Method of treatment
阅读说明：本技术 治疗方法 (Method of treatment ) 是由 P·赫佐格 Z·马克斯 N·伯克 S·S·林 N·德维尔德 N·曼干 A·马修斯 于 2018-01-30 设计创作，主要内容包括：本发明涉及使用干扰素ε(IFNε)治疗疾病的方法,其中所述IFNε包括组合物中各种天然的、合成的和重组IFNε。(The present invention relates to methods of treating diseases using interferon epsilon (IFN epsilon), including various natural, synthetic and recombinant IFN epsilon in compositions.)
1. A method for inhibiting a cancer cell in a subject, the method comprising exposing the cancer cell to an amount of interferon epsilon (IFN epsilon) or a functional natural or synthetic variant or hybrid form thereof or an inducer of IFN epsilon expression or IFN epsilon activity effective to indirectly or indirectly induce apoptosis of cancer cell proliferation, motility, and/or migration.
2. The method of claim 1, wherein the IFN epsilon is derived from a species homologous to the species of the subject being treated.
3. The method of claim 1, wherein the IFN epsilon is derived from a species heterologous to the species of the subject being treated.
4. The method of claim 1 or 2 or 3, wherein the subject is a human.
5. The method of claim 4 wherein the IFN epsilon is selected from the group consisting of an inducer of recombinant human IFN epsilon or IFN epsilon expression; an inducer of recombinant non-human IFN epsilon or Ifn epsilon expression; and hybrids between human and non-human IFN epsilon.
6. The method of claim 5 wherein said IFN epsilon is a hybrid between human and murine IFN epsilon.
7. The method of any one of claims 1 to 6, wherein the cancer cells are derived from epithelial tissue, connective tissue, glandular tissue, embryonic tissue, hematopoietic cells, lymphoid tissue or bone marrow or cells from which such cells are derived.
8. The method of claim 7, wherein the cell is a cancer cell from ovary, uterus, oviduct, endometrium, placenta, breast, testis, prostate, brain, stomach, liver, spleen, pancreas, thymus, colon, lung, kidney, heart, thyroid, or smooth muscle.
9. The method of claim 8, wherein the cell is an ovarian cancer cell.
10. The method of claim 9, wherein the ovarian cancer cells are low-grade to high-grade serous cancer cells.
11. The method of claim 10, wherein the ovarian cancer cells are higher serous carcinoma cells.
12. The method of any one of claims 1-11, wherein the IFN epsilon or variant, hybrid, or inducer is used in combination with another anti-cancer agent.
13. The method of claim 12, wherein the anti-cancer agent is selected from the group consisting of chemotherapeutic agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, steroids, sex hormones or hormone-like drugs, alkylating agents, nitrogen mustards, nitrosoureas, hormone agonists, and microtubule inhibitors.
14. The method of any one of claims 1-13, wherein the amount of IFN epsilon or the variant or hybrid is from 10 IU/dose to 10 IU/dose6IU/dose.
Use of an IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or an inducer of IFN epsilon expression or IFN epsilon activity in the preparation of a medicament for treating cancer in a subject.
16. An inducer of IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or IFN epsilon expression or IFN epsilon activity for use in treating cancer in a subject.
17. The use of claim 15 or the IFN epsilon or functional natural or synthetic variant or hybrid form thereof or inducer of IFN epsilon expression or IFN epsilon activity of claim 36 in which the IFN epsilon is derived from a species homologous to the species of the subject to be treated.
18. The use of claim 15 or the IFN epsilon or functional natural or synthetic variant or hybrid form thereof or inducer of IFN epsilon expression or IFN epsilon activity of claim 16 in which the IFN epsilon is derived from a species heterologous to the species of the subject to be treated.
19. The use of claim 16 or 17 or 18 or an inducer of IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or IFN epsilon expression or IFN epsilon activity, wherein the subject is a human.
20. The use of claim 19 or an inducer of IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or IFN epsilon expression or IFN epsilon activity, wherein the IFN epsilon is selected from the group consisting of recombinant human IFN epsilon; recombinant non-human IFN epsilon; and hybrids between human and non-human IFN epsilon.
21. The use of claim 20 or an inducer of IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or IFN epsilon expression or IFN epsilon activity, wherein the IFN epsilon is a hybrid between human and murine IFN epsilon.
22. The use of any one of claims 15 to 21 or IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or an inducer of IFN epsilon expression or IFN epsilon activity, wherein the cancer is a cancer of epithelial tissue, connective tissue, glandular tissue, embryonic tissue, hematopoietic cells, lymphoid tissue or bone marrow.
23. The use of claim 22 or an IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or an inducer of IFN epsilon expression or IFN epsilon activity, wherein said cancer is located in ovary, uterus, oviduct, endometrium, placenta, breast, testis, prostate, brain, stomach, liver, spleen, pancreas, thymus, colon, lung, kidney, heart, thyroid, or smooth muscle.
24. The use of claim 23 or IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or an inducer of IFN epsilon expression or IFN epsilon activity, wherein the cancer is ovarian cancer.
25. The use of claim 24 or an inducer of IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or IFN epsilon expression or IFN epsilon activity, wherein the ovarian cancer is a higher serous carcinoma.
26. Use or an inducer of IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or expression of IFN epsilon or activity of IFN epsilon wherein said use is an adjuvant for another anti-cancer agent.
27. The use of claim 26 or an inducer of IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or IFN epsilon expression or IFN epsilon activity, wherein the anti-cancer agent is selected from the group consisting of chemotherapeutic agents, antimetabolites, anti-tumor antibiotics, mitotic inhibitors, steroids, sex hormones or hormone-like drugs, alkylating agents, nitrogen mustards, nitrosoureas, hormone agonists and microtubule inhibitors.
28. An agent for the treatment of cancer, said agent comprising an inducer of IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or expression of IFN epsilon or activity of IFN epsilon and one or more carriers, adjuvants and/or excipients.
29. The formulation of claim 28, wherein the cancer is a cancer in ovary, uterus, fallopian tube, endometrium, placenta, breast, testis, prostate, brain, stomach, liver, spleen, pancreas, thymus, colon, lung, kidney, heart, thyroid, or smooth muscle.
30. The formulation of claim 29, wherein the cancer is ovarian cancer.
31. The formulation of any one of claims 21 to 30, in combination with an anti-cancer agent.
32. The formulation of claim 31, wherein the anti-cancer agent is selected from the group consisting of an anti-metabolite, an anti-tumor antibiotic, a mitotic inhibitor, a steroid, a sex hormone or hormone-like drug, an alkylating agent, a nitrogen mustard, a nitrosourea, a hormonal agonist.
Technical Field
The present invention relates to the field of cancer therapy and formulations therefor.
Background
Bibliographic details of the publications cited by authors in this specification are collected alphabetically at the end of the specification.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as, an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Cancer is a complex, multifaceted cellular disorder. It can lead to debilitating disease levels with potentially significant morbidity and mortality. The economic cost of cancer treatment in the healthcare sector is enormous, not to mention the emotional burden on individuals and families. Much effort has been devoted to understanding cancer biology and the endogenous and exogenous factors that impede its development. Despite the tremendous progress made over decades, further research is crucial in order to fully understand this disease.
For example, ovarian cancer is a heterogeneous disease that is complex involving many molecularly distinct tumors that are produced not only by ovarian cells, but also by cells of the fallopian tubes and/or surrounding tissues (Jayson et al (2014) the Lancet384(9951): 1376-88). Many women are diagnosed for the first time when advanced disease has been reached, and more than half of those who respond to treatment relapse and die within 5 years (AIHW. (2010) cancer 52 Cat No. cann 48).
The vast majority of ovarian cancers are of Epithelial Origin (EOC) and have the fourth highest mortality rate from female cancers (Jayson et al (2014) supra). EOC is classified based on histological subtypes, including mucinous, clear cell, endometrioid, and serous cancers, each associated with a different morphology, mutation profile, cell of origin, and prognosis. Serous carcinoma is the most commonly diagnosed EOC, and there is increasing evidence that EOC comes from the secretory epithelial lining of the distal fallopian tubes. Standard treatment options, surgical resection and platinum-based chemotherapy are generally ineffective because many women with advanced disease are not surgical candidates and chemosensation results in increased recurrence rates (Jayson et al (2014) supra).
Extensive molecular profiling analysis of ovarian Cancer has shown that mutations in the BRCA1/2 gene significantly increase the risk of higher serous carcinoma (HGSC), the most common and fatal EOC (Bowtell et al (2010) Nature Rev Cancer10(11): 803-8). Both BRCA1 and BRCA2 are documented Interferon (IFN) regulatory genes (IRGs) and play an important role in the pathway of homologous recombination repair of DNA (Venkitaraman (2014) Science 343(6178):1470-5), whose somatic and germline mutations contribute to overall chromosomal instability. Molecular profiling analysis has also found that higher serous carcinomas (HGSCs) with higher expression of immune-related genes such as CD8A, granzyme B and CXCL9, termed immune subtypes, exhibit optimal overall survival (Tothill et al (2008) Clin Cancer res.14(16): 5198-. Furthermore, although The role of hormones in ovarian cancer tumorigenesis remains unclear, there is evidence that poor prognosis in Progesterone Receptor (PR) -negative patients is not associated with Estrogen Receptor (ER) expression (Sieh et al (2013) The Lancet Oncology 14(9):853-62), with triple-negative breast cancer (TNBC) or estrogen receptor positive/progesterone receptor negative (ER)+/PR-) Breast cancer patients with cancerSimilar to the poor prognosis reported (Thakkar and Mehta (2011) Oncoloist 16(3): 276-85). There are currently many unknown drivers of both cancers, both of which are related to common elements of oncogene and tumor suppressor gene expression, hormone sensitivity, and immune cell involvement.
There is a need to further investigate the role of immune induction in modulating the development and treatment of ovarian cancer as well as other cancer types.
This is particularly the case with respect to the interaction between innate and adaptive immunity. The innate immune response represents a response to pre-existing, intrinsic, first-line and rapidly-induced defenses of the pathogen and to homeostatic cues (Mangan et al (2007) Eur J Immunol 37(5): 1302-12; Smith et al (2007) J Immunol 178(7): 4557-66). This is mediated by resident cells such as macrophages, Natural Killer (NK) and epithelial cells. Adaptive immune responses include recognition and response to antigens mediated by antibody-secreting B and T helper and effector lymphocytes, where the elicited response is gradual and specific. The adaptive response is formed by the innate system (sculpte). In the reproductive tract, the two arms of the immune system must balance the presence of an allogeneic fetus, which essentially contains "foreign" proteins and controls harmful pathogens, such as viruses and bacteria. It must also maintain homeostasis in the context of the cyclic hormonal environment and structural changes that occur in the mucosa.
Innate and adaptive immune cells of the Female Reproductive Tract (FRT) produce cytokines and chemokines and thereby influence a variety of reproductive processes including sperm migration, fertilization, implantation, endometrial remodeling, and immune responses to infection or other attacks (Salamonsen et al (2007) Semin Reprod Med 25(6): 437-44).
In its simplest form, the innate response includes physicochemical barriers such as mucus secretions, pH and redox status. In its most complex form, it is represented by an innate immune response that senses the pathogen and initiates a series of reactions within minutes, ultimately producing products such as antibacterial defensins, NOS enzymes, chemokines that recruit and activate inflammatory cells, and cytokines that regulate cell behavior. One class of inducers with pleiotropic activity is the type I Interferons (IFNs).
Clinical trials using type I IFN, particularly IFN α and IFN β, for ovarian Cancer have not given an impressive picture, mainly because of the dose-limiting toxicity that prevents high-dose therapy in advanced disease, as is the case with other solid tumors (Berek et al (1985) Cancer Res.45: 4447-53; Willemse et al (1990) Eur J Cancer oncol26(3): 353-8; Markman et al (1992) Gynecol Oncol.45(1): 3-8; Frasci et al (1994) Eur Jcancer30(7): 946-50; Bruzzone et al (1997) Gynecol Oncol.65(3): 499-505; Moore et al (1995) Gynecol 267.59 (2): 267-72; Berek et al (1999) Gynecol.75 (1): 10-75; Marek et al (1995) Gynecol 2004-59 (2): 10-72; Berek et al (1985) have been informed about the efficacy of successful treatment of ovarian Cancer using IFN-3653, Berek et al (1985) as a therapeutic mechanism that is nonetheless important to interfere with IFN-3653 and to treat ovarian Cancer.
IFN epsilon (IFN epsilon) is a type I IFN (Fung et al (2013) Science 339(123): 1088-.
Interestingly, unlike other type I IFNs, which remain at undetectable levels until pathogen induction, IFN epsilon has been found to be constitutively expressed predominantly in FRT organs such as uterus, cervix, vagina and ovaries. IFN epsilon is produced by the luminal and glandular epithelial cells of FRT and is not altered in the absence of hematopoietic cells.
In addition, IFN epsilon regulation is different from other type I IFN, unlike Ifn α and Ifn β, murine Ifn epsilon expression is essentially unchanged in response to pathogenic stimuli.
In contrast, IFN epsilon levels varied significantly at each stage of the murine estrus cycle, with expression levels during estrus 30-fold higher than during estrus, which is the expression pattern reflected in human tissue during the menstrual cycle. This suggests that unlike other type I IFNs, IFN epsilon is hormone regulated.
There is a need to investigate the role of IFN epsilon in cancer biology.
Disclosure of Invention
The nucleotide and amino acid sequences are represented by the sequence identification number (SEQ ID NO). The SEQ ID NO corresponds numerically to the sequence identifiers <400>1(SEQ ID NO:1), <400>2(SEQ ID NO:2), etc. An overview of the sequence identifiers is provided in table 2. The sequence listing is provided after the claims.
The invention is based, in part, on the determination that IFN epsilon has an inhibitory effect on cancer cells. Such inhibition includes inducing cancer cell death, directly or indirectly, by including apoptotic processes, and preventing includes slowing or inhibiting the development, proliferation, motility, and/or migration of cancer cells. The IFN epsilon may act directly on the cancer cells, or it may induce an immune response that is effected via the production of a particular cell type or regulator or other factor that in turn induces a cytotoxic or cytostatic effect on the cancer cells, or via cells in a matrix or component in the tumor cell environment. Although the present invention has been elucidated after studying ovarian cancer, the findings apply to other cancers of the Female Reproductive Tract (FRT) in any mammal, particularly humans, as well as to cancers elsewhere in female or male subjects.
Accordingly, the present invention provides methods of inhibiting the viability, growth, development and spread of cancer cells in a subject, including humans. This includes arresting, including slowing or inhibiting, the development, proliferation, movement, and migration of cancer cells.
Accordingly, taught herein are methods for inhibiting a cancer cell in a subject, the method comprising exposing the cancer cell to an amount of interferon epsilon (IFN epsilon) or a functional natural or synthetic variant or hybrid form thereof or an inducer of IFN epsilon expression or IFN epsilon activity effective to directly or indirectly induce apoptosis of the cancer cell or inhibit proliferation, motility, and/or migration of the cancer cell. This can lead to local growth and invasion of cancer cells and a reduction in their transfer to other parts of the body. By "exposure" in relation to cancer cells is meant direct or indirect exposure of the cancer cells or via other cells or components.
Further embodied herein are methods for treating a subject having cancer, the method comprising administering to the subject an effective amount of an IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or an inducer of IFN epsilon expression or IFN epsilon activity for a time and under conditions sufficient to directly or indirectly induce apoptosis of cancer cells or inhibit cancer cell proliferation, motility, and/or remission. This includes preventing cancer cell growth and development.
The specification describes the use of an inducer of IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or of IFN epsilon expression or IFN epsilon activity in the manufacture of a medicament for the treatment of cancer in a subject. In one embodiment, taught herein are inducers of IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or IFN epsilon expression or IFN epsilon activity for use in the treatment of cancer in a subject. The medicament comprises an anti-cancer vaccine comprising IFN epsilon or a variant or hybrid thereof or an inducer as a major active ingredient, or wherein it acts as an adjuvant for another anti-cancer agent. Examples of other anti-cancer agents that may be used in combination with IFN epsilon or variants or hybrids thereof or an inducer include chemotherapeutic agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, steroids, sex hormones or hormone-like drugs, alkylating agents, nitrogen mustards, nitrosoureas, hormone agonists and microtubule inhibitors. The recombinant cell can also be engineered to produce IFN epsilon or a variant, hybrid, or inducer thereof, or a recombinant virus engineered to direct an infected cell to produce IFN epsilon, a variant, hybrid, or inducer thereof. Engineered IFN epsilon includes IFN epsilon produced by optimizing codon expression and/or optimizing therapeutic activity.
An agent for the treatment of cancer, which agent comprises an inducer of IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or expression of IFN epsilon or activity of IFN epsilon and one or more carriers, adjuvants and/or excipients. IFN epsilon or functional natural or synthetic variants thereof or hybrid forms thereof may also be used as vaccine adjuvants in combination with anti-cancer agents or cancer cell modulating molecules.
Abbreviations used herein are defined in table 1.
TABLE 1
Abbreviations
Abbreviations
Definition of
EOC
Of epithelial origin
ER
Estrogen receptors
FCS
Fetal bovine serum
FRT
Female genital tract
HGSC
Advanced serous carcinoma
HuIFNε
Human interferon epsilon
IFN
Interferon
IFNε
Interferon epsilon
IRG
Interferon modulationNode gene
Ifnε
Gene encoding IFN epsilon
LGSC
Low grade serous carcinoma
MuIFNε
Mouse interferon epsilon
PEC
Peritoneal exudate cells
PR
Progesterone receptors
TNBC
Triple negative breast cancer
Drawings
Some graphs contain color representations or entities. Color photographs may be obtained from the patentee at the time of filing or from the appropriate patent office. If obtained from the patent office, a fee may be charged.
Figures 1A to C are diagrams showing that IFN epsilon and IFN β induce Interferon Regulatory Genes (IRG) in ID8 cells the figure shows a 3 hour dose response induced by CXCL10(a), lfit1(B) and Isg15(C) of 10-1000IU/ml IFN epsilon (left panel shown in black) and IFN β (right panel shown in grey) gene expression was measured by qRT-PCR, expression calculated by dCT normalized to 18s and relative expression shown here was determined in relation to expression at T0 data is shown as mean +/-SEM of 3 independent experiments, each experiment was performed in three technical parallel experiments (technical triplicate) significance was determined by student T-test p < 0.0001.
Figures 2A to E are graphs showing the regulation of genes involved in cancer-related biological functions.graphs show the expression of Bcl-2(a), Ccne1(B), Cdc20(C), Tap1(D) and Casp1(E) in response to stimulation with 1000IU/ml of IFN epsilon (middle bar) or IFN β (right bar). data are shown as the mean +/-SEM of 3 independent experiments, each experiment was performed in three technical replicates.significance was determined by student T-test,. p <0.05,. p <0.01,. p <0.001,. p < 0.0001.
FIGS. 3A and B are graphs showing mean cell index measurements at 30 minute intervals over 72 hours of treatment of ID8 cells with interferon-correlation of cell numbers-showing that IFN ε (A) but not IFN β (B) inhibited ID8 cell proliferation-graphs showing that ID8 cells treated with 100-.
Figures 4A to C are graphs showing IFN-induced inhibition of growth of ID8 cells, ID8 cells were plated onto electrode-coated 96-well E plates to measure cell impedance, cells were serum starved for 24 hours, then treated with 0-1000IU/ml of (a) IFN epsilon, or (B) IFN β for 48 hours, cell indices (a measure of CI-impedance) were normalized to treatment time and doubling time was calculated 48 hours after treatment using RTCA software (C) the slope of the growth curve (representing proliferation rate) was also calculated from normalized Cl to 48 hours after treatment using RTCA software, data representing N ═ 3 independent experiments, performed in four technical parallel experiments, data represented as the mean ± SD of N ═ 3 independent experiments, analyzed using two-way ANOVA (2-way ANOVA) and Sidak multiple comparison test, < 0.0001.
Figure 5 is a graph showing that IFN epsilon treatment inhibited cell migration of ID8 cells. ID8 cells were treated with 1-100IU/ml of IFN epsilon or buffer control and migration was measured 12 hours after treatment. Fetal Calf Serum (FCS) was used as a chemoattractant. Serum Free Medium (SFM) was used as negative control. Data represent one independent experiment, performed in triplicate with three techniques, and are expressed as mean ± SD of the technique replicates. Significance was determined using one-way ANOVA and Tukey multiple comparisons; p < 0.05; p, 0.01; p < 0.001; p < 0.0001.
Figures 6A to D are graphs showing that IFN epsilon treatment induced apoptosis of ID8 cells. The data show annexin V/PI staining analysis of ID8 cells treated with 40-400IU/ml IFN epsilon for 4 hours compared to PBS and buffer treated controls. H2O2Used as a positive control. (A) A living cell; (B) necrotic cells; (C) early apoptosis; (D) late apoptosis. Data represent N-3 independent experiments, performed in two technical replicates, and are expressed as mean ± SD of technical replicates. Significance was determined using student T test; p, 0.05; p<0.01。
Figure 7 is a graphical representation of the intensity of IFN epsilon staining in benign human epithelial and serous cancer samples. Immunohistochemical staining of IFN epsilon expression in human control epithelial Low (LG) and High (HG) Serous Carcinoma (SC) samples was analyzed using positive pixel analysis in Imagecope software to quantify staining intensity in epithelial-derived tissue components. Data are presented as intensity scores for each sample stained in two technical parallel experiments. Data are presented as a dot plot of n-30 samples from control and epithelium from low (n-6) and advanced serous carcinoma samples (n-70), with the average represented by a bar. Data were analyzed using a Mann-Whitney test alone, p <0.01, p < 0.001.
Fig. 8A to E are graphical representations of late stage disseminated ovarian cancer from a primary tumor metastasis that is established directly. WT and Ifn epsilon deficient mice indicate advanced primary tumors and metastatic ovarian cancer 13 weeks after intravesicular ID8 injection. A-B) left ovaries and spleens from non-tumor and ID8 injected mice were weighed; C) draining ascites fluid from the peritoneum; and E) measuring the red blood cell count; D) the number of metastases on the peritoneal wall was recorded. Data show each genotype. n-3 tumor free and n-6 ID8 injected mice and analyzed using unpaired student T-test p < 0.05.
FIGS. 9A-D are diagrams showing recombinant IFN epsilon in vivo modulation of peritoneal immune cell populations by intraperitoneal injection, three times per week for 8 weeks with healthy C57BL/6 wild type mice (6 to 8 weeks old) of recombinant murine IFN epsilon or IFN β (at 500 IU/dose), peritoneal exudate cells collected in PBS by peritoneal lavage and analyzed using flow cytometry for immune cell populations including A) CD45+CD8+A T cell; B) CD45+CD4+A T cell; C) CD45+CD11b+Ly6C + inflammatory monocytes; and D) CD45+CD4+PD1+T cells. Data are expressed as mean +/-SEM of n-5 mice per group, analyzed using unpaired student T-test,. p<0.05，**p<0.01。
Fig. 10A to C are graphs showing that IFN epsilon inhibits the development of malignant ascites in a stained ovarian cancer model a) images show the volume of ascites shed from the peritoneum of
FIGS. 11A-C are graphs showing changes in inflammatory cytokine levels in tumor-bearing mice treated with IFN ε or IFN β the images show the concentration of MCP-1(A), IL6(B) and IL-10(C) in peritoneal drainage from
figure 12 is a diagram showing that recombinant IFN epsilon modulates peritoneal immune cell populations in a stained ovarian cancer model. C57BL/6 wild type mice (6 to 8 weeks old) were injected intraperitoneally with ID8 cells and intraperitoneallyTreatment with recombinant murine IFN epsilon or IFN β (at 500 IU/dose), three times per week for 8 weeks peritoneal exudate cells were collected via peritoneal lavage in PBS and immune cell populations were analyzed using flow cytometry+/-SEM, n-5 mice per group, analyzed using unpaired student T-test, p<0.05；**p<0.01。
Fig. 13A to D are graphs showing growth and ascites development in murine cancers of Epithelial Origin (EOC) treated with recombinant interferon. A) The body weight of the mice was monitored over 8 weeks after ID8 cell injection and the percent body weight gain for each treatment group relative to the mean of all mice on
Figures 14A to D are graphs showing the effect of IFN on systemic anemia, peritoneal hemorrhage and splenomegaly in murine EOC. A) Clinical signs of anemia in mice 8-weeks after ID8 cell injection included pallor of the hind paw, which was graded as 0-normal perfusion, 1-mild pallor, 2-extreme pallor. B) Peritoneal lavage was performed with 5ml PBS and for bleeding grading, 0-no bleeding to 3-profuse bleeding, dark red and completely opaque fluids. C) Cell counts, including Red Blood Cell (RBC) counts were performed on Peritoneal Exudate Cells (PEC). D) Spleen weights from
Figures 15A to F are graphs showing the effect of tumor burden in murine EOC treated with recombinant IFN epsilon.A) The degree of mesenteric tumor burden was graded from 0-no macroscopic disease to 4-massive tumor formation, evident by large sub-nodal septal tumor masses and unclear tumor deposits throughout the mesentery. B) Macroscopic tumor deposits attached to the peritoneal wall were counted. These include tumors of different sizes. C) Macroscopic tumor deposits attached to the diaphragm were counted. These include tumors of different sizes. D) Macroscopic tumor deposits attached to liver lobes were counted. E) The free floating spheroids were counted. F) Surface area measurement of the largest representative tumor nodule per mouse. Data are presented as mean values+/-SEM, each group of n-3-5 mice. Significance was determined using unpaired student T-test<0.001，**p<0.01，*p<0.05。
Figures 16A and B are the nucleotide and amino acid sequences of human and murine IFN epsilon including optimized expression sequences (optimized codon usage).
Detailed Description
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any other element or integer or method step or group of elements or integers or method steps.
As used in this specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cancer cell" includes a single cancer cell, as well as two or more cancer cells; reference to "IFN epsilon" includes a single IFN epsilon molecule, as well as two or more IFN epsilon molecules; reference to "the present disclosure" includes both individual and multiple aspects taught by the present disclosure; and so on. Aspects of the teachings and implementations herein are included within the term "invention". Any variants and derivatives contemplated herein are included in the "forms" of the present invention. All aspects of the invention are realized within the breadth of the claims.
The present invention teaches the use of interferon epsilon (IFN epsilon) in the treatment of cancer in a subject. This includes functional natural or synthetic variants or hybrid forms of IFN epsilon. Further taught herein is the use of inducers of Ifn epsilon expression or Ifn epsilon activity in the treatment of cancer. Thus, an IFN epsilon, or a functional natural or synthetic variant or hybrid form thereof, may act directly on cancer cells, or may act indirectly via innate or adaptive immune cells or modulators or IFN epsilon-induced processes or via stromal cells or environmental components in tumor cells.
Thus, herein is achieved the use of the following for the direct or indirect inhibition of cancer cells:
(i) a naturally purified IFN epsilon;
(ii) recombinant IFN epsilon, including IFN epsilon produced by optimized expression;
(iii) a functional natural variant of IFN epsilon;
(iv) functional synthetic variants of IFN epsilon, including optimization for activity;
(v) a hybrid of two or more IFN epsilon from different species; and/or
(vi) An inducer of Ifn epsilon expression or IFN epsilon activity,
the present invention may treat cancer using any one of (i) to (vi), i.e., an agent selected from (i) to (vi), or using a combination of two or more of (i) to (vi). References to inducers of Ifn epsilon expression or Ifn epsilon activity include agents that upregulate promoter activity, optimize regulatory control to provide elevated levels of Ifn epsilon, and agents that enhance Ifn epsilon activity.
Treatment of cancer includes inhibition of single or multiple cancer cells. This includes any one or more of directly or indirectly inducing apoptosis of the cancer cells, directly or indirectly acting as a cytotoxic agent, directly or indirectly inhibiting replication, growth, development, motility, proliferation, survival and/or migration of the cancer cells, and/or directly or indirectly inducing leukocyte recruitment of the cancer cells. The treatment may enhance anti-cancer activity via stromal cells or environmental components of tumor cells.
In addition, the IFN epsilon or a functional natural or synthetic variant or inducer thereof can directly or indirectly prevent local growth or invasion of the cancer cells and/or prevent metastasis of the cancer cells elsewhere in the subject, including in regions distant from the original focus of cancer cell development.
The present invention stems in part from the study of ovarian cancer. However, the anti-cancer effects of IFN epsilon are applicable to any of a range of cancers, including cancers derived from epithelial tissues, connective tissues, glandular tissues, embryonic tissues, blood-borne cancers, and cancers including hematopoietic cells, lymphoid tissues, and bone marrow or cells from which such cells are derived. The present invention is not limited to the treatment of any type of cancer or organ or anatomical compartment or region affected by cancer. Thus, the invention extends to the treatment of cancer from any one of ovary, uterus, fallopian tube, endometrium, placenta, breast, testis, prostate, brain, stomach, liver, spleen, pancreas, thymus, colon, lung, kidney, heart, thyroid and smooth muscle. This is not intended to be an exhaustive list but rather represents the types of cancer that can be treated by IFN epsilon or functional natural or synthetic variants or hybrids thereof or inducers of IFN epsilon expression or IFN epsilon activity.
However, in one embodiment, the invention extends to cancers that affect the Female Reproductive Tract (FRT), such as but not limited to ovarian cancer. As noted above, IFN epsilon or a functional natural or synthetic variant or hybrid form thereof may act directly on cancer cells that induce any one or more of apoptosis, cytotoxicity, senescence, lysis or other forms of cell death, or may delay, inhibit or otherwise inhibit cell growth, proliferation, replication, development, migration or motility. The IFN epsilon, or functional natural or synthetic variant or hybrid form thereof, may also act indirectly on cancer cells that induce any one or more of apoptosis, cytotoxicity, senescence, lysis, or other forms of cell death, or may delay, inhibit, or otherwise prevent cell growth, proliferation, replication, development, migration, or movement. Without limiting the invention to any theory or mode of action, indirect activity includes induction of innate and adaptive immune modulators and processes. IFN epsilon may also act via stromal cells or environmental components surrounding cancer cells or tissues.
Subjects receiving treatment include human and non-human mammals. Non-human animals include those useful in animal models. Such animals include mice, rats, guinea pigs, hamsters, rabbits, pigs, and larger non-human animals. Other animals included herein are companion animals (e.g., dogs and cats), and equine animals, including horses, Przewalski horses (Przewalski horse), zebras, and donkeys. "horses" include thoroughbred, warm-blooded horses, quart horses (Quarter horse) and standard horses. Captive wild animals such as badgers (Tasmanian devil) may also be the subject of treatment and are included in the present invention. Thus, the present invention is useful in human and veterinary medicine as well as a research tool.
Reference to a human subject includes a human of any gender or age. In one embodiment, the human is a female with a cancer that affects FRT, such as, but not limited to, ovarian cancer.
Although it is not intended to limit the scope of the present invention to any type of cancer, it relates to carcinomas, sarcomas, adenocarcinomas, blastomas, leukemias, lymphomas, and myelomas. The term "cancer" should not be construed as distinct from "tumor", and both terms are used herein to refer to the same cell type. Regardless of the stage classification, the cancer may be of any grade and any stage. Thus, a cancer may be a solid tumor or derived from blood or lymph or bone marrow and may be defined in terms of cell type, location, tumor size, degree of local, regional or distant metastasis. For example, in the case of ovarian cancer, this may be high or low or intermediate grade serous, mucinous, clear cell or endometrioid.
Accordingly, embodied herein is a method for inhibiting a cancer cell in a subject, the method comprising exposing the cancer cell to an amount of interferon epsilon (IFN epsilon) or a functional natural or synthetic variant or hybrid form thereof or an inducer of IFN epsilon expression or IFN epsilon activity effective to indirectly or indirectly induce apoptosis, survival, proliferation, motility, and/or migration of the cancer cell.
Further embodied herein are methods for treating a subject having cancer, the method comprising administering to the subject an effective amount of IFN epsilon or a functional natural or synthetic variant or hybrid form thereof or an inducer of IFN epsilon expression or IFN epsilon activity for a time and under conditions sufficient to induce apoptosis of cancer cells or inhibit cancer cell proliferation, motility, and/or migration.
Taught herein is the use of an IFN epsilon or a functional natural or synthetic variant thereof or a hybrid form thereof or an inducer of IFN epsilon expression or IFN epsilon activity in the preparation of a medicament for treating cancer in a subject.
Further taught herein are inducers of IFN epsilon or functional natural or synthetic variants or hybrid forms thereof or IFN epsilon expression or IFN epsilon activity for the treatment of cancer in a subject.
An IFN epsilon or a functional natural or synthetic variant or hybrid form thereof may also be used as an adjuvant with an anti-cancer agent such as a chemotherapeutic agent, another type I interferon such as IFN α or IFN β, or another biomolecule.
Thus, embodied herein is a method for inhibiting a cancer cell in a subject, the method comprising exposing the cancer cell to an amount of interferon epsilon (IFN epsilon), or a functional natural or synthetic variant or hybrid form thereof, or an inducer of IFN epsilon expression or IFN epsilon activity, effective to indirectly or indirectly induce apoptosis, survival, proliferation, motility, and/or migration of the cancer cell, in combination with another anti-cancer agent.
Further embodied herein are methods for treating a subject having cancer, the method comprising administering to the subject an effective amount of IFN epsilon, or a functional natural or synthetic variant or hybrid form thereof, or an inducer of IFN epsilon expression or IFN epsilon activity, in combination with another anti-cancer agent, for a time and under conditions sufficient to induce apoptosis of cancer cells or inhibit proliferation, motility, and/or migration of cancer cells.
Taught herein is the use of IFN epsilon or a functional natural or synthetic variant thereof or a hybrid form thereof or an inducer of IFN epsilon expression or IFN epsilon activity in combination with another anti-cancer agent in the preparation of a medicament for the treatment of cancer in a subject. The drug may be a single entity or a combination of pharmaceutically effective agents used in combination with each other.
Reference to another anti-cancer agent includes, but is not limited to, a chemotherapeutic agent, an anti-metabolite, an anti-tumor antibiotic, an inhibitor of mitotic toxicity, a steroid, a sex hormone or hormone-like drug, an alkylating agent, a nitrogen mustard, a nitrosourea, and/or a hormonal agonist. The anti-cancer agent may further comprise microtubule immune cells or products thereof.
Examples of chemotherapeutic agents include actinomycin, daunorubicin, doxorubicin (adriamycin), idarubicin, and mitoxantrone or platinum-based agents. Antimetabolites are substances that interfere with human chemical processes, such as the production of proteins, DNA, and other chemicals required for cell growth and reproduction; in cancer therapy, antimetabolites disrupt DNA production, which in turn prevents cell division. Examples include azaserine, D-cycloserine, mycophenolic acid, trimethoprim, 5-fluorouracil, capecitabine, methotrexate, gemcitabine, cytarabine (ara-C) and fludarabine.
Antitumor antibiotics interfere with DNA by blocking enzymes and mitosis or altering membranes surrounding cells. These agents may act at all stages of the cell cycle. Therefore, they are widely used for various cancers. Examples of antitumor antibiotics include actinomycin, daunorubicin, doxorubicin (adriamycin), idarubicin, and mitoxantrone.
Mitotic inhibitors are plant alkaloids and other compounds derived from natural products. They may inhibit or prevent mitosis or inhibit enzymes used to produce proteins required for cell proliferation. These play a role during the M phase of the cell cycle. Examples of mitotic inhibitors include paclitaxel, docetaxel, etoposide (VP-16), vinblastine, vincristine, and vinorelbine.
Steroids are natural and synthetic hormones that can be used to treat some types of cancer (lymphoma, leukemia, and multiple myeloma) as well as other diseases. They can kill cancer cells or slow their growth. Examples include prednisone and dexamethasone.
Sex hormones or hormone-like drugs alter the action or production of female or male hormones. They are useful for slowing the growth of breast, prostate and endometrial cancers, which typically grow in response to hormone levels in the body. Examples include antiestrogens (tamoxifen, fulvestrant), aromatase inhibitors (anastrozole, letrozole), progestins (megestrol acetate), antiandrogens (bicalutamide, flutamide) and LHRH agonists (leuprorelin, goserelin).
Alkylating agents act directly on DNA to prevent cancer cells from replicating. As a class of drugs, these drugs are not phase-specific (in other words, they act at all stages of the cell cycle). These drugs are active against chronic leukemia, non-hodgkin's lymphoma, hodgkin's disease, multiple myeloma, and certain cancers of the lung, breast and ovary. Examples of alkylating agents include busulfan, cisplatin, carboplatin, chlorambucil, cyclophosphamide, ifosfamide, Dacarbazine (DTIC), dichloromethyldiethylamine (mechlorethamine), and melphalan.
Nitrogen mustard in the form of its crystalline hydrochloride is used as a medicament for the treatment of hodgkin's disease, non-hodgkin's lymphoma and brain tumors. Nitrogen mustards cause mutations in the genetic material of cells, disrupting mitosis or cell division. The cells differ in their susceptibility to nitrogen mustards, with rapidly proliferating tumor and cancer cells being most sensitive; the bone marrow that produces red blood cells is also sensitive and suppression of red blood cell production is a common side effect of nitrogen mustard therapy. Nitrogen mustards also suppress the immune response (see immunity). Other classes include aromatic nitrogen mustards melphalan and chlorambucil, cyclophosphamide, HN1, bis- (2-chloroethyl), ethylamine; HN2, bis- (2-chloroethyl), methylamine and HN3, tris- (2-chloroethyl), amines.
Nitrosoureas function in a similar manner to alkylating agents. They interfere with enzymes that help repair DNA. These drugs are able to travel to the brain and are therefore useful in the treatment of brain tumors as well as non-hodgkin's lymphoma, multiple myeloma and malignant melanoma. Examples of nitrosoureas include carmustine (BCNU) and lomustine (CCNU).
Hormone agonists include leuprolide (Lupron, Viadur, Eligard) for prostate cancer, goserelin (Zoladex) for breast and prostate cancer, and triptorelin (tresstar) and nafarelin acetate (Synarel) for ovarian and prostate cancer.
Microtubule inhibitors include the "vinca" alkaloids, taxanes and benzimidazoles.
Inducing expression of Ifn epsilon or activity of Ifn epsilon involves the use of an inducer of Ifn epsilon. Such agents include both proteinaceous and non-proteinaceous agents. These agents may interact with the regulatory regions of the gene (including mature or precursor forms of IFN epsilon) or modulate the expression of upstream molecules that subsequently modulate IFN epsilon expression or expression product activity. Thus, contemplated herein are agents that directly or indirectly induce or modify Ifn epsilon expression and/or Ifn epsilon activity.
In another example, TGF β may be used, similarly, bioinformatic analyses have identified glucocorticoid receptor response elements and Ets factor binding elements within the IFN epsilon promoter, putative transcription factor binding sites for BRCA1 have also been identified in the human Ifn epsilon promoter, therefore, molecules that activate transcription via these sites, such as Elf3 and Elf5, may be used to up-regulate Ifn epsilon expression.
The inducer agent used according to aspects of the invention may take any suitable form. For example, a protein agent may or may not be glycosylated, phosphorylated or dephosphorylated to varying degrees, and/or may comprise a series of other molecules used with, linked to, associated with, or otherwise associated with a protein, such as an amino acid, lipid, carbohydrate, or other peptide, polypeptide, or protein. Similarly, non-protein molecules may also take any suitable form. Both the proteinaceous and non-proteinaceous agents described herein can be linked, bound, or otherwise associated with any other proteinaceous or non-proteinaceous molecule. For example, in one embodiment of the invention, the agent is associated with a molecule that allows it to target a localized region.
The term "expression" refers to the transcription and/or translation of a nucleic acid molecule. Reference to an "expression product" refers to the product resulting from the transcription and translation of a nucleic acid molecule.
"variants" of the molecules described herein include naturally occurring or synthetically prepared fragments, portions (part) or derivatives. Non-natural sources include, for example, recombinant or synthetic sources. By "recombinant source" is meant that the source of the cells from which the IFN epsilon is harvested has been genetically altered. This may occur, for example, to increase or otherwise enhance the production rate and throughput of that particular cell source. Portions or fragments include, for example, the active region of IFN epsilon. The derivatives may be derived from insertions, deletions or substitutions of amino acids. Amino acid insertion derivatives include amino and/or carboxy terminal fusions as well as intrasequence insertions of single or multiple amino acids. An inserted amino acid sequence variant is one in which one or more amino acid residues are introduced at a predetermined site in the protein, although random insertion in combination with appropriate screening of the resulting product is also possible. Deletion variants are characterized by the removal of one or more amino acids from the sequence. A substituted amino acid variant is one in which at least one residue in the sequence has been removed and a different residue inserted at its position. Addition of amino acid sequences includes fusion with other peptides, polypeptides or proteins as detailed above.
Variants also include fragments with specific epitopes or portions of the entire IFN epsilon protein fused to peptides, polypeptides or other proteins or non-protein molecules. Analogs of the molecules contemplated herein include, but are not limited to, glycosylation variants, side chain modifications, incorporation of unnatural amino acids and/or derivatives thereof during peptide, polypeptide, or protein synthesis, and the use of cross-linking agents, as well as other methods of imposing conformational constraints on the protein molecule or analog thereof.
"variants" or "mutants" of an IFN epsilon are understood to mean molecules that exhibit at least some of the functional activity (i.e., direct or indirect anti-cancer activity) of an IFN epsilon, which are variants or mutants of IFN epsilon. Variations or mutations may take any form and may be natural or non-natural. In one embodiment, the nucleic acid has been subjected to codon optimization to enhance expression, and/or the IFN epsilon protein may comprise amino acid changes in order to optimize activity. In one embodiment, the variant is a hybrid of two or more IFN epsilon molecules. For example, aspects of IFN epsilon derived from a subject species receiving treatment may be modified to incorporate IFN epsilon from another species, or vice versa. In one example, a murine IFN epsilon has higher activity on human cells than a human IFN epsilon. Thus, hybrid mouse IFN epsilon (and vice versa) can be generated incorporating elements of human IFN epsilon to render it non-immunogenic.
Reference to IFN epsilon or a nucleic acid thereof includes reference to SEQ ID NO:28 or 32 or at least 80% similar to SEQ id no: 27. 29 or 31 has a protein sequence with at least 80% identity. Reference to at least "80%" includes 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. Variants of the nucleic acid encoding IFN epsilon comprise SEQ ID NO: 27. 29 or 31 (see also figure 16).
The term "similarity" as used herein includes the exact identity between compared sequences at the nucleotide or amino acid level. In the case of non-identity at the nucleotide level, "similarity" includes differences between sequences that result in different amino acids that are related to each other at the structural, functional, biochemical and/or conformational level. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are related to each other at the structural, functional, biochemical and/or conformational level. In one embodiment, the nucleotide and sequence comparisons are performed at the identity level rather than at the similarity level.
The terms used to describe a sequence relationship between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence similarity", "sequence identity", "percentage of sequence similarity", "percentage of sequence identity", "substantially similar", and "substantially identical". The "reference sequence" is at least 12, but often 15 to 18, usually at least 25 or more, such as 30, monomeric units, including nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence that is similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence), and (2) a sequence that is different between the two polynucleotides, a sequence comparison between the two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. "comparison window" refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. For optimal alignment of the two sequences, the comparison window may include additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not include additions or deletions). Optimal alignment of sequences for alignment over a comparison window can be performed by computerized implementation of an algorithm (GAP, BESTFIT, FASTA, and TFASTAin the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group,575Science Drive Madison, Wis., USA), or by inspection and optimal alignment produced by any of a variety of methods of choice (i.e., producing the highest percent homology over the comparison window). Reference may also be made to the BLAST family of programs disclosed, for example, in Altschul et al (1997) Nucl. acids. Res.25: 3389. A detailed discussion of sequence analysis can be found In Ausubel et al, 19.3 Block (In: Current Protocols In molecular biology, John Wiley & Sons Inc.1994-1998).
The terms "sequence similarity" and "sequence identity" as used herein refer to the degree of identity or functional or structural similarity over the comparison window with respect to nucleotide basis or amino acid to amino acid basis sequences. Thus, for example, a "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, gin, Cys, and Met) occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. For purposes of the present invention, "sequence identity" will be understood as the "percent match" calculated by the DNASIS computer program (version2.5for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, Calif., USA) using standard default values used in the reference manual accompanying the Software. Similar comments apply to sequence similarity.
The present invention relates to variants of Ifn epsilon nucleic acid molecules. Typically, the variant will still hybridize to the Ifn epsilon sequence under low stringency conditions.
Variants include chemical and functional equivalents of IFN epsilon, including molecules that exhibit any one or more of the functional activities of IFN epsilon (i.e., direct or indirect anti-cancer activity), which functional equivalents may be derived from any source, such as chemically synthesized or identified via a screening process such as natural product screening. For example, chemical or functional equivalents can be designed and/or identified using well known methods such as high throughput screening of combinatorial chemical or recombinant libraries or natural product screening.
For example, libraries comprising small organic molecules can be screened, wherein organic molecules having a large number of substituents of a particular parent group are used. General synthetic protocols may follow published procedures (e.g., Bunin et al (1994) Proc. Natl. Acad. Sci. USA,91: 4708-691 4712; DeWitt et al (1993) Proc. Natl. Acad. Sci. USA,90: 6909-6913). Briefly, in each successive synthesis step, one of a plurality of different selected substituents is added to each of the selected tube sets in the array, the selection of the tube sets being such as to produce all possible permutations of the different substituents used to generate the library. One suitable arrangement is outlined in U.S. patent No. 5,763,263. Another arrangement includes a fragment-based drug design.
There is currently a wide range of interest in using combinatorial libraries of random organic molecules to search for biologically active compounds (see, e.g., U.S. patent No. 5,763,263). Ligands discovered by screening libraries of this type can be used to mimic or block natural ligands or naturally occurring ligands that interfere with biological targets. In the context of the present invention, they can be used, for example, as starting points for the development of IFN epsilon analogs which exhibit properties such as more potent pharmacological effects. IFN epsilon or functional portions thereof according to the invention can be used in combinatorial libraries formed by various solid or solution phase synthetic methods (see, e.g., U.S. Pat. No. 5,763,263 and references cited therein). Millions of new chemical and/or biological compounds can be routinely screened in less than a few weeks using techniques such as that disclosed in U.S. patent No. 5,753,187. Of the large number of compounds identified, only those compounds that exhibit the appropriate biological activity were further analyzed.
With respect to high throughput library screening methods, oligomeric or small molecule library compounds capable of specific interaction with a selected biological agent, such as a biomolecule, macromolecular complex or cell, are screened using combinatorial library devices, which can be readily selected by one skilled in the art from well known methods such as those described above. In this method, each member of the library is screened for the ability to specifically interact with the selected agent. In practicing the method, the biopharmaceutical agent is drawn into the tube containing the compound and allowed to interact with the individual library compounds in each tube. The interaction is designed to produce a detectable signal that can be used to monitor for the presence of a desired interaction.
Analogs of IFN epsilon contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or derivatives during peptide, polypeptide or protein synthesis, and the use of cross-linking agents and other methods of imposing conformational constraints on the analogs. The particular form this modification may take will depend on whether the subject molecule is a proteinaceous molecule or a non-proteinaceous molecule. The nature and/or suitability of a particular modification can be routinely determined by one skilled in the art.
As described above, the present invention relates to a formulation wherein the IFN epsilon is a hybrid between human and murine IFN epsilon. Formulations administered alone or in combination with another anti-cancer agent of the invention, which include an inducer of IFN epsilon or a functional natural or synthetic variant or hybrid thereof or expression of IFN epsilon or activity of IFN epsilon, may also be referred to as pharmaceutical compositions. Such formulations may be prepared by any convenient method. The components of the formulation are expected to exhibit anti-cancer activity when administered in amounts that depend on the particular situation. IFN epsilon or variants, hybrids or inducers suitable for achieving anticancer activityThe amount of (a) is defined as a "therapeutically effective dose" or "effective amount". The dosage regimen and effective amount for this use, i.e., the "dosing regimen", will depend upon a variety of factors including the stage of the disease or disorder, the severity of the disease or disorder, the general state of the patient's health, the patient's physical condition, age, pharmaceutical formulation, and concentration of the active agent (e.g., IFN epsilon), among others. The mode of administration is also considered when calculating the dosage regimen for the patient. The dosage regimen must also take into account pharmacokinetics, i.e., absorption rate, bioavailability, metabolism, clearance, etc., of the pharmaceutical composition. See, e.g., Egleton (1997) Peptides 18: 1431-; langer (1990) Science 249: 1527-. A wide range of dosages may be applicable. The dosage regimen may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals, or the dose may be proportionally reduced as indicated by the exigencies of the situation. In one example, each subject may be administered from 1 to 3 times per week in an amount of 10 Ul/dose to 1,000,000 Ul/dose. Exemplary dosage regimens include 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100/dose, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000/dose or 10IU3、104、105、106IU/dose. This may be 1, 2, 3, 4, 5, 6 or 7 times per week. The dose may also be calculated based on IU/kg body weight of the subject. In one embodiment, the dosage is administered by any convenient means.
Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (which are water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions, or may be in the form of creams or other forms suitable for topical administration. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. For example, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. In addition, the active agent can be coupled to poly-L-lysine (ply L lysine) or pegylated.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The formulations may be administered in a convenient manner, such as by oral, intraperitoneal, intravenous, subcutaneous, inhalation, suppository routes or implantation (e.g., using slow release molecules). The formulations may be administered in the form of pharmaceutically acceptable non-toxic salts, such as acid addition salts, or metal complexes with, for example, zinc, iron, and the like (considered salts for purposes of this application). Exemplary such acid addition salts are hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is administered in tablet form, the tablet may contain a binder, such as tragacanth, corn starch or gelatin; disintegrating agents, such as alginic acid; and lubricating agents, such as magnesium stearate.
The IFN epsilon of the invention, or variants, hybrids, or inducers thereof, may be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. The pharmaceutically acceptable carrier may comprise a physiologically acceptable compound, for example, which acts to stabilize, or increase or decrease the rate of absorption or clearance of the pharmaceutical composition of the invention. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce clearance or hydrolysis of peptides or polypeptides, or excipients or other stabilizers and/or buffers. Detergents may also be used to stabilize or increase or decrease the absorption of pharmaceutical compositions comprising liposome carriers. Pharmaceutically acceptable carriers and formulations for peptides and polypeptides are known to those skilled in the art and are described in detail in the scientific and patent literature.
As described above, IFN epsilon may also be added as an adjuvant to another anti-cancer agent. In this regard, "drug" includes IFN epsilon or variants or hybrids thereof, alone or in combination with another anti-cancer agent.
The solid formulations may be for enteral (oral) administration. They may be formulated, for example, as pills, tablets, powders or capsules. For solid compositions, conventional non-toxic solid carriers can be used and include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable non-toxic composition is formed by incorporating any of the commonly used excipients, such as those carriers previously listed. Non-solid formulations may also be used for enteral administration. The carrier may be selected from various oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients include, for example, starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, polyethylene glycol, water and ethanol.
The compositions of the present invention may be protected from digestion when administered orally. This can be achieved by complexing the composition with a composition to render it resistant to acidic and enzymatic hydrolysis, or by packaging the molecules in a suitable resistant carrier such as liposomes. Methods for protecting compounds from digestion are well known in the art, see, e.g., Fix (1996) PharmRes.13: 1760-; samanen (1996) J.Pharm.Pharmacol.48: 119-135; us patent 5,391,377 describes lipid compositions for oral delivery of therapeutic agents (liposome delivery is discussed in further detail below).
The compositions of the present invention may also be administered in a sustained delivery or sustained release mechanism, which may deliver the formulation internally. For example, biodegradable microspheres or capsules or other biodegradable polymer configurations capable of sustained delivery of peptides can be included in the formulations of the invention (see, e.g., Putney (1998) nat. Biotechnol.16: 153-157).
For inhalation, the compositions of the present invention may be delivered using any system known in the art, including dry powder aerosols, liquid delivery systems, air jet nebulizers, propellant systems, and the like. See, e.g., Pattern (1998) Biotechniques16: 141-143; for example, Dura Pharmaceuticals (San Diego, Calif.), Aradigm (Hayward, Calif.), Aerogen (Santa Clara, Calif.) products for polypeptide macromolecules and inhalation delivery systems, inhalation therapy systems (San Carlos, Calif.), and the like. For example, the IFN epsilon formulation may be administered in the form of an aerosol or mist. For aerosol administration, the formulation may be supplied in a finely divided form together with the surfactant and propellant. In another aspect, the device for delivering the formulation to the respiratory tissue is an inhaler in which the formulation is vaporized. Other liquid delivery systems include, for example, air jet sprayers.
The IFN epsilon may also be formulated into pharmaceutically acceptable compositions suitable for pulmonary or respiratory delivery to a patient. Specific formulations include dry powders, liquid solutions or suspensions suitable for aerosolization, and propellant formulations suitable for use in Metered Dose Inhalers (MDI). The preparation of such formulations is well described in the patent, scientific and medical literature, and the following description is intended to be exemplary only.
Liquid formulations of IFN epsilon for use in nebulizer systems can include components that enhance or maintain chemical stability, including chelating agents, protease inhibitors, isotonic modifying agents, inert gases, and the like.
For use in metered dose inhalers, the IFN epsilon of the invention is dissolved or suspended in a suitable aerosol propellant such as a chlorofluorocarbon (CFC) or Hydrofluorocarbon (HFC). Suitable CFCs include trichlorofluoromethane (propellant 11), dichlorotetrafluoroethane (propellant 114) and dichlorodifluoromethane (propellant 12). Suitable HFCs include tetrafluoroethane (HFC-134a) and heptafluoropropane (HFC-227).
In one embodiment, for incorporation into an aerosol propellant, the IFN epsilon of the present invention is processed into inhalable particles as described below for dry powder formulations. The particles are then suspended in a propellant, usually coated with a surfactant to enhance their dispersion. Suitable surfactants include oleic acid, sorbitan trioleate and various long chain diglycerides and phospholipids.
Such aerosol propellant formulations may further include lower alcohols, such as ethanol (up to 30% by weight) and other additives to maintain or enhance chemical stability and physiological acceptability.
Dry powder formulations typically comprise IFN epsilon in a dry, usually lyophilized form, having a specific size within a preferred range for deposition in the alveolar region of the lung. Inhalable powders of IFN epsilon in this preferred size range may be produced by various conventional techniques such as jet milling, spray drying, solvent precipitation, and the like. The dry powder can then be administered to a patient in a conventional Dry Powder Inhaler (DPI) that uses an inspiratory breath to disperse the powder into an aerosol cloud or in an air-assisted device that uses an external power source to disperse the powder into the aerosol cloud. In the above description, reference to "IFN epsilon" includes variants, hybrids and inducers thereof.
In preparing the pharmaceutical formulations of the present invention, various modifications may be used and manipulated to alter pharmacokinetics and biodistribution. Many methods for altering pharmacokinetics and biodistribution are known to those of ordinary skill in the art.
In one embodiment, induction of expression of Ifn epsilon is achieved by directly affecting expression of Ifn epsilon. This can be achieved by introducing the gene comprising Ifn epsilon directly into the cancer cells in the solid tumor of the construct, which will allow to induce the level of Ifn epsilon or active variants thereof upon expression or even de novo expression, thus generating the biological function for which it is directed. Thus, recombinant cellular or viral means can be used to produce IFN epsilon or variants, hybrids, or inducers thereof at or near cancer cells or within cancer cells.
The invention further contemplates combinations of methods of treating cancer. For example, IFN epsilon therapy or treatment by variants or hybrids of IFN epsilon or inducers may be used in combination with surgical or chemical ablation of cancer or organs or tissues affected by cancer.
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