This application contains a Sequence Listing in computer readable form submitted herewith as an ASCII text file (having a file name of “AE_02_SEQ_11-12-2021_TO-FILE.TXT”, a file creation date of Nov. 12, 2021 and a file size of 12,249 bytes), which is hereby incorporated by reference herein.
Throughout this application various publications are referred to in parentheses or superscript. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Cancer remains a major health concern in the U.S. and abroad. In 2011, there were an estimated 13,397,159 people living with cancer in the United States. Based on age-adjusted data from 2007-2011, the number of new cases of cancer per year was 460.4 per 100,000 men and women. The number of deaths per year was 173.8 per 100,000 men and women. Approximately 40.4 percent of men and women are expected to be diagnosed with cancer at some point during their lifetime, based on 2009-2011 data. It is expected that annual cancer cases will rise from 14 million in 2012 to 22 million within the next 2 decades (World Cancer Report 2014).
Success of cancer immunotherapy is hindered by two major problems. One problem is that tumor-associated antigens (TAA), used in cancer vaccines, are often self-antigens that are overexpressed or mutated in tumor cells compared to normal cells. The T cells in the thymus have been taught earlier in life not to react to self-antigens, and therefore it is difficult to induce strong T cell responses to TAA. The other problem is that most cancer patients are old, and the elderly react less efficiently to vaccines than young adults. This is often due to lack of naïve T cells (only generated at young age, and are used during life) that react for the first time to a new antigen and are responsible for the generation of memory T cells upon repeated exposures with the same antigen. The present invention addresses both of these problems and the need for improved treatments for cancers and in particular for improved treatments for metastases.
The present invention provides methods of treating tumors in a subject, and/or reducing or preventing metastasis of tumors in a subject, comprising administering to the subject an attenuated bacteria that expresses a recall antigen in an amount effective to treat the tumor, and/or to reduce or prevent metastasis of the tumor.
Also provided are pharmaceutical compositions and cancer vaccines comprising an attenuated bacteria that expresses a recall antigen.
The present invention provides a method of treating a tumor in a subject, and/or reducing the incidence or likelihood of metastasis of a tumor in a subject, comprising administering to the subject an attenuated bacteria that expresses a recall antigen in an amount effective to treat the tumor, and/or to reduce the incidence or likelihood of metastasis of the tumor.
The bacteria can be, for example, one or more of Listeria monocytogenes, Salmonella thyphimurium, Vibrio cholera, Clostridium, and Bifidobacterium breve. In a preferred embodiment, the bacteria are Listeria monocytogenes. The bacteria are attenuated to reduce or eliminate virulence. As used here, attenuated Listeria, for example, is denoted as Listeriaat.
As used herein, a recall antigen is an antigen to which a subject has previously been exposed earlier in life. Recall antigens can include, for example, antigens used for childhood vaccinations, such as tetanus toxoid, measle virus, and poliovirus antigens. Most individuals have been vaccinated and boosted with these antigens during childhood, resulting in memory T cells that circulate in their blood stream for life. These memory T cells can be reactivated at any age, even in a tumor microenvironment.
Examples of recall antigens that can be used include, but are not limited to, an epitope or an fragment containing one or more immunodominant epitopes of one or more of tetanus toxoid, measle virus, and polio virus. In a preferred embodiment, the antigen is a tetanus toxoid fragment containing one or more immunodominant epitopes.
This principle is not only applicable to childhood antigens, but to almost any immunogenic antigen that patients have seen earlier in life. For example, up to 70% of all women acquire a Candida albicans infection earlier or later in life (1), which expresses highly immunogenic proteins including heat-shock protein (Hsp)70(2). On the other hand, flu virus is less suitable because of their continuous antigenic drift. Basically, the number of immunogenic antigens to be used for this approach is unlimited.
As an example, shown below are the Tetanus toxoid (TT) (aa position 856-1313) amino acid (upper case) (SEQ ID NO:5) and DNA (lower case) (SEQ ID NO:6) sequence cloned into Listeria (see Experimental Details). The underlined and bold portions of the DNA sequence represent primer sequences used for cloning TT into Listeria. The underlined and bold portions of the amino acid sequence represent CD8 epitopes in TT immunodominant in the Panc-02 model (C57B16 mice). The portion of the amino acid sequence in italics and bold font represents the CD8 epitope in TT immunodominant in the 4T1 model (BALB/c mice).
tcaacaccaattccattt
tcttattctaaaaatctggattgttgggttgataatgaagaa
Also as an example, shown below are the poliovirus (PV) (aa position: 49-273 in VP1) amino acid (SEQ ID NO:7) and DNA (SEQ ID NO:8) sequence cloned into Listeria. The underlined and bold portions of the DNA sequence represent primer sequences used for cloning PV VP1 into Listeria. The portions of the amino acid sequence in italics and bold font represent CD8 epitopes in PV VP1 immunodominant in the 4T1 model (BALB/c mice/H2-d haplotype).
ag
gtcaaggtcagagtctagc
atagagtctttcttcgcgcggggtgcatgcgtg
gatggtacgcttacaccc
3′
As a further example, shown below are the measlevirus (MV) amino acids (Nucleocapsid aa position: 38-351) amino acid (SEQ ID NO:9) and DNA (SEQ ID NO:10) sequence cloned into Listeria. The underlined and bold portions of the DNA sequence represent primer sequences used for cloning of the MV sequence into Listeria. The portions of the amino acid sequence in italics and bold font represent CD8 epitopes in MV immunodominant in the 4T1 model (AKR mice/H2-k haplotype).
tcc
tcaattaccactcgatccagacttctggaccggttggtcaggttaattggaaacccg
The tumor can be, for example, a tumor of one or more of the pancreas, ovary, uterus, neck, head, breast, prostate, liver, lung, kidney, neurones, glia, colon, testicle, or bladder. The tumor can be an inoperable tumor.
Preferably, prior to administration to the subject, the bacteria are cultured in yeast medium.
The method can further comprise administering Cytosine-phosphate-Guanine (CpG) to the subject as an adjuvant.
In one embodiment, prior to administration of bacteria to the subject, the subject is screened for their major histocompatibility complex (MHC) 1 haplotype and administered an antigen for which the subject shows a CD8 T cell recall response.
In one embodiment, prior to administration of bacteria to the subject, an epitope of the antigen is administered to the subject to generate memory T cells to the antigen. This method will be less effective in older subjects who have fewer naïve T cells than younger subjects.
Bacteria can be administered by different routes to the subject. For example, bacteria can be administered systemically to the subject, such as for example, by intravenous administration. Bacteria can be administered by direct injection to a tumor site in the subject. Myeloid-derived suppressor cells (MDSCs) can be used to deliver attenuated bacteria to the microenvironment of both primary and metastatic neoplastic lesions, where the attenuated bacteria spread from MDSCs into tumor cells (see, e.g., 10). The infected tumor cells then become a target for activated immune cells.
Preferably, the subject receives repeated administrations of attenuated bacteria that expresses the recall antigen. For example, the administration may be daily or every other day, for a period of several days until a satisfactory therapeutic outcome is achieved.
As used herein, “treating” a tumor means that one or more symptoms of the disease, such as the tumor itself, metastasis thereof, vascularization of the tumor, or other parameters by which the disease is characterized, are reduced, ameliorated, placed in a state of remission, or maintained in a state of remission. “Treating” a tumor also means that one or more hallmarks of the tumor may be eliminated or reduced by the treatment. Non-limiting examples of such hallmarks include uncontrolled degradation of the basement membrane and proximal extracellular matrix, migration, division, and organization of the endothelial cells into new functioning capillaries, and the persistence of such functioning capillaries. Preferably, the method is effective to reduce tumor growth and/or size.
As used herein, reducing or preventing metastasis of a tumor means that any of the symptoms of the disease, such as the metastases, the extent of spread thereof, the vascularization of the metastases or other parameters by which the disease is characterized are reduced, ameliorated, prevented, placed in a state of remission, maintained in a state of remission, or eliminated. Preferably, the method is effective to reduce metastases. The method can reduce the incidence or likelihood of metastasis of a tumor.
The method can further comprise administering to the subject a chemotherapeutic agent that reduces the number of myeloid-derived suppressor cells (MDSCs). Such chemotherapeutic agents include, for example, gemcitabine, Vitamin A derivates, Amiloride, CpG oligodeoxynucleotide (CpG ODN), Docetaxel, 5-Fluorouracil, GW2580, Sildenafi and Sinitinib (3, 4).
The subject can be a mammal. In different embodiments, the mammal is a mouse, rat, cat, dog, horse, donkey, mule, sheep, goat, cow, steer, bull, livestock, primate, monkey, or preferably a human. The human can be of different ages, such as for example, a person 60 years of age or older.
Also provided is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and attenuated bacteria that expresses a recall antigen. The bacteria can be, for example, one or more of Listeria monocytogenes, Salmonella thyphimurium, Vibrio cholera, Clostridium, and Bifidobacterium breve. The recall antigen can be, for example, an epitope of one or more of tetanus toxoid, measle virus, and polio virus.
Examples of acceptable pharmaceutical carriers include, but are not limited to, additive solution-3 (AS-3), saline, phosphate buffered saline, Ringer's solution, lactated Ringer's solution, Locke-Ringer's solution, Krebs Ringer's solution, Hartmann's balanced saline solution, and heparinized sodium citrate acid dextrose solution. The pharmaceutically acceptable carrier used can depend on the route of administration. The pharmaceutical composition can be formulated for administration by any method known in the art, including but not limited to, oral administration, parenteral administration, intravenous administration, transdermal administration, intramuscular administration, intranasal administration, direct injection into a tumor site, and administration through an osmotic mini-pump.
Also provided is a cancer vaccine comprising attenuated bacteria that expresses a recall antigen. The bacteria can be, for example, one or more of Listeria monocytogenes, Salmonella thyphimurium, Vibrio cholera, Clostridium, and Bifidobacterium breve. The recall antigen can be, for example, an epitope of one or more of tetanus toxoid, measle virus, and polio virus.
This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.
Listeria constructs were developed that express antigens with immunodominant epitopes of childhood recall antigens tetanus toxoid (TT), measle virus (MV), and poliovirus (PV). Repeated immunizations with Listeria-TT in mice with memory T cells to the TT nearly completely eliminates metastases in mice with metastatic breast cancer cancer without side effects. Listeria-TT combined with gemcitabine in mice with pancreatic cancer was even more effective that Listeria-TT alone, most likely because gemcitabine reduces immune suppression through the elimination of myeloid-derived suppressor cells (MDSCs).
Development of the Listeriaat-TT856-1313 vaccine. The Listeria-TT vaccine was developed as described below. The TT856-1313 (62 kDa) was cloned as a fusion-protein with a truncated Listeriolysin O (LLO)(48 kDa) in the Listeriaat vector (pGG34) under the control of the LLO promoter (P), and a myc sequence for detection of the TT protein (
Generation of CD8 T cell responses to immunodominant epitope in TT856-1313 protein. It was tested whether the TT856-1313 protein induced CD8 T cell responses to the immunodominant epitope of TT856-1313. For this purpose, BALB/cByJ mice were immunized three times with TT856-1313 protein and CpG. Two days after the last immunization, mice were euthanized and white blood cells were restimulated with an immunodominant peptide GYNAPGIPL1228-1236 (SEQ ID NO:1) within the TT856-1313 protein (7). CD8 T cells were activated against the immunodominant T1228-1236 epitope in blood of BALB/cByJ mice (
Vaccination with Listeriaat-TT856-1313 is highly effective against metastases in breast cancer model 4T1. The efficacy of the Listeria-TT856-1313 vaccine was tested against metastatic breast cancer in the 4T1 model. First, memory T cells to the immunodominant CD8 T cell epitope were generated with TT856-1313 protein and CpG. Then, 4T1 tumor and metastases were generated by injection of the 4T1 cell line into the mammary fat pad, and Listeriaat-TT vaccinations were administered every other day for two weeks after the tumor size had reached 5 mm. Listeriaat-TT856-1313 was highly effective against the metastases (
Listeria-TT and gemcitabine is highly effective against metastases and tumors in mice with pancreatic cancer. In clinical trials of Listeria-recall antigens in patients with pancreatic cancer, the patients would be expected to be treated with gemcitabine. Therefore, it was tested whether gemcitabine affected Listeria-recall antigen immunizations. Since gemcitabine is known for eliminating MDSCs, which are a major contributor to immune suppression, it was expected that gemcitabine will reduce immune suppression and the recall antigens can do their job better.
Listeria was starved in saline for 30 min, and subsequently cultured in yeast medium (keeps Listeria alive but Listeria does not replicate) for 60 min. This treatment allowed the injection of 107 CFU of Listeria every day instead of 104 CFU every day. Then, Listeria-TT was tested in combination with gemcitabine (Gem). Mice with pancreatic cancer were treated with gemcitabine ip (1.2 mg/300 μl per dose; every 3rd day, starting day 3 after tumor cell injection), followed by Listeria-TT ip starting on day 10 after tumor cell injection (107 CFU every day for 4 days, followed by a rest period of 3 days, followed by another 3 injections with 107 CFU of Listeria-TT). All mice were euthanized on day 21. Untreated mice will die between day 21-28 after tumor cell injection in this highly aggressive pancreatic cancer model. The combination of gemcitabine and Listeria-TT eliminated metastases completely and primary tumors nearly completely (
Table 2 Shows T cell responses in the Panc-02 mice treated with Listeria-TT and Gemcitabine. The T cell responses in Listeria-TT and Gemcitabine is far better than in the separate groups.
Table 3 Shows T cell responses in the KPC mice treated with Listeria-TT and Gemcitabine. The T cell responses in Listeria-TT and Gemcitabine is far better than in the separate groups.
Table 4 shows that the combination of Listeria-TT and Gemcitabine reduces inhibitory cytokines produced by MDSC and TAM, and improve expression levels of CD80 involved in T cell stimulation.
Panc-02 mice with advanced pancreatic cancer were treated with one high and multiple low doses of Listeria-TT (LM-TT) and GEM as described in
KPC mice with advanced pancreatic cancer were treated with one high and multiple low doses of Listeria-TT (LM-TT) and GEM as described in
Panc-02 mice with advanced pancreatic cancer were treated with one high and multiple low doses of Listeria-TT (LM-TT) and GEM as described in
The success of cancer immunotherapy has been hindered by two major problems. One problem is that tumor-associated antigens (TAA), used in cancer vaccines, are often self-antigens that are overexpressed or mutated in tumor cells compared to normal cells. The T cells in the thymus have been taught earlier in life not to react to self-antigens, and therefore it is difficult to induce strong T cell responses to TAA. The other problem is that most cancer patients are old, and the elderly react less efficiently to vaccines than young adults. This is often due to lack of naïve T cells (only generated at young age, and are used during life) that react for the first time to a new antigen and are responsible for the generation of memory T cells upon repeated exposures with the same antigen. None of the vaccines currently available avoids the need of naïve T cells at an older age, and none of the vaccines allow delivery of highly immunogenic recall antigens directly into tumor cells by live attenuated bacteria.
The present approach overcomes the problem of poorly immunogenic antigens in cancer vaccination by using highly immunogenic recall antigens, and at the same time avoids the need of naive T cells in older age. The present procedure involves reactivating memory T cells to foreign highly immunogenic antigens to which most individuals have been exposed during childhood when plenty of naïve T cells are available, such as tetanus toxoid (TT), measle virus (MV), polio virus (PV) antigens, and by the selective delivery of these antigens into tumor cells by an attenuated non-toxic and non-pathogenic bacterium, such as Listeria monocytogenes. These memory T cells will now kill infected tumor cells presenting the highly immunogenic antigens. In previous studies, Listeria has been used for the selective delivery of anticancer agents to the tumor microenvironment and into tumor cells of metastases and tumors. Listeria was effectively cleared by the immune system in normal tissue but not in the heavily immune-suppressed microenvironment of metastasis and primary tumor (10, 11).
However, immune suppression may not be completely overcome by this treatment. This problem can be resolved by combining the Listeria-recall antigens with a chemotherapeutic, such as gemcitabine, that reduces the number of myeloid-derived suppressor cells (MDSCs). MDSCs are the most important contributor to immune suppression in the tumor microenvironment.
All together, these results are very impressive. Also, the mechanism that Listeria-induced ROS improves gemcitabine sensitivity is very important because most clinicians don't want to stop gemcitabine treatment in pancreatic cancer patients. And most important, the combination therapy is effective against advanced pancreatic cancer, and can work at young and old age, because elderly patients lack naïve T cells (required to develop memory T cells). With the present approach, one immediately reactivates memory T cells to tetanus toxoid antigens, measle virus antigens and poliovirus antigens, and avoids the need of naïve T cells at older age. Since the Listeria with the recall antigens selectively infect tumor cells in vivo, the memory T cells that circulate in blood for life can now kill the infected tumor cells. These memory T cells were generated during childhood with the childhood vaccines.
The most obvious uses of the present invention are treatment of types of cancer for which there are practically no effective treatments, such as pancreatic cancer, which is almost always detected in metastatic form, or ovarian cancer. Good candidates also include cancers for which surgery to remove the primary tumor is often not an option because of tumor location, such as head and neck cancers or inoperable hepatocellular carcinoma. A third cohort of patients that would be expected to benefit from such therapy are patients with various types of metastatic disease, which is recurrent or refractory to standard treatments, such as for example lung and colon cancers as well as breast cancer.
This application is a divisional application of U.S. patent application Ser. No. 15/568,491, filed Oct. 23, 2017, which is a U.S. national stage entry under 35 U.S.C. § 371 of PCT International Patent Application No. PCT/US2016/029283, filed Apr. 26, 2016, which claims the benefit of U.S. Provisional Patent Application No. 62/153,728, filed Apr. 28, 2015, the contents of each of which are hereby incorporated by reference herein.
Number | Date | Country | |
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62153728 | Apr 2015 | US |
Number | Date | Country | |
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Parent | 15568491 | Oct 2017 | US |
Child | 17527488 | US |