WO2015131176A1

WO2015131176A1 - Compositions, methods, and kits for treatment of cancer

Info

Publication number: WO2015131176A1
Application number: PCT/US2015/018265
Authority: WO
Inventors: Eckhard R. Podack; Taylor Schreiber
Original assignee: Podack Eckhard R; Taylor Schreiber
Priority date: 2014-02-28
Filing date: 2015-03-02
Publication date: 2015-09-03

Abstract

Methods, kits and compositions are provided for cancer, wherein the treatment administering to a patient in need thereof a combination of a modified heat shock protein with: (a) one or more checkpoint inhibitors; (b) one or more T cell co-stimulators; or (c) one or more checkpoint inhibitors and one or more T cell co-stimulators.

Description

COMPOSITIONS, METHODS, AND KITS FOR TREATMENT OF CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Serial No. 61/946,609, filed February 28, 2014. TECHNICAL FIELD

This document relates to methods for treating cancer by administering a combination of agents.

BACKGROUND

Cancer is a disease in which abnormal cells divide without proper control and are able to invade other tissues, spreading to other parts of the body through the blood and lymphatic systems. There are more than 100 different types of cancer, most named for the organ or type of cell in which they start. Despite many advances in the field, cancer remains one of the leading causes of morbidity and death in developed nations. There are many treatments presently available for cancer, including chemotherapy, radiation therapy, surgical intervention, and the use of various therapeutic agents including, for example, natural products, derivatives of natural products, and synthetic compounds. Although many of the molecular mechanisms of tumor genesis have now been revealed, standard treatment of most aggressive tumors continues to be surgical resection, chemotherapy, and radiotherapy. While increasingly successful, each of these treatments still causes numerous undesired side effects.

According to the American Cancer Society (ACS), lung cancer is one of the leading causes of cancer-related mortality in the United States. In its 2014 "Cancer Facts and Figures" publication, the ACS estimated that there would be 224,210 new cases of lung cancer diagnosed in the United States alone in 2014, with almost 160,000 lung cancer-related deaths.

Non-Small Cell Lung Cancer (NSCLC) is one of the most deadly types of cancer. NSCLC refers to an epithelial lung cancer other than small cell lung cancer (SCLC). NSCLC typically is largely insensitive to chemotherapy and radiation therapy, as compared to SCLC. While patients with resectable NSCLC may be successfully treated with surgery (or with surgery followed by chemotherapy), those patients with non- resectable NSCLC often are treated with radiation therapy and/or chemotherapy.

Patients with advanced metastatic disease may achieve improved survival and reduction of symptoms following treatment with one or more of chemotherapy, targeted agents, and other therapies.

The annual incidence of NSCLC in the United States exceeds 135,000 (out of a total of 170,000 patients with all types of lung cancer). NSCLC, after metastasis or recurrence, is almost uniformly fatal, with a five-year survival of <5%. The annual mortality rate from lung tumors is higher than that from colon, breast, and prostate carcinoma combined. Results of treatment with chemotherapy for NSCLC disease are poor. Phase III trials have typically demonstrated response rates of 15% to 30%, with a median survival of less than one year. A meta-analysis of clinical studies randomizing metastatic NSCLC patients between best supportive care and chemotherapy concluded that the mean potential gain in survival was only six weeks. Many new drugs and combinations have been reported for use in NSCLC, but these regimens have resulted in a complete response in less than 10% of patients, with a minimal to modest impact on survival.

Although the prognosis for patients with cancer has improved, it is clear that new approaches are needed to increase the fraction of patients cured. Because of a lack of effective treatments for cancer and the toxicity and side effects associated with existing therapies, a need exists for new therapies in the treatment of cancer.

SUMMARY

The present application provides compositions and methods for the treatment of cancer. More particularly, the inventors have developed novel compositions containing a combination of active agents, and a method for treating cancer that includes administering the active agents in combination. Thus, this document is based at least in part on the development of treatments that include administering to a patient a composition containing multiple active agents. For example, this document is based in part on the discovery that administration to a patient of a modified heat shock protein in combination with (a) one or more checkpoint inhibitors, (b) one or more T cell co- stimulators, or (c) one or more checkpoint inhibitors and one or more T cell co- stimulators leads to a surprising increase in the expansion of tumor specific CD8+ T cells, which, in turn, provides effective cancer treatment. Without being bound by a particular mechanism, the mechanism for the apparent synergy may be through the expansion of such T cells. The compositions and methods provided herein may be very valuable in the treatment of cancer, as their use may provide greater efficacy and/or potency, resulting in improved therapeutic response, diminished side effects, or both, as compared to using traditional cancer therapeutics.

In one aspect, this document features a method of treating a cancer in a human subject, where the method includes administering to the subject a therapeutically effective amount of a vaccine containing a plurality of host cells, each of the host cells co-expressing at least one tumor antigen and a heat shock protein modified to be secreted from each of the host cells, and a composition containing one or more checkpoint inhibitors.

In another aspect, this document features a method of treating a cancer in a human subject, where the method includes administering to the subject a therapeutically effective amount of a vaccine containing a plurality of host cells, each of the host cells co-expressing at least one tumor antigen and a heat shock protein modified to be secreted from each of the host cells, and a composition containing one or more T-cell co- stimulators.

In another aspect, this document features a method of treating a cancer in a human subject, where the method includes administering to the subject a therapeutically effective amount of (a) a vaccine containing a plurality of host cells, each of the host cells co-expressing at least one tumor antigen and a heat shock protein modified to be secreted from each of the host cells, (b) a composition containing one or more checkpoint inhibitors, and (c) a composition containing one or more T-cell co- stimulators.

With respect to any of the methods provided herein, the cancer can be selected from the group consisting of lung cancer, renal cell carcinoma, bladder cancer, colorectal cancer, melanoma, sarcoma, breast cancer, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, and nasopharyngeal cancer. The cancer can be a carcinoma or adenocarcinoma. In some embodiments, the cancer can be non-small cell lung cancer ( SCLC).

The survival time of the subject can be increased over the expected survival time for other subjects having the same type and stage of cancer. The method can further include the step of analyzing CD8 T lymphocytes in the blood of the subject both before and after treatment.

The host cells can be cancer cells. In some embodiments, the cancer in the subject can be a lung cancer and the host cells can be lung cancer cells. For example, the cancer in the subject can be a non-small cell lung cancer and the host cells can be non- small cell lung cancer cells. The host cells can be allogeneic to the subject, and/or can be irradiated before administration of the vaccine. In some embodiments, the host cells can be administered intradermally.

When included, the one or more T-cell co-stimulators can be selected from the group consisting of B7-1, B7-2, B7-h/B7rp- l, CD48, GITR, ICAM-1, ICAM-2, ICAM- 3, LFA-1, LFA-2, LFA-3, VLA-1, VCAM-1, CD30 Ligand (CD30L), CD40 Ligand (CD40L), 4- IBB Ligand (4-1BBL), OX40 ligand, CD70, CD24, LIGHT, and other cell adhesion proteins and other cell surface proteins that can activate T cell co-stimulatory pathways through T cell surface proteins. In some embodiments, the one or more T-cell co-stimulators can be OX40 ligand, or can be 4- IBB Ligand.

When included, the one or more checkpoint inhibitors can be selected from the group consisting of nivolumab (MDX-1 106; BMS-936558), BMS-936559,

pembrolizumab (lambrolizumab; MK3475), pidilizumab (CT-01 1), AMP-224 and MEDI4736 (PD1 antibodies), ipilmumab and tremelimumab (CTLA4 antibodies), MPDL3280A and MDX-1 105 (PDL1 antibodies), IMP321 (LAG3 antibody), and MGA271 (B7-H3 antibody). In some embodiments, the one or more checkpoint inhibitors can be selected from the group consisting of CTLA-4, PD-1, PD-L1, LAG-3, IDO, TGF-beta, and TIM-3.

In another aspect, this document features a composition containing a vaccine that contains a plurality of host cells, each of the host cells co-expressing at least one tumor antigen and a heat shock protein modified to be secreted from each of the host cells, and one or more checkpoint inhibitors.

In a further aspect, this document features a composition containing a vaccine that contains a plurality of host cells, each of the host cells co-expressing at least one tumor antigen and a heat shock protein modified to be secreted from each of the host cells, and one or more T-cell co-stimulators.

In still another aspect, this document features a composition containing (a) a vaccine that contains a plurality of host cells, each of the host cells co-expressing at least one tumor antigen and a heat shock protein modified to be secreted from each of the host cells, (b) one or more checkpoint inhibitors, and (c) one or more T-cell co-stimulators.

The compositions featured herein can be for use in treating a cancer in a human subject. The cancer can be selected from the group consisting of lung cancer, renal cell carcinoma, bladder cancer, colorectal cancer, melanoma, sarcoma, breast cancer, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, and nasopharyngeal cancer. In some embodiments, the cancer can be a carcinoma or adenocarcinoma. In some embodiments, the cancer can be NSCLC.

The host cell can be a cancer cell. For example, the host cells can be non-small cell lung cancer cells. In some embodiments, the host cells can be irradiated.

When included, the one or more T-cell co-stimulators can be selected from the group consisting of B7-1, B7-2, B7-h/B7rp- l, CD48, GITR, ICAM-1, ICAM-2, ICAM- 3, LFA-1, LFA-2, LFA-3, VLA-1, VCAM-1, CD30 Ligand (CD30L), CD40 Ligand (CD40L), 4- IBB Ligand (4-1BBL), OX40 ligand, CD70, CD24, LIGHT, and other cell adhesion proteins and other cell surface proteins that can activate T cell co-stimulatory pathways through T cell surface proteins. In some embodiments, the one or more T-cell co-stimulators can be OX40 ligand, or 4- IBB Ligand.

When present, the one or more checkpoint inhibitors can be selected from the group consisting of nivolumab (MDX-1 106; BMS-936558), BMS-936559,

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph plotting survival of mice that were subjected to adoptive transfer of OT-I and OT-II cells at day -2, followed by injection of B16-ova cells at day 0, therapeutic vaccination with control or T cell co-stimulatory antibody, with or without GITR, at day 9, and treatment with antibody only at days 13 and 17.

FIG. 2 is a graph plotting OT-I/total CD8+ in mice that were subjected to adoptive transfer of OT-I and OT-II cells at day -2, followed by injection of B 16-ova cells at day 0, therapeutic vaccination with control or T cell co-stimulatory antibody, with or without GITR, at day 9, and treatment with antibody only at days 13 and 17.

FIG. 3 is a graph plotting survival of mice that were subjected to adoptive transfer of OT-I and OT-II cells at day -2, followed by injection of B 16-ova cells at day 0, therapeutic vaccination with control or T cell co-stimulatory antibody, with or without OX40, at day 9, and treatment with antibody only at days 13 and 17.

FIG. 4 is a graph plotting OT-I/total CD8+ in mice that were subjected to adoptive transfer of OT-I and OT-II cells at day -2, followed by injection of B 16-ova cells at day 0, therapeutic vaccination with control or T cell co-stimulatory antibody, with or without OX40, at day 9, and treatment with antibody only at days 13 and 17.

FIG. 5 is a graph plotting survival of mice that were subjected to adoptive transfer of OT-I and OT-II cells at day -2, followed by injection of B 16-ova cells at day 0, therapeutic vaccination with control or T cell co-stimulatory antibody, with or without 4-1BB, at day 9, and treatment with antibody only at days 13 and 17.

FIG. 6 is a graph plotting OT-I/total CD8+ in mice that were subjected to adoptive transfer of OT-I and OT-II cells at day -2, followed by injection of B 16-ova cells at day 0, therapeutic vaccination with control or T cell co-stimulatory antibody, with or without 4- IBB, at day 9, and treatment with antibody only at days 13 and 17.

DETAILED DESCRIPTION

As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the terms "treat" and "treatment" are used interchangeably and are meant to indicate a postponement of development of a disorder and/or a reduction in the severity of symptoms that will or are expected to develop. The terms further include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying metabolic causes of symptoms.

As used herein, "individual" (as in the subject of the treatment) includes human beings and non-human animals, including both mammals and non-mammals. Mammals include, for example, humans, non-human primates (e.g., apes and monkeys), cattle, horses, sheep, and goats. Non-mammals include, for example, fish and birds.

The expression "effective amount" or "therapeutically effective amount," in connection with the treatment of a patient suffering from cancer, refers to the amount of a composition, or of each active agent, that inhibits the growth of cancer cells that are proliferating at an abnormally high rate, or induces apoptosis of such cells, reduces the proportion of abnormal cells, or that maintains the disease in a state of complete or partial remission, or slows the progression of the disease.

Treatment of Cancer

It is thought that the dominant immune cell that can kill cancer cells is the T cell, specifically the CD8+ cytotoxic T cell. There are four main elements (priming, activation/proliferation, migration to the tumor site, and tumor cell killing) for an antitumor cytotoxic T cell response. This document describes the surprising discovery that numbers of CD8+ cells can be increased by administering a combination of agents to the patient, thereby providing effective treatments for cancer.

As provided herein, modified heat shock proteins are administered to a patient in combination with one or both of:

(a) a checkpoint inhibitor; and

(b) a T-cell co-stimulator

to treat a hyperproliferative disease. In some embodiments, the hyperproliferative disease is cancer. In some embodiments the cancer is selected from the group of cancers consisting of lung cancer, renal cell carcinoma, bladder cancer, colorectal cancer, melanoma, sarcoma, breast cancer, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, and nasopharyngeal cancer. In some embodiments the cancer is a carcinoma or adenocarcinoma. In some embodiments the cancer is NSCLC.

The compositions and methods described herein may be employed in therapy to individuals (e.g., animals, including mammals such as humans) suffering from cancer. The compositions and methods are believed to be effective against cancer at any stage of the disease, and to retard progression of the disease. It also is believed that the compositions and methods will be effective to maintain the disease in complete or partial remission following treatment that has been effective in attaining such remission. Modified Heat Shock Proteins

Heat shock proteins are a class of functionally related proteins involved in the folding and unfolding of other proteins. Heat shock protein expression is increased when cells are exposed to elevated temperatures or other stressors. Heat shock proteins include gp96, also referred to as 90kDa beta member 1 (HSP90B1), endoplasmin, grp94, and ERp99 (a chaperone protein that in humans is encoded by the HSP90B 1 gene).

Modified heat shock proteins, as well as means of administering modified heat shock proteins to patients, are described in Schreiber et al, J. Immunol. 2012,

189(7):331 1-3318; U.S. Publication No. 2008/0026012; and U.S. Publication No.

2011/0287057; each of which are incorporated herein by reference in their entirety.

In some embodiments, the modified heat shock protein is conjugated to one or more peptides. For example, in some embodiments, a modified heat shock protein can be conjugated to one or more (e.g., two, three, four, or more than four) cancer antigens.

The modified heat shock proteins provided herein are modified such that they are secreted by the cells in which they are expressed, and can be easily purified from the cell culture medium. In some embodiments, a modified hsp lacks a segment of the polypeptide that signals retention of the hsp in the endoplasmic reticulum (ER). Such a peptide signal is found in hsps that remain in the ER, such as but not limited to gp96. In some embodiments, the retention signal is disabled by deletion, or by substitution with a peptide that does not function as a signal. In addition, in some embodiments, the modified hsp includes a peptide tag that facilitates recovery and purification. The peptide tag can be fused to any portion of the hsp that is not involved in binding antigenic peptide, such as for example, the carboxyl terminus. In some embodiments, the amino acids of a retention signal that usually is located at the carboxyl terminus of an hsp can be replaced by a peptide tag. Further, if the hsp resides naturally in the cytoplasm, a leader peptide can be added to direct its translocation across the ER membrane for secretion.

The peptide that causes an hsp to remain in the endoplasmic reticulum (ER) typically is located at the carboxyl terminal of the hsp, and has the sequence, Xaa-Asp- Glu-Leu (or XDEL) (Munro and Pelham, Cell, 1987, 48:899-907). The term "retention peptide" is used herein to refer to this tetrapeptide sequence, which is Lys-Asp-Glu-Leu (KDEL) in most mammalian hsps. The retention peptide sequences in Saccharomyces cerevisiae and in Schizosaccharomyces pombe are His-Asp-Glu-Leu (HDEL) and Ala- Asp-Glu-Leu (ADEL), respectively (Pidoux and Armstrong, EMBO J., 1992, 1 1 : 1583- 1591).

The modifications present in modified hsps as provided herein can be produced by various methods known in the art. The manipulations that result in their production can occur at the gene or protein level, but manipulations at the gene level can be particularly useful. For example, the cloned coding region of an hsp can be modified by any of numerous recombinant DNA methods known in the art (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d ed., 1990, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and Ausubel et al, in Chapter 8 of Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley Interscience, New York). It will be apparent from the following discussion that substitutions, deletions, insertions, or any combination thereof are introduced or combined to arrive at final nucleotide sequence encoding a modified hsp. Alternatively, a modified hsp can be chemically synthesized. For example, a peptide corresponding to a portion of an hsp that contains the desired modifications can be synthesized using a peptide synthesizer.

In some embodiments, one or more modified heat shock proteins can be administered to a patient via a vaccine. This document provides pharmaceutical compositions and medicaments that include or use as an active agent cells expressing one or more tumor-associated antigens and secreting a heat shock protein (e.g., a secreted form of gp96). The cells may be from one or more human tumor cell lines developed from tumors explanted from a patient (e.g., a single tumor cell line, or multiple tumor cell lines of the same cancer type or different cancer types), or may be from a human cell line (e.g., HEK293) not derived from a cancer, but engineered to express one or more tumor-associated antigens. The cells can be irradiated (e.g., with at 1C

least 2000; 4000; 6000; 8000, 10,000; or 12,000 rad) to prevent their replication, while allowing the heat shock protein to be secreted for at least 1, 2, 3, 4, 5, 6, or 7 days. The cells also may be engineered to express another marker (e.g., a human MHC protein). Cells for use in a vaccine can be stored frozen and reconstituted just before use in a sterile, pharmaceutically acceptable liquid such as USP grade saline or a buffered salt solution. A list of pharmaceutically acceptable carriers, as well as pharmaceutical formulations, can be found in Remington's Pharmaceutical Sciences, a standard text in this field, and in USP-NF (the United States Pharmacopeia's official compendium in a combined volume with the National Formulary). Other substances may be added to the compositions (e.g., human serum albumin and/or DMSO), and other steps can be taken to stabilize and/or preserve the compositions, and/or to facilitate their administration to a subject.

Vaccine Administration

In some embodiments, the modified heat shock proteins are administered so as to provide a number of cells to the patient that secrete at least 100 to 9000 (e.g., 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 100 to 1000, 500 to 3000, 1000 to 5000, or 4000 to 9000) mg/ml/day of the secreted form of the hsp in in vitro culture. The number of cells in each dose may range from 100,000 to 100,000,000 (e.g., about 100,000; 250,000; 500,000; 750,000; 1,000,000; 2,000,000; 5,000,000; 10,000,000; 20,000,000; 50,000,000; or 100,000,000 + 1-20, 10, or 5%, or 100,000 to 1,000,000; 500,000 to 2,000,000; 1,000,000 to 10,000,000; or 10,000,000 to 100,000,000). The dose may be given repeatedly, e.g., hourly, daily, semiweekly, weekly, bi-weekly, triweekly, or monthly.

As a non-limiting example of an administration protocol, on each visit for therapy (e.g., every week or every other week) a clinical evaluation of the cancer and of toxicity is conducted. Blood samples for immunological evaluation are obtained on Day 1 of each course before vaccination is given. Patients with evidence of stable disease or responding NSCLC, and acceptable toxicity (autoimmune <grade 2, and grade ~2 for other body systems) upon completion of the first course of vaccination are treated with an additional course at the same dose and schedule. A third course at the same dose and schedule is given provided that the patient has evidence of stable disease or responding

) NSCLC, and acceptable toxicity (autoimmune <grade 2, and grade ~2 for other body systems) on completion of the second course.

Checkpoint inhibitors

Checkpoint inhibitors include compounds that inhibit PD1 receptor, PDL1 (PD1 receptor ligand), CTLA-4 receptor, LAG3 receptor, or TIM3, as discussed by, e.g., Pardoll, Nat. Rev. Cancer, 2012, 12:252-264; and Creelan, Cancer Control, 2014, 21(l):80-89. Examples of checkpoint inhibitors include, without limitation, nivolumab (MDX-1106; BMS-936558), BMS-936559, pembrolizumab (lambrolizumab; MK3475), pidilizumab (CT-011), AMP-224 and MEDI4736 (PD1 antibodies), ipilmumab and tremelimumab (CTLA4 antibodies), MPDL3280A and MDX-1105 (PDL1 antibodies), IMP321 (LAG3 antibody) and MGA271 (B7-H3 antibody).

In some embodiments, the checkpoint inhibitors are one or more of CTLA-4, PD- 1, PD-L1, LAG-3, IDO, TGF-beta, and TIM-3.

T-cell co-stimulators

T-cells, including cytotoxic T-lymphocytes, are a component of effective human immune responses to tumors, viral infections and, other infectious diseases. T-cells destroy neoplastic or virally infected cells through recognition of antigenic peptides presented by MHC class I molecules on the surfaces of target cells. Activation of T-cells is dependent upon coordinate signaling through antigen receptors and co-stimulator receptors on T-cell surfaces.

Useful T cell co-stimulators include, for example, those that are known and have been discussed in the field. See, for example Carreno et al, Annu. Rev. Immunol, 2002, 20:29; Nakamura et al, J. Exp. Med., 2001, 194:629; Kochli et al, Immunol. Lett, 1999,

65: 197; Wolthers et al, Eur. J. Immunol, 1996, 26: 1700; Takasaki et al, Intern. Med.,

1999, 38: 175; Weintraub et al, J. Immunol, 1997, 159:4117; Weintraub et al, Clin.

Immunol, 1999, 91 :302; Hwang et al, J. Exp. Med., 2000, 191 : 1137; Sabzevari et al, J.

Immunol, 2001, 166:2505; Lorber et al, J. Immunol, 1982, 128:2798; and Hudrisier et al, J. Immunol, 2001, 166:3645, each of which is incorporated by reference in its entirety.

Any suitable T cell co-stimulator can be used in the methods provided herein, including, but not limited to, B7 family co-stimulators (e.g., B7-1, B7-2, and B7-h/B7rp- 1), CD48, GITR, ICAM-1, ICAM-2, ICAM-3, LFA-1, LFA-2, LFA-3, VLA-1, VCAM- 1, CD30 Ligand (CD30L), CD40 Ligand (CD40L), 4-1BB Ligand (4-1BBL), OX40 ligand, CD70, CD24, LIGHT, and other cell adhesion proteins and other cell surface proteins that can activate T cell co-stimulatory pathways through T cell surface proteins.

Administration of Therapy

As described herein, anti-cancer therapy can be achieved by administering a combination of a modified heat shock protein with:

(a) one or more checkpoint inhibitors;

(b) one or more T cell co-stimulators; or

(c) one or more checkpoint inhibitors and one or more T cell co-stimulators. The combination of a heat shock protein with one or both of a checkpoint inhibitor or a T cell co-stimulator may further include, or be used in combination with, other drugs, such as anti-proliferative compounds or drugs to control side-effects (e.g., anti-emetic agents).

In some embodiments, the combination of a modified heat shock protein with one or both of a checkpoint inhibitor or a T cell co-stimulator can be formulated and administered as two or more separate compositions, at least one of which contains at least one modified heat shock protein, and at least one of which contains at least one checkpoint inhibitor and/or at least one T cell co-stimulator. The separate compositions may be administered by the same or different routes, administered at the same time or different times, and administered according to the same schedule or on different schedules, provided the dosing regimen is sufficient to bring about the desired antiproliferative effect in the patient. When the agents are administered in serial fashion, it may prove practical to intercalate administration of the two drugs, wherein a time interval, for example a 0.1 to 48 hour period, separates administration of the two drugs.

In some embodiments, when treatment includes a modified heat shock protein and both a checkpoint inhibitor and a T cell co-stimulator, three separate compositions may be used. Thus, in some embodiments, each separate composition can contain only one of the active agents (e.g., one composition provides the modified heat shock protein, one composition provides the checkpoint inhibitor, and one composition provides the T- cell co-stimulator). In some embodiments, each separate composition can contain two or more of the active agents (e.g., one composition provides a modified heat shock protein and a checkpoint inhibitor, one composition provides a checkpoint inhibitor and a T-cell co-stimulator, and one composition provides a modified heat shock protein and a T-cell co-stimulator).

Alternatively, according to some embodiments, the combination of a heat shock protein with one or both of a checkpoint inhibitor or a T cell co-stimulator can be co- formulated and used as part of a single pharmaceutical composition or dosage form. In such compositions, the active agents may together comprise from 0.1 to 99.99 weight percent of the total composition. The compositions can be administered by any route and according to any schedule that is sufficient to bring about the desired therapeutic effect in the patient.

Routes of administration include enteral, such as oral, and parenteral, such as intravenous, intra-arterial, intradermal, intramuscular, intranasal, rectal, intraperitoneal, subcutaneous, and topical routes. In some embodiments, a composition providing the modified heat shock protein are administered intradermally. In some embodiments, a composition containing one or both of the checkpoint inhibitor(s) and the T cell co- stimulator can be administered intravenously.

It will be appreciated that "administered" means the act of making the active agents available to the patient such that a physiological effect is realized. Thus, in some embodiments the instillation of active agents in the body of the patient is in a controlled or delayed release formulation, with systemic or local release of the active agents occurring at a later time and/or over a prolonged time interval. Accordingly, in some embodiments, a depot of a first active agent may be administered to the patient and the composition comprising the other agent(s) may be administered prior to, subsequent to, or during the systemic release of the first agent.

In embodiments where the compositions are administered by injection, the needle size should be selected to minimize shear to protect the integrity of the cells (e.g., depending on the application, larger than 14, 16, 18, 20, 22, or 24 gauge). The compositions can be administered in multiple injections (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 45, 50, 2 to 6, 5 to 10, 8 to 14, 12 to 20, 18 to 30, or 25 to 50 injections) or by continuous infusion (e.g., using a pump) at multiple sites (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 2 to 4, 3 to 5, 4 to 6, 5 to 10, or 8 to 14 sites). In some embodiments, cutaneous injections are performed at multiple body sites to reduce the extent of local skin reactions. On a given vaccination day, the patient can receive the assigned total dose of cells administered from one syringe in 3 to 5 separate intradermal injections of the dose (e.g., at least 0.4 ml, 0.2 ml, or 0.1 ml) each in an extremity spaced at least about 5 cm (e.g., at least 4.5, 5, 6, 7, 8, 9, or 10 cm) at needle entry from the nearest neighboring injection. On subsequent vaccination days, the injection sites may be rotated to different limbs in a clockwise or counter-clockwise manner.

By "pharmaceutically acceptable carrier" is meant any carrier, diluent or excipient that is compatible with the other ingredients of the formulation and not deleterious to the recipient. The active agents, whether as separate compositions or a combined composition, may be formulated into dosage forms according to standard practices in the field of pharmaceutical preparations. See, e.g., Remington's

Pharmaceutical Sciences, 18th Ed., 1990, Alphonso Gennaro, ed., Mack Publishing Co., Easton, PA. Suitable dosage forms may include, for example, tablets, capsules, solutions, parenteral solutions, troches, suppositories, or suspensions.

For parenteral administration, the active agents may be mixed with a suitable carrier or diluent such as water, an oil (particularly a vegetable oil), ethanol, saline solution, aqueous dextrose (glucose) and related sugar solutions, glycerol, or a glycol such as propylene glycol or polyethylene glycol. Solutions for parenteral administration can contain a water-soluble salt of the active agents. Stabilizing agents, antioxidant agents and preservatives also may be added. Suitable antioxidant agents include sulfite, ascorbic acid, citric acid and its salts, and sodium EDTA, for example. Suitable preservatives include, e.g., benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol. The composition for parenteral administration may take the form of an aqueous or nonaqueous solution, dispersion, suspension, or emulsion.

For oral administration, the active agents may be combined with one or more solid inactive ingredients for the preparation of tablets, capsules, pills, powders, granules or other suitable oral dosage forms. For example, the active agent may be combined with one or more excipients, such as fillers, binders, humectants, disintegrating agents, solution retarders, absorption accelerators, wetting agents, absorbents, or lubricating agents. According to one tablet embodiment, the active agent may be combined with carboxymethylcellulose calcium, magnesium stearate, mannitol, and starch, and then

Ϊ formed into tablets by conventional tableting methods.

For intravenous or intradermal administration, the composition can be formulated in an aqueous solution.

The specific doses of the active agents employed in the compositions and methods to obtain the therapeutic benefit will, of course, be determined by the particular circumstances of the individual patient. Such circumstances include the size, weight, age and sex of the patient, the nature and stage of the disease, the aggressiveness of the disease, and the route of administration.

The doses selected also will depend on the particular compound being used and the route and frequency of administration. In general, suitable doses for human administration range from about 5 to about 400 mg/m², (e.g., about 50, 100, 200, or 300 mg/m², about 10 to 100 mg/m², or about 10, 20, 30, 50, 60, 85, or 100 mg/m²).

Typically, treatment can be given weekly, or every two, three, or four weeks, with individual treatments including an infusion of one or more doses (e.g., up to about seven daily bolus doses).

When one or more modified heat shock proteins are used in combination with one or more checkpoint inhibitors and/or one or more T-cell co-stimulators in the compositions and methods described herein, it is envisaged that the dose of the active agents used may be comparable to those that have been found safe and effective with the compound alone or in other combinations with other agents. However, the ability to use lower doses of the active agents in the combination is envisaged, and may be necessary due to the surprising enhancement of efficacy observed in the combination as compared to when the active agents are used alone. Kits

This document also provides kits for carrying out the therapeutic regimens described herein. Such kits can include, in one or more containers, therapeutically or prophylactically effective amounts of the active agents in pharmaceutically acceptable form. In some embodiments, a kit can further include, in a container, a pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.), preferably sterile, for reconstituting the complex to form a solution for injection purposes. In some embodiments, a kit can further include a needle or syringe, preferably packaged in sterile It

form, for injecting the complex, and/or a packaged alcohol pad. Instructions optionally can be included for administration of the active agents.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

An exemplary experiment included tumor inoculation (B16-ova) to mice containing both OT-I and OT-II T cells, followed by therapeutic vaccination with or without a checkpoint inhibitor or T cell co-stimulatory antibody. At Day -2, an adoptive transfer of a mixed population of 2 million OT-II and 1 million OT-I was performed. At day 0, subcutaneous injection of 250,000 B16-ova cells in the flank of each mouse was performed. At day 9, each mouse was treated with 3T3-ova-gp96 or control +/- antibody (100 ug per antibody). At days 13 and 17, this treatment was repeated with antibody only. OT-I, OT-II and Treg were monitored by daily bleeds along with tumor area. Survival was measured when the tumor diameter exceeded 15 mm in the longer of two perpendicular measurements.

GITR

Post tumor inoculation, % survival was measured after treatment with or without GITR as shown in FIG. 1. Control animals reached 0% survival first, at about 21 days. Animals treated with GITR reached 0% survival at about 22 days. Animals treated with 3T3-ova-gp96 plus GITR reached 0% survival at about 28 days.

Post tumor inoculation, OT-I/total CD8+ was measured as shown in FIG. 2. Data for the control animals are shown in the lowest trace at the time point between 10 and 15 days. The GITR data are shown as the highest trace at the time point at about 11 days. The 3T3-ova-gp96 data are shown as the highest trace at the 20 day point.

OX40

Post tumor inoculation, % survival was measured after treatment with or without OX40, as shown in FIG. 3. Control animals reached 0% survival first, at about 21 days. Animals treated with OX40 reached 0% survival at about 30 days, as did animals treated with 3T3-ova-gp96 + OX40 data. Animals treated with 3T3-ova-gp96 alone reached 0% survival at about 28 days. 1^"

Post tumor inoculation, OT-I/total CD8+ was measured as shown in FIG. 4. Data for control animals are shown in the lowest trace at the time point between 15 and 20 days. The OX40 data are shown in the trace that hits about 2 on the y-axis at the 13 day time point. The 3T3-ova-gp96 data are shown in the trace that hits about 2 on the y-axis at the 20 day time point. The 3T3-ova-gp96 +OX40 data are shown in the highest (dotted) trace at the 15 day time point.

4-1BB

Post tumor inoculation, % survival was measured after treatment with or without 4-1BB, as shown in FIG. 5. Control animals reached 0% survival at about 21 days.

Animals treated with 4- IBB reached 0% survival at about 30 days, as did animals treated with 3T3-ova-gp96 + 4-1BB. Mice treated with 3T3-ova-gp96 alone reached 0% survival at about 27 days.

Post tumor inoculation, OT-I/total CD8+ was measured as shown in FIG. 6. The control data is the black line. The 4- IBB data are shown in the lowest trace at about the 12 day time point. The 3T3-ova-gp96 data are shown in the highest trace at the 15 day time point. The 3T3-ova-gp96 +4-1BB data are shown in the (dotted) trace that hits about 3 at about 13 days.

These data indicate that expansion of tumor specific CD8+ T cells was increased in both magnitude and duration as a result of the combination treatment.

All references discussed herein are incorporated by reference. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a cancer in a human subject, the method comprising administering to the subject a therapeutically effective amount of:

a vaccine comprising a plurality of host cells, each of the host cells co-expressing at least one tumor antigen and a heat shock protein modified to be secreted from each of the host cells; and

a composition comprising one or more checkpoint inhibitors.

2. A method of treating a cancer in a human subject, the method comprising administering to the subject a therapeutically effective amount of:

a composition comprising one or more T-cell co-stimulators.

3. A method of treating a cancer in a human subject, the method comprising administering to the subject a therapeutically effective amount of:

a vaccine comprising a plurality of host cells, each of the host cells co-expressing at least one tumor antigen and a heat shock protein modified to be secreted from each of the host cells;

a composition comprising one or more checkpoint inhibitors; and

a composition comprising one or more T-cell co-stimulators.

4. The method of any one of claims 1-3, wherein the cancer is selected from the group consisting of lung cancer, renal cell carcinoma, bladder cancer, colorectal cancer, melanoma, sarcoma, breast cancer, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, and nasopharyngeal cancer.

5. The method of any one of claims 1-3, wherein the cancer is a carcinoma or adenocarcinoma.

6. The method of any one of claims 1-3, wherein the cancer is non-small cell lung cancer ( SCLC).

7. The method of any one of claims 1-3, wherein a survival time of the subject is increased over the expected survival time for other subjects having the same type and stage of cancer.

8. The method of any one of claims 1-3, further comprising the step of analyzing CD8 T lymphocytes in the blood of the subject both before and after treatment.

9. The method of any one of claims 1-3, wherein the host cell is a cancer cell.

10. The method of any one of claims 1-3, wherein the cancer in the subject is a lung cancer and the host cells are lung cancer cells.

1 1. The method of any one of claims 1-3, wherein the cancer in the subject is a non-small cell lung cancer and the host cells are non-small cell lung cancer cells.

12. The method of any one of claims 1-3, wherein the host cells are allogeneic to the subject.

13. The method of any one of claims 1-3, wherein the host cells are irradiated before administration of the vaccine.

14. The method of any one of claims 1-3, wherein the host cells are administered intradermally.

15. The method of claim 2 or claim 3, wherein the one or more T-cell co-stimulators is selected from the group consisting of B7-1, B7-2, B7-h/B7rp-l, CD48, GITR, ICAM-1, ICAM-2, ICAM-3, LFA-1, LFA-2, LFA-3, VLA-1, VCAM-1, CD30 Ligand (CD30L), CD40 Ligand (CD40L), 4- IBB Ligand (4-1BBL), OX40 ligand, CD70, CD24, LIGHT, and other cell adhesion proteins and other cell surface proteins that can activate T cell co-stimulatory pathways through T cell surface proteins.

16. The method of claim 2 or claim 3, wherein the one or more T-cell co-stimulators is OX40 ligand.

17. The method of claim 2 or claim 3, wherein the one or more T-cell co-stimulators IBB Ligand.

18. The method of claim 1 or claim 3, wherein the one or more checkpoint inhibitors is selected from the group consisting of nivolumab (MDX-1 106; BMS-936558), BMS-936559, pembrolizumab (lambrolizumab; MK3475), pidilizumab (CT-01 1), AMP -224 and

MEDI4736 (PD1 antibodies), ipilmumab and tremelimumab (CTLA4 antibodies),

MPDL3280A and MDX-1105 (PDLl antibodies), IMP321 (LAG3 antibody), and MGA271 (B7-H3 antibody).

19. The method of claim 1 or claim 3, wherein the one or more checkpoint inhibitors is selected from the group consisting of CTLA-4, PD-1, PD-L1, LAG-3, IDO, TGF-beta, and TIM-3.

20. A composition comprising:

one or more checkpoint inhibitors.

21. A composition comprising:

one or more T-cell co-stimulators.

22. A composition comprising:

one or more checkpoint inhibitors; and

one or more T-cell co-stimulators.

23. The composition of any one of claims 20-22, wherein the composition is for use in treating a cancer in a human subject.

24. The composition of claim 23, wherein the cancer is selected from the group consisting of lung cancer, renal cell carcinoma, bladder cancer, colorectal cancer, melanoma, sarcoma, breast cancer, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, and

nasopharyngeal cancer.

25. The composition of claim 23, wherein the cancer is a carcinoma or adenocarcinoma.

26. The composition of claim 23, wherein the cancer is NSCLC.

27. The composition of any one of claims 20-22, wherein the host cell is a cancer cell.

28. The composition of any one of claims 20-22, wherein the host cells are non-small cell lung cancer cells.

29. The composition of any one of claims 20-22, wherein the host cells are irradiated.

30. The composition of claim 20 or claim 21 , wherein the one or more T-cell co- stimulators is selected from the group consisting of B7-1, B7-2, B7-h/B7rp-l, CD48, GITR, ICAM-1, ICAM-2, ICAM-3, LFA-1, LFA-2, LFA-3, VLA-1, VCAM-1, CD30 Ligand (CD30L), CD40 Ligand (CD40L), 4-1BB Ligand (4-1BBL), OX40 ligand, CD70, CD24, LIGHT, and other cell adhesion proteins and other cell surface proteins that can activate T cell co-stimulatory pathways through T cell surface proteins.

31. The composition of claim 20 or claim 21 , wherein the one or more T-cell co- stimulators is OX40 ligand.

32. The composition of claim 20 or claim 21, wherein the one or more T-cell co- stimulators is 4- IBB Ligand.

33. The composition of claim 20 or claim 22, wherein the one or more checkpoint inhibitors is selected from the group consisting of nivolumab (MDX-1 106; BMS-936558), BMS-936559, pembrolizumab (lambrolizumab; MK3475), pidilizumab (CT-01 1), AMP-224 and MEDI4736 (PD1 antibodies), ipilmumab and tremelimumab (CTLA4 antibodies), MPDL3280A and MDX-1105 (PDLl antibodies), IMP321 (LAG3 antibody), and MGA271 (B7-H3 antibody).

34. The composition of claim 20 or claim 22, wherein the one or more checkpoint inhibitors is selected from the group consisting of CTLA-4, PD-1, PD-L1, LAG-3, IDO, TGF-beta, and TIM-3.