groups of mice (n=5/group) were studied, including mice receiving no treatment, anti-OX40 alone (1.0 mg/kg), 1-MT alone, anti-OX40 and 1-MT, cancer vaccine alone, cancer vaccine and anti-OX40 (1.0 mg/kg), cancer vaccine and 1-MT, and cancer vaccine, anti-OX40, and 1-MT.

[0093] Mice receiving the cancer vaccine, anti-OX40 agonist antibody (1 mg/kg), and 1-MT significantly inhibited and/or delayed tumor growth in a tumorigenic mouse model (FIG. 5B: panel h), compared to other groups of mice, including mice receiving cancer vaccine and anti-OX40 (FIG. 5B: panel 0 or cancer vaccine and 1-MT (FIG. 5B: panel g). Of the mice receiving cancer vaccine, anti-OX40 agonist antibody, and 1-MT, two mice showed a complete reduction in tumor volume 35 days after injection of TC-1 tumor cells. Mice receiving the cancer vaccine, anti-OX40 agonist antibody (1 mg/kg) (red), and MT-1 had increased survival compared to other groups of mice, including mice receiving cancer vaccine and anti-OX40 (blue) or cancer vaccine and 1-MT (green) (FIG. 5C). Up to 80% survival was observed in these mice at up to about 55 days after injection of TC-1 tumor cells. In contrast, 20% mice receiving cancer vaccine and anti-OX40 agonist antibody were alive up to about 55 days after injection of TC-1 tumor cells, consistent with previous results (see, e.g., FIG. 1B). The inhibition of tumor growth and survival of mice were correlated (FIG. 5D). Thus, the addition of 1-MT to treatment with cancer vaccine and anti-OX40 led to a significant increase in therapeutic potency of treatment (e.g., 60% vs. 20% complete regression). The results presented herein demonstrate that use of OX40 agonists and IDO inhibitors in combination with cancer vaccines has the potential to increase the efficacy of cancer vaccines.

[0094] The results described herein above were carried out using the following materials and methods.

[0095] Mice (C57BL6; female, 6-8 weeks old) were obtained from Jackson Laboratory (Bar Harbor, Me.) and kept under pathogen-free conditions.

[0096] TC-1 cells that were derived by co-transfection of human papilloma-virus strain 16 (HPV16) early proteins 6 and 7 (E6 and E7) and activated ras oncogene to primary C57BL/6 mouse lung epithelial cells were obtained from ATCC (Manassas, Va.). TC-1 cells were grown in RPMI 1640 supplemented with 10% FBS, penicillin and streptomycin (100 U/ml each) and L-glutamine (2 mM) at 37 C. with 5% CO.sub.2.

[0097] Vaccine consisting of CTL epitope from E7 antigen (E7.sub.49-57, a 9-mer peptide (RAHYNIVTF)), mixed with PADRE 13-mer T helper epitope (aKChaVAAWTLKAAa) (both from Celtek Bioscience (Nashville, Tenn.)) and QuilA adjuvant (Brenntag, Denmark). Anti-OX40 antibody (clone OX86) was provided by Medimmune. 1-Methyl-D-Tryptophan (1-MT) was obtained from Sigma-Aldrich (St. Louis, Mo.).

[0098] In the experiments where analysis of tumor growth and survival were the endpoint, mice (n=5/group) were implanted with 70,000 TC-1 cells on day 0. Anti-OX40 antibody (1 mg/kg, i.p.) was injected either on day 4 or day 10 after tumor implantation. On day 10, when all mice had tumors of 3-4 mm in diameter, animals from appropriate groups were injected with vaccine (E7-100 g/mouse, PADRE-20 g/mouse, QuilA-10 g/mouse) s.c. Mice from proper groups were supplied with 1-MT in drinking water (2 mg/ml) also starting day 10 after tumor implantation throughout the experiment. Mice were treated with vaccine weekly throughout the experiment; anti-OX40 antibody was given twice a week. Tumors were measured every 3-4 days using digital calipers, and tumor volume was calculated using the formula V=(W.sup.2L)/2, whereby V is volume, L is length (longer diameter), and W is width (shorter diameter). In these experiments, mice were sacrificed when they became moribund, tumors were ulcerated, or tumor volume reached 1.5 cm.sup.3.

Other Embodiments

[0099] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0100] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0101] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.