Patent Application
20220106381
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 2, 2020, is named OPB-00103_SL.txt and is 10,463 bytes in size.
The present invention relates to peptides that inhibit the interaction of CD40 and CD154, and the use of such compounds in modulating T-cell activity and in treating disease.
Inflammation normally occurs in response to infection by invading micro-organisms. This inflammatory response is beneficial because it is an important part in localizing the infecting agent for removal by the immune system. However, in autoimmunity there is no infection, yet severe inflammation is present. The inflammation in this case, referred to as aseptic chronic inflammation, is detrimental since it destroys normal tissues. The results of this aseptic inflammation are life-altering and in some cases life-threatening. Moreover, as with acute inflammation, this process is mediated by immune cells, including T-cells.
A major concern for modern medicine is how to control aseptic, chronic inflammation (ACI) such as that which occurs during autoimmune diseases, as well as how to control acute inflammation resulting from trauma. Inflammation, both chronic and acute, leads to tissue degeneration and eventual loss of function of major organs. ACI is not limited to a single disease, but is instrumental in numerous autoimmune diseases including, but not limited to type 1 diabetes, multiple sclerosis, systemic lupus erythematosa, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, chronic obstructive pulmonary disease including types of autoimmune asthma, atherosclerosis, vasculitis, hypertension, thyroiditis including Hashimoto's and Graves diseases, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome, acute lung injury, and ACI associated with organ transplantation.
Autoimmune disorders are classified into two types: organ-specific (directed mainly at one organ) and non-organ-specific (widely spread throughout the body). Examples of organ-specific autoimmune disorders are insulin-dependent Type 1 diabetes which affects the pancreas; Hashimoto's thyroiditis and Graves' disease, which affect the thyroid gland; pernicious anemia, which affects the blood; Addison's disease, which affects the adrenal glands; chronic active hepatitis, which affects the liver; myasthenia gravis which affects the muscle; and multiple sclerosis, which affects tissue of the nervous system. An example of a non-organ-specific autoimmune disorders is rheumatoid arthritis. Autoimmune diseases are often chronic, debilitating, and life-threatening. The National Institutes of Health (NIH) estimates that up to 23.5 million Americans suffer from autoimmune disease and that the prevalence is rising. It has been estimated that autoimmune diseases are among the ten leading causes of death among women in all age groups up to 65 years.
Acute inflammation, as observed during trauma or sepsis, is also immune cell mediated. While all of the molecular mediators in this process have not yet been identified, a prominent role for T cells, macrophages/monocytes, neutrophils etc., is strongly implicated. Therefore, a means to modulate these cell types would necessarily control the inflammatory response.
A unique T-cell subset has been shown to be instrumental in the development of autoimmune disease. These cells are phenotypically characterized as CD4loCD40+ (Waid, D. M., et al. (2004.) Euro. J. of Immunol. 34:1488; Vaitaitis, G. M., et al. (2003). Cutting Edge, J. Immunol. 170:3455; Wagner, D. H., Jr., et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 99:3782; Wagner, D. H., Jr., et al. (1999). Intl. J. of Mole. Med. 4:231) and are referred to as Th40 cells. CD40 expression typically is associated with antigen presenting cells and the majority of prior art describes CD40 as being expressed on B cells, macrophages, monocytes etc. However, CD40 proteins are also expressed on T-cells (Waid, D. M., et al. (2004). Eur. J. of Immunol. 34:1488; Vaitaitis, G. M., et al. (2003). Cutting Edge, J. Immunol. 170:3455; Wagner, D. H., Jr., et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 99:3782; Wagner, D. H., Jr., et al. (1999). Intl. J. of Mole. Med. 4:231; Bourgeois, C., et al. (2002). Science. 297:2060; Fanslow, W. C., et al. (1994). J. of Immunol. 152:4262; Ramsdell, F., et al. (1994). J. of Immunol. 152:2190; Grabstein, K. H., et al. (1993). J. of Immunol. 150:3141; Armitage, R. J., et al. (1993). Sem. in Immunol. 5:401; Cooper, C. J., et al. (2004). J. Immunol. 173:6532). While Th40 cells comprise a proportion of the peripheral CD4+ compartment in naive, non-autoimmune mice (Waid, D. M., et al. (2004). Eur. J. of Immun. 34:1488; Wagner, D. H., Jr., et al. (1999). Intl. J. of Mole. Med. 4:231), and in humans (Waid. D. M, et al. (2007). Clin. Immunol. 124:138), this proportion is drastically expanded to as much as 50% of the CD4+ compartment in autoimmune prone mice (Waid, D. M., et al. (2004). Eur. J. of Immunol. 34:1488; Wagner, D. H., Jr., et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 99:3782; Wagner, D. H., Jr., et al. (1999). Intl. J. of Mole. Med. 4:231) and humans (Waid. D. M., et al. (2007). Clin. Immunol. 124:138). These T-cells do not express early activation markers and occur in the naive phenotype of non-challenged mice. In diabetic NOD mice, Th40 cells occur at exaggerated levels in spleen, lymph nodes and the pancreas, even prior to diabetes onset (Waid, D. M., et al. (2004). Eur. J. of Immunol. 34:1488; Wagner, D. H., Jr., et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 99:3782). An elevated number and percentage of these T-cells is seen in peripheral blood of type 1 diabetic patients when compared to non-autoimmune controls and type 2 diabetic patients (Waid. D. M , et al. (2007). Clin. Immunol. 124:138).
The observed increase in Th40 cells could mean that those T-cells are antigen responsive or that CD40 expression is activation induced. Furthermore, several diabetogenic T cell clones are CD40+ (Wagner, D. H., Jr., et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 99:3782). Purified primary Th40 cells from diabetic NOD mice and from pre-diabetic NOD (12—weeks of age) mice successfully transfer type 1 diabetes to NOD.scid recipients, directly demonstrating pathogenicity of that T cell subset (Waid, D. M., et al. (2004). Eur. J. of Immunol. 34:1488; Wagner, D. H., Jr., et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 99:3782). It has been shown that Th40 cells infiltrate islet beta cells destroying insulin production thus suggesting islet antigen specificity (Waid, D. M., et al. (2004). Eur. J. of Immunol. 34:1488; Wagner, D. H., Jr., et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 99:3782). It has also been shown that Th40 cells are required for diabetes transfer. Peripheral (spleen and regional lymph node) T-cells that were CD40 depleted, then CD25, Treg, depleted were not capable of transferring diabetes to Scid recipients. Even though Tregs were removed, if the autoaggressive CD40+ T cells subset is absent, disease transfer does not occur.
While Th40 cells are important in the development of autoimmunity, another important factor is expression of the CD40—Ligand, CD154. CD154 is temporally induced on activated T cells in response to CD3/TCR stimulation (Lederman, S., et al. (1992). J. of Exp. Med. 175:1091). CD154 expression has also been demonstrated to be present on platelets, monocytes, basophils, eosinophils, dendritic cells, fibroblasts, smooth muscle, and endothelial cells (Russo, S., et al. (2003). J. Immunol. 171:5489; Stumpf, C., et al. (2003). Eur. J. Heart Fail. 5:629; Schonbeck, U., et al. (2001). Cell Mol. Life Sci. 58:4). CD154 is a member of the tumor necrosis factor (TNF) super-family and a soluble form of CD154 (sCD154) has been described (Russo, S., et al. (2003). J. Immunol. 171:5489; Stumpf, C., et al. (2003). Eur. J. Heart Fail. 5:629; Toubi, E., et al. (2004). Autoimmunity 37:457). Therefore, sCD154 may act like a cytokine (Stumpf, C., et al. (2003). Eur. J. Heart Fail. 5:629). Even though CD154 has not been genetically linked in T1D studies, sCD154 is significantly elevated in T1D and may play a role in the disease process (Varo, N., et al. (2003). Circulation 107:2664; Cipollone, F., et al. (2005). Diabetologia 48:1216; Devaraj, S., et al. (2006). Diabetes 55:774). The importance of CD40-CD154 interaction in autoimmunity has been established (Wagner, D. H., Jr., et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 99:3782; Kobata, T., et al. (2000). Rev. Immunogenet. 2:74; Homann, D., et al. (2002). Immunity. 16:403; Goodnow, C. C. (2001). Lancet 357:2115; Balasa, B., et al. (1997). J. of Immunol. 159:4620). Blocking CD40-CD154 interaction prevents collagen induced arthritis (Durie, F. H., et al. (1993). Science. 281:1328), experimental autoimmune encephalitis (Howard, L. M., et al. (2004). Autoimmunity. 37:411), prostatitis (Grossman, M. E., et al. (2001). J. Immunother. 24:237), and importantly type 1 diabetes in the NOD mouse model (Balasa, B., et al. (1997). J. of Immunol. 159:4620). In the diabetes model it was essential to administer a CD154 blocking antibody to NOD mice at 3-weeks of age; at 9-weeks, blocking antibodies had no effect on diabetes prevention (Balasa, B., et al. (1997). J. of Immunol. 159:4620).
Previous work has also demonstrated that the Th40 cell subset induces RAG1 and RAG2 transcription, translation and nuclear translocation (Vaitaitis, G. M., et al. (2003). Cutting Edge, J. Immunol. 170:3455) when CD40 is engaged. CD3 engagement does not induce RAG1 or RAG2 in T cells (Vaitaitis, G. M., et al. (2003). Cutting Edge, J. Immunol. 170:3455). Subsequent to RAG1/RAG2 induction, CD40-mediated TCR revision occurs in peripheral T cells (Vaitaitis, G. M., et al. (2003). Cutting Edge, J. Immunol. 170:3455). CD40 induction of TCR revision is RAG dependent. T cells isolated from a TCR-Tg mouse undergo TCR revision when CD40 engaged, but T cells from the TCR-Tg.RAG−/− mouse do not TCR revise when CD40 engaged (Wagner, D. H., Jr., et al. (1999). Intl. J. of Mole. Med. 4:231).
Multiple treatment options have been put forward to address and control both chronic and acute inflammation. Many approaches use non-steroidal anti-inflammatory drugs (NSAIDS) that attack the production of leukotrienes and prostaglandins, cellular products that cause localized inflammation. Other approaches use more powerful immunosuppressant drugs such as cyclophosphamide, methotrexate and azathioprine that suppress the immune response and stop the progression of the disease. Still other treatments involve the use of monoclonal antibodies designed to alter the immune responses to self-tissues, as occurs during autoimmune diseases. However, all of these treatments often have severe, long-term side effects.
Thus, there exists a need in the art for safer and more effective methods for treatment and prevention of autoimmune diseases. The present invention addresses this need by describing a novel method for treatment of autoimmune diseases.
The present developments provide a novel method for modulating inflammation, and, inflammation that arises as a result of an autoimmune disease. The developments are based on the knowledge that interaction of CD40-ligand (CD154 protein) with CD40 protein expressed on T-cells (Th40 cells), is important in the development of autoimmune disease. The developments are also based on the elucidation of the critical residues in CD40 and CD154 that are important for this interaction. The present developments relate to blocking the interaction between a CD40 protein and a CD154 protein through the use of small peptides that interact with the CD40 protein at a site where the CD154 protein would normally bind. The present developments also relate to using such peptides to reduce the level of Th40 cells, thereby reducing the severity of disease. Finally, the present developments also relate to novel methods for detecting Th40 cells.
One embodiment of the present developments is a peptide that interacts with a CD40 protein in such a manner as to modulate inflammation. Preferred peptides are those that are less than 25 amino acids in length, and that bind to a CD40 protein, thereby inhibiting its interaction with a CD154 protein. Preferred peptides are those that comprise a portion of the CD40 binding site from a CD154 protein. One embodiment of the present developments is a method to inhibit the interaction between a CD40 protein and a CD154 protein, the method comprising contacting the CD40 protein with a peptide that interacts with the CD40 protein. Preferred peptides interact with the CD40 protein at the CD154—binding site. Preferably such peptides are less than 20 amino acids in length. Even more preferred peptides are those consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 3−10.
One embodiment of the present developments is a method to modulate inflammation in an animal or a culture of cells, the method comprising administering to said animal, or said cells, a peptide that interacts with a CD40 protein in such a manner as to modulate inflammation. Preferred peptides are those that interact with the CD40 protein at the CD154-binding site, thereby modulating inflammation. Preferred peptides modulate inflammation by reducing the level of Th40 cells to no more than 25% of the total T-cell population. Such methods can be used to treat autoimmune diseases such as diabetes.
Another embodiment of the present developments is a means to detect autoagressive T-cells, the method comprising contacting a population of T-cells with a peptide that binds the CD40 protein and detecting the CD-40 bound peptide.
One embodiment of the present developments is a method to identify a patient at risk for developing an autoimmune disease, the method comprising obtaining a sample containing T-cells from a patient to be tested, contacting the sample with a peptide that binds the CD40 protein, detecting the CD-40 bound peptide, and determining the level of Th40 cells from the amount of CD40 bound, wherein a level of Th40 cells greater than 25% of the total T-cell population indicates the patient is at risk for developing an autoimmune disease.
The present invention is based on the discovery that a unique subset of T-cells, which express CD40 protein, and thus are referred to as Th40 cells, is instrumental in autoimmune inflammation. Moreover, involvement of Th40 cells in the autoimmune process is dependent on the interaction between CD40 protein expressed on the surface of the T-cell, and CD154 protein. Interaction of CD40 and CD154 results in activation signals being delivered between the cells, and subsequent activation of the Th40 cell. Such activation results in propagation of the Th40 cell and an increase in inflammation (e.g., an increase in the number of immune cells and immunoregulatory molecules, present in the system). Accordingly, inhibition of the CD40/CD154 interaction may modulate Th40 cell activity, and thereby affect inflammation. Thus, the present invention relates to peptides that affect the interaction between a CD40 protein and a CD154 protein, thereby modulating inflammation. In particular, the present invention relates to peptides that affect the interaction between CD40 protein expressed on the surface of a T-cell, and a CD154 protein, thereby affecting T-cell activity and modulating inflammation. The invention also relates to methods of using such peptides to modulate inflammation and to treat autoimmune disease. The present invention also encompasses the use of such peptides to detect Th40 cells.
Before the present invention is further described, it is to be understood that this invention is not strictly limited to particular embodiments described, as such may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the claims.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It should further be understood that as used herein, the term “a” entity or “an” entity refers to one or more of that entity. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. As such, the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably. Similarly, the terms “comprising”, “including” and “having” can be used interchangeably.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or, use of a “negative” limitation.
Furthermore, as used herein the term animal refers to a vertebrate, preferably a mammal, more preferably a human. Suitable mammals on which to use the methods of the present invention include but are not limited farm animals, sports animals, pets, primates, mice, rats, horses, dogs, cats, and humans. The term animal can be used interchangeably with the terms subject or patient.
One embodiment of the present invention is a peptide that interacts with a CD40 protein in such a manner as to modulate inflammation. As used herein, the terms interact, interaction, and the like, mean that two molecules come into sufficient physical proximity such that they cause a modulation of inflammation. One type of interaction is a binding interaction. In such an interaction the peptide associates with CD40 to form a complex. An example of complex formation is the association of an antigen with an antibody. According to the present invention, binding of a peptide of the present invention to a CD40 protein can be reversible (e.g., non-covalent binding interactions) or non-reversible (e.g., covalent binding interactions). Moreover, a reversible interaction can be strong or weak, the strength of the interaction being determined by the forces (e.g., ionic charges, hydrogen binding, van der Walls interactions, etc.) exerted by each protein on the other protein in the complex. Factors affecting the strength of an interaction between two molecules are known to those skilled in the art. One useful measure of the strength of binding between two molecules, such as a peptide and a protein, is the dissociation constant (Kd). Preferred peptides of the present invention are those that bind to a CD40 protein with a Kd of no more than about 1×10−6 M, about 1×10−7 M, or about 1×10−8 M. Particularly preferred peptides are those having a Kd of less than about 1×10−9 M. In one embodiment, a peptide of the present invention binds to a CD40 protein with a Kd of less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than 1 nM. Methods of measuring and analyzing binding interactions between a peptide and a CD40 protein are known by those of skill in the art.
As used herein, to modulate inflammation means to change the level of Th40 cells present in an animal, or in a culture of T-cells. As used herein, the terms level, number, count and concentration can be used interchangeably. Modulation of inflammation can mean an increase or decrease in the number of Th40 cells present in the inflammatory environment. Consequently, modulation can be referred to as positive or negative. Positive modulation (also referred to as up-regulation) of inflammation refers to an increase in the number of Th40 cells in the inflammatory environment. Negative modulation (also referred to as down-regulation) of inflammation refers to a reduction in the number of Th40 cells present in the inflammatory environment. A preferred peptide is one that down-regulates inflammation, thereby reducing the number of Th40 cells present in the inflammatory environment. Positive and negative modulation of inflammation may or may not result in a change in the type and amount of immunoregulatory molecules present in the inflammatory environment.
It will be appreciated by those skilled in the art that both a cell culture system and the immune system of an animal comprise basal levels of immune cells and immunoregulatory molecules. The phrases basal level and normal level can be used interchangeably. With regard to the immune system of an animal, as used herein, the basal level of a type of immune cell (e.g., Th40 cell), or a immunoregulatory molecule, refers to the average number of that cell type, or immunoregulatory molecule, present in a population of individuals considered healthy (i.e., free of metabolic, autoimmune, or infectious disease). With regard to a cell culture system, as used herein, the basal level of a type of immune cell, or an immunoregulatory molecule, refers to the average level of that cell type, or immunoregulatory molecule, present in a population of cells that is non-activated. Those skilled in the art are capable of determining if a T-cell, or a population of such cells, is activated. For example, the expression of CD69, CD25 and/or CD154 proteins by a cell indicates that the cell has been activated.
The basal level of a cell or molecule can be a specific amount (e.g., a specific concentration) or it can encompass a range of amounts. Basal levels, or ranges, of immune cells and immunoregulatory molecules are known to those in the art. For example, in a healthy individual, the normal level of CD4+ T-cells present in human blood is 500−1500 cells/ml. Variability in this measurement can result from differences in the method used to determine the cell count. Furthermore, normal levels of cells can also be reported as a percentage of a total cell population. For example, in a healthy individual, Th40 cells make up less than 25% of the total T cell population. Thus, as used herein, the term inflammation refers to an inflammatory environment in which Th40 cells make up greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45% , greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, or greater than about 80% of the total T-cell population. Moreover, a preferred peptide of the present invention is one that reduces the level of Th40 cells to less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 27%, or equal to about 25% of the total T-cell population. Methods of measuring different types of T-cells in the T-cell population are known to those skilled in the art. Furthermore, a novel method for detecting Th40 cells using peptides of the present invention is disclosed herein.
As used herein, the phrase inflammatory environment refers to the overall population of immune cells, and related immunoregulatory molecules, that are present in a culture of cells, or in the body of an animal. As such, the phrase inflammatory environment encompasses the types, and/or the relative amounts of immune cells and immunoregulatory molecules (e.g., cytokines) present in a culture of cells, or in an animal, which are involved in affecting an inflammatory reaction. Examples of cells encompassed by the term inflammatory environment include, but are not limited to, T cells, neutrophils, macrophages, granulocytes, and the like. The inflammatory environment relates to cells and molecules that mediate both acute and chronic inflammation. It will be appreciated by those skilled in the art that the inflammatory environment refers to the system to which peptides of the present invention are administered. In one embodiment, the system is a cell culture system. In one embodiment, the system is a whole animal.
A preferred peptide of the present invention is one that selectively interacts with a CD40 protein in solution, as determined using an assay such as an immunosorbent assay, or on the surface of a T-cell. As used herein, the terms selectively, selective, specific, and the like, indicate the peptide has a greater affinity for a CD40 protein than it does for proteins unrelated to the CD40 protein. More specifically, the terms selectively, selective, specific, and the like indicate that the affinity of the peptide for CD40 is statistically significantly higher than its affinity for a negative control (e.g., an unrelated protein such as albumin) as measured using a standard assay (e.g., ELISA). Suitable techniques for assaying the ability of a peptide to selectively interact with a CD40 protein are known to those skilled in the art. Such assays can be in vitro or in vivo assays. Examples of useful assays include, but are not limited to, an enzyme-linked immunoassay, a competitive enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, an agglutination assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, an immunoblot assay (e.g., a western blot), a phosphorescence assay, a flow-through assay, a chromatography assay, a polyacrylamide gel electrophoresis (PAGE)-based assay, a surface plasmon resonance assay, a spectrophotometric assay, a particulate-based assay, an electronic sensory assay and a flow cytometric assay. Methods of performing such assays are well known to those skilled in the art. In one embodiment, an assay can be performed using cells in culture, or it can be performed in a whole animal. Assays can be designed to give qualitative, quantitative or semi-quantitative results, depending on how they are used and the type of result that is desired.
One embodiment of the present developments is a peptide that interacts with a CD40 protein in such a manner as to affect the interaction of the CD40 protein with a CD154 protein, thereby modulating inflammation. The effect of the peptide on the CD40/CD154 interaction can be positive or it can be negative. For example, the peptide can interact with the CD40 protein in such a manner that the strength of the interaction between the CD40 protein and a CD154 protein is increased. Alternatively, the peptide can interact with the CD40 protein such that the strength of the interaction between the CD40 protein and a CD154 protein is decreased. Methods of measuring the strength of binding between the peptide and a CD40 protein are known to those skilled in the art. A preferred peptide of the present invention is one that reduces the strength of the interaction between a CD40 protein and a CD154 protein. Preferred peptides of the present invention reduce the strength of binding between a CD40 protein and a CD154 protein by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. A particularly preferred peptide is one that completely inhibits binding of CD40 to CD154. Complete inhibition of binding between CD40 and CD154 means that when a peptide of the present invention is brought into proximity with a CD40 protein and a CD154 protein under conditions that would normally allow the interaction of CD40 and CD154, no such interaction occurs, and activation signals are not stimulated in the CD40-expressing cell. Consequently CD40/CD154 mediated modulation of inflammation does not occur. In one embodiment, the peptide interacts with the CD40 protein in such a manner as to reduce the level of inflammation in the system. In one embodiment, the peptide interacts with the CD40 protein in such a manner as to inhibit the development of inflammation in the system.
While peptides of the present invention can interact with any site on the CD40 protein, preferred peptides of the present invention interact with the CD40 protein at a location that overlaps with the CD154 binding site. In one embodiment, a peptide of the present invention interacts with the CD40 protein at the CD154 binding site. An example of such a peptide is a CD40 ligand competitive antagonist. As used herein, peptides that interfere with, or inhibit, the binding of a CD154 protein to a CD40 protein are referred to as small interfering peptides (SIPs). As used herein a small interfering peptide is a peptide that, through physio-chemical properties, interferes with the interaction of a CD40 protein with a CD154 protein, thereby preventing activation signals from being delivered to the CD40-bearing cell, thus limiting the activation of the CD40-bearing cell, and consequently, inflammation. As demonstrated herein, the consequences of such interference are prevention of T-cell activation and propagation, and a prevention or reduction of inflammation.
A peptide useful for practicing methods of the present developments should be of a size sufficient to interact with CD40 protein in such a manner as to modulate inflammation. It is understood by those skilled in the art that preferred peptides are relatively short since they are easier and less expensive to produce. Preferred peptides may be those that are less than 20 amino acids in length. A preferred peptide may be one that is 6, 13 or 15 amino acids in length. In one embodiment, the peptide consists of an amino acid selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQID NO:9 and SEQ ID NO:10. The sequences of such peptides are shown below in Table 1.
Interaction of a CD40 protein and a CD154 protein has been shown to occur at particular regions within each protein. The inventor has now shown that, surprisingly, a peptide comprising only a short portion of the CD154 region that interacts with CD40 is capable of binding to a CD40 protein, thereby modulating inflammation. Thus one embodiment of the present invention is a peptide that comprises at least a portion of the amino acid sequence of a CD154 protein such that the peptide interacts with CD40 protein in such a manner as to modulate inflammation. In one embodiment, interaction of the peptide with CD40 protein results in negative modulation of inflammation. In one aspect, the peptide comprises at least a portion of SEQ ID NO:1 or SEQ ID NO:2. In a preferred aspect, the peptide is as short as possible yet comprises enough of the CD154 protein to allow interaction with a CD 40 protein in such a manner as to modulate inflammation. In one embodiment, a peptide of the present invention comprises 6, 13 or 15 contiguous amino acids from SEQ ID NO:1 or SEQ ID NO:2, and interacts with CD40 in such a manner as to modulate inflammation. A preferred peptide comprises a core sequence consisting of lysine-glycine-tyrosine-tyrosine (KGYY; SEQ ID NO:3), which corresponds to amino acids 142−145 of SEQ ID NO:1 and amino acids 143−146 of SEQ ID NO:2. Useful peptides can comprise additional regions of sequence from SEQ ID NO:1 or SEQ ID NO:2 that are adjacent to the core sequence, so long as the peptide is capable of modulating inflammation. In one embodiment of the present invention, a peptide comprises at least one sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQID NO:9 and SEQ ID NO:10, so long as the peptide interacts with CD40 protein in such a manner as to modulate inflammation. In one embodiment of the present developments, a peptide consists of a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQID NO:9 and SEQ ID NO:10.
While peptides of the present invention can consist entirely of sequences that are responsible for the interaction of the peptide with a CD40 protein, they may additionally contain amino acid sequences that do not interact with a CD40 protein, but which have other useful functions. Any useful, additional amino acid sequence can be added to the CD40-interacting sequence, so long as the additional sequences do not have an unwanted affect on the ability of the CD40 interacting sequence to interact with a CD40 protein. For example, in addition to the amino acid sequence responsible for interacting with a CD40 protein, a peptide of the present invention can contain amino acid sequences that are useful for visualizing or purifying the peptide. Such sequences act as labels (e.g., enzymes) or tags (antibody binding sites). Examples of such labels and tags include, but are not limited to, B-galactosidase, luciferase, glutathione-s-transferase, thioredoxin, HIS-tags, biotin tags, and fluorescent tags. Other useful sequences for labeling and tagging proteins are known to those of skill in the art.
Likewise, peptides of the present invention can be modified, so long as such modification does not significantly affect the ability of the peptide to modulate inflammation. Such modifications can be made, for example, to increase the stability, solubility or absorbability of the protein. Examples of such modifications include, but are not limited to pegylation, glycosylation and chemical modification of the peptide.
Peptides of the instant invention can be obtained from nature (e.g., obtained from plants, animals or microorganisms) or they can be produced in a laboratory (e.g., recombinantly or synthetically). Preferred peptides are those that are synthesized. Also encompassed are peptides that are combinations of natural and synthetic molecules. General methods for producing and isolating recombinant or synthetic peptides are known to those skilled in the art. It should be noted that, as used herein, an isolated, or biologically pure, molecule, is one that has been removed from its natural milieu. As such the terms isolated, biologically pure, and the like, do not necessarily reflect the extent to which the protein has been purified.
As has been described herein, interaction of the CD40 protein and the CD154 protein are necessary for involvement of Th40 cells in autoimmune inflammation. Consequently, inhibition of the interaction between a CD40 and CD154 protein using peptides of the present invention is a useful method of affecting autoimmune inflammation. Thus one embodiment of the present invention is a method to reduce the interaction between a CD40 protein and a CD154 protein comprising introducing into an environment containing a CD40 protein and a CD154 protein a peptide that interacts with the CD40 protein in such a manner as to reduce the interaction between the CD40 protein and the CD154 protein. In one aspect of the invention, the peptide reduces the interaction between the CD40 protein and the CD154 protein by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In one embodiment, the peptide reduces the interaction between the CD40 protein and the CD154 protein by a factor of at least 10, at least 100, at least 1,000, at least 10,000. Methods of measuring the strength of the interaction between the CD40 protein and the CD154 protein have been discussed previously, and are also know to those of skill in the art.
One embodiment of the present invention is a method to modulate inflammation comprising contacting a CD40 protein with a peptide that interacts to the CD40 protein in such a manner as to modulate inflammation. In one aspect of the invention, interaction of the peptide with the CD40 protein increases the number of Th40 cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In one embodiment, interaction of the peptide with the CD40 protein increases the number of Th40 cells by a factor of at least 10, at least 100, at least 1,000, at least 10,000. One aspect of the present invention is a method to reduce inflammation in a patient, the method comprising administering a peptide of the present invention to the patient. In one embodiment, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. In one embodiment, the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. In a preferred embodiment, interaction of the peptide with the CD40 protein decreases the number of Th40 cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In another embodiment, interaction of the peptide with the CD40 protein decreases the number of Th40 cells by a factor of at least 10, at least 100, at least 1,000, at least 10,000. In a preferred embodiment, the level of Th40 cells is reduced so that Th40 cells comprise no more than about 20%, about 25%, about 30%, about 35%, or about 40% of the total T-cell population.
Peptides and methods of the present invention are suitable for use in cell culture as well as for treating a patient. As used herein the term patient refers to any animal in need of such treatment. The animal can be a human or a non-human animal. A preferred animal to treat is a mammal. A peptide can be administered or applied per se, or as pharmaceutical compositions. A peptide of the present invention, or a pharmaceutical composition thereof, can be administered to a patient by a variety of routes, including, but limited to, by injection (e.g., intravenous, intramuscular, subcutaneous, intrathecal, intraperitoneal), by inhalation, by oral (e.g., in a pill, tablet, capsule, powder, syrup, solution, suspension, thin film, dispersion or emulsion.), transdermal, transmucosal, pulmonary, buccal, intranasal, sublingual, intracerebral, intravaginal rectal or topical administration or by any other convenient method known to those of skill in the art.
The amount of a peptide of the present invention and/or a pharmaceutical composition thereof that will be effective can be determined by standard clinical techniques known in the art. Such an amount is dependent on, among other factors, the patient being treated, including, but not limited to the weight, age, and condition of the patient, the intended effect of the compound, the manner of administration and the judgment of the prescribing physician. A peptide of the present invention, or a pharmaceutical composition thereof, can be administered alone or in combination with one or more other pharmaceutical agents, including other compounds of the present invention. The specific pharmaceutical composition depends on the desired mode of administration, as is well known to the skilled artisan.
Because the inventors have discovered that Th40 cells are intimately involved in the development of autoimmune diseases, the peptides and methods disclosed herein can be used to affect inflammation resulting from such diseases. Thus, one embodiment of the present invention is a method to treat autoimmune disease in a patient in need of such treatment, the method comprising administering to a patient a peptide that interacts with the CD40 protein, thereby reducing inflammation. In one embodiment the peptide interacts with the CD40 protein in such a manner as to affect the interaction of CD40 and CD154, thereby reducing inflammation. In a preferred embodiment, interaction of the peptide with the CD40 protein reduces the number of Th40 cells in a patient to a level equal to that observed in subjects that do not have autoimmune disease. The present invention is suitable for treating any patient having an autoimmune disease the development of which is dependent on Th40 cells. More specifically, peptides of the present invention are suitable for reducing the level of Th40 cells in such patients. In a preferred embodiment, a peptide of the present invention reduces the level of Th40 cells in a patient suffering from an autoimmune disease to no more than about 25% of the total T-cell population. Examples of such disease include, but are not limited to, asthma, type 1 diabetes; multiple sclerosis; systemic lupus erythematosa; rheumatoid arthritis; Crohn's disease; inflammatory bowel disease; chronic obstructive pulmonary disease (COPD) including types of autoimmune asthma; atherosclerosis; vasculitis; hypertension; thyroiditis including Hashimoto's and Graves diseases; primary biliary cirrhosis; Paget's disease; Addison's disease; acute respiratory distress syndrome, acute lung injury; ACI associated with organ transplantation; hypertension, etc.
One example of a disease that is particularly amenable to treatment using a peptide of the present invention is diabetes. In diabetes, the body's production of, or response to, insulin is impaired. Consequently cells are unable to utilize glucose in the blood, and the levels of this sugar become elevated. Mice are considered diabetic when their blood glucose level is greater than 250 mg/dl for three consecutive days. In humans, a normal, average blood glucose level is 60−110 mg/dl. However diabetics have blood glucose levels of at least 130 mg/dl, and usually much higher. Thus, one embodiment of the present invention is a method to prevent diabetes in an individual at risk for developing diabetes, the method comprising administering to the individual a peptide of the present invention. Such risk can result from familial factors (e.g., inheritance) or from other factors, such as the physical condition of the individual. Methods of risk assessment are known to those skilled in the art. In one embodiment, the peptide is administered at a time when the individual's blood glucose level is from about 60 mg/dl to about 110 mg/dl. In one embodiment, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, so long as the peptide can down-regulate inflammation. In one embodiment, the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
The inventors have also shown that, surprisingly, peptide of the present developments can be used to reverse the disease process in individuals already showing signs of diabetes. Thus, one aspect of the present developments is a method to reverse diabetes comprising administering to a patient diagnosed as having diabetes, a peptide of the present developments. In one embodiment, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, so long as the peptide can down-regulate inflammation. In one embodiment, the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. As used herein the phrase to reverse diabetes means to reduce the blood glucose level of a diabetic individual to a level comparable to that observed in a non-diabetic individual. As noted above, the blood glucose level of a non-diabetic subject is from about 60 mg/dl to about 110 mg/dl. Thus, one aspect of the present invention is a method to reduce the blood glucose level in a patient diagnosed as having diabetes to less than 110 mg/dl, and preferably between 60 mg/dl and 110 mg/dl.
As has been described, peptides of the present developments selectively bind to a CD40 expressing cell. Consequently, peptides of the present invention may be used to identify Th40 cells. Thus, one embodiment of the present invention is a method to detect Th40, said method comprising contacting a T-cell population with a peptide of the present invention. In a preferred embodiment, the peptide is labeled with a detectable marker, such as, for example, luciferase or alkaline phosphatase. Such detection can be performed using assay techniques known to those skilled in the art. In general, an assay for detecting Th40 cells using a peptide of the present invention comprises (a) obtaining a sample of cells; (b) contacting a peptide of the present invention with said cells under condition suitable to allow binding of the peptide to Th40 cells, if present; (c) washing said cells using conditions that disrupt non-specific interactions, and that remove unbound peptide; and (d) detecting peptide bound to cells. Detection of bound peptide can be achieved directly or indirectly. For example, direct detection can be achieved using a peptide labeled using a detectable marker, as disclosed herein. Following the wash step listed above, the cells are then simply screened for the presence of detectable marker. The presence of detectable marker in the cell sample indicates the presence of Th40 cells. Alternatively, indirect detection involves the use of a second molecule, such as an antibody, that binds to the peptide. In an indirect detection assay, following the wash step listed above, a detection molecule that binds the peptide is added to the cell sample. The detection molecule is labeled with a detectable marker. After washing away unbound detection molecule, the cells are screened for the presence of detectable marker. The presence of detectable marker in the cell sample indicates the presence of Th40 cells. It should be understood that the assays described herein are meant as examples of useful assays, and other assay techniques can be employed. Suitable assay techniques are known to those skilled in the art, and are also disclosed in, for example, Molecular Cloning: A Laboratory Manual, Sambrook, J., Fritsch, E. F., and Maniatis, T, Cold Spring Harbor Laboratory Press; 2nd Edition (December 1989). All referenced cited herein are incorporated herein in their entirety.
The assay technology described above can also be used to identify other molecules that affect the interaction of a CD40 protein with a CD514 protein. Examples of such molecules include, but are not limited to, proteins, peptides and small molecules. For example, assays can be designed that test the ability of molecules to compete with a peptide of the present invention for binding to a Th40 cell. For instance, a peptide labeled with a detectable marker, can be mixed with a test molecule and a population of cells known to contain Th40 cells, under conditions that allow binding of the peptide to the Th40 cells. Following an appropriate incubation period, the cells are washed to remove unbound peptide, and the cells screened for the presence of detectable marker. Alternatively, the labeled peptide could be bound to Th40 cells first, and after a wash step to remove unbound peptide, the test molecule could be added to the cells containing bound peptide. Following an incubating period and a wash step to remove unbound molecule, or released peptide, the cells are screened for the presence of detectable marker. In either case, absence of the detectable marker in the cell sample indicates the test molecule is able to compete with the peptide for binding to the Th40 cells, while presence of the detectable marker would indicate the test molecule does not inhibit binding of the peptide to Th40 cells. Inhibition of binding need not be 100%, as such assay would also be useful for identifying molecules that partially inhibit binding of the peptide to Th40 cells. It is understood by those skilled in the art that such assays would involve the use of positive controls (e.g., unlabeled peptide) and negative controls (e.g., a protein/molecule that is known not to bind to Th40 cells).
Because increased levels of Th40 cells are associated with the development of autoimmune disease, the present invention can be used to identify patients at risk for developing autoimmune disease. Thus, one embodiment of the present invention is a method to identify a patient at risk for developing an autoimmune disease. In one embodiment, patients at risk for developing an autoimmune disease are identified by obtaining a sample from a patient to be tested, contacting the T-cell portion said sample with a peptide of the present invention, and determining the level of Th40 cells present in the sample, wherein a level of Th40 cells greater than about 25% of the total T-cell population indicates the patient is at risk for developing an autoimmune disease. In one embodiment, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, so long as the peptide binds to the CD40 protein. In one embodiment, the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. In a preferred embodiment the peptide is labeled with a suitable detectable marker such as, for example, luciferase or alkaline phosphatase.
The present invention also comprises kits useful for practicing the methods disclose herein. One embodiment is a kit for modulating inflammation in an animal or in cells in culture, the kit comprising a peptide that interacts with a CD40 protein in such a manner as to modulate inflammation. In one embodiment, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, so long as the peptide can down-regulate inflammation. In one embodiment, the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. Another embodiment is a kit for determining the level of Th40 cells, the kit comprising a peptide that interacts with a CD40 protein, and means for detecting CD40-bound peptide. Kits can also contain associated reagents and components, such as, but not limited to, buffers, labels, containers, inserts, tubing, vials, syringes, and the like.
The following examples are provided for the purpose of illustration and are not intended to limit the scope of the present invention.
This Example demonstrates the effect of various peptide fragments of CD154 on CD4/CD8 ratios and the development of diabetes in NOD mice.
Peptides were designed based on the amino acid sequence of mouse CD154 protein (SEQ ID NO:1) in the SwissPro database. The peptides were then ordered from New England Peptide. The lyophilized peptides were suspended in sterile saline at 1 mg/ml. 100 ug of a specified peptide was then injected into the tail vein of 8−9 week-old NOD mice. Control mice received 100 ul of sterile saline. This is well before the onset of diabetes, but after damage to pancreatic islets has begun. Three days after the initial injection, another 100 ug of peptide (or 100 ul of saline in the case of the Control mice) was injected into the tail vein. Mice were then injected with peptide (or saline) on a weekly basis. At 10 weeks of age, mice were monitored for diabetes, as indicated by a blood glucose level greater than 250 mg/dL for three consecutive days. The results of this study are shown in
The results demonstrate that treatment with a peptide unrelated to the CD154 protein did not reduce the development of diabetes in NOD mice. In contrast, treatment of mice with a 15-mer peptide derived from the CD154 protein prevented the onset of diabetes. Further, both the 6-mer and 10-mer peptides derived from the CD154 protein had significant effects on the development of diabetes. In addition, the data demonstrate that the 15-mer peptide did not result in compromise of the immune system, as determined by the CD4/CD8 ratio.
This Example demonstrates the effect of the 15-mer peptide on hyperglycemia in newly diabetic NOD mice.
Six mice from that had received the 6-mer peptide in Example 1, and that had subsequently developed diabetes, were injected intravenously with 100 ug of the 15-mer peptide. These mice were then given weekly injections of the 15-mer peptide into their tail veins, and their blood glucose levels monitored twice-weekly. The 15-mer peptide was administered for a total of ten weeks, after which the treatment was stopped. The results of this study are shown in
This study demonstrates that injection of the 15-mer peptide into already diabetic mice can reverse hyperglycemia. It also demonstrates that cessation of the treatment results in return of hyperglycemia within 7 weeks.
This study demonstrates the ability of the 15-mer peptide to bind to Th40 cell and B cells.
Total lymphocytes were isolated from 9 week-old NOD mice. The lymphocytes were incubated with anti-CD, anti-CD8, and an FITC-labeled 15-mer peptide, and then analyzed by flow cytometry. Cells were gated for CD4 (both CD4hi and CD4lo populations were included) and CD4 versus the 15-mer peptide. The results of this analysis are shown in
B cells were isolated from the spleens of NOD mice. Sorted MHC-II+ (major histocompatibility complex) cells were purified from total lymphocytes. Cells were stained with FITC-labeled 15 mer peptide, anti-CD40, and B cell markers CD19 and CD21. MHC-II+ cells were gated for CD19+ and CD21+ and then 15-mer peptide versus Cd40 antibody was measured. The results of this study are shown in
This study shows that a substantial majority, 90% of CD40+ T-cells, also bind the 15-mer peptide, thereby demonstrating that the 15-mer peptide is highly specific for CD40+ cells. It also shows that while 90% of B cells were CD40 positive, only 8% of B cells bound the 15-mer peptide.
This example demonstrates the level of CD40 positive cells in the blood of type-I diabetic subjects and non-diabetic (control) subjects.
1 ml of whole blood was obtained from each individual and incubated with biotin-conjugated, 15-mer peptide. The cells were then exposed to horse radish peroxidase (HRP)-avidin, washed and the absorbance at 405 nm determined using a spectrophotometer. The results of this study are shown in
This study demonstrates that blood cells from patients having type-I diabetes had higher 15-mer peptide binding activity than cells from non-diabetic controls.
This example demonstrates the level of insulin granulation observed in the pancreas of NOD mice treated with either the 15-mer peptide or a peptide from ovalbumin.
At the onset of diabetes, six NOD mice were injected with 100 ug/ml of the 15-mer peptide (SEQ ID NO:9), resulting in the reversal of hyperglycemia in 80% of the recipients. Six weeks after reversal of hyperglycemia, mice were sacrificed, and the pancreas removed for analysis. The pancreas was fixed, sectioned and then stained using an aldehyde/fuschsin stain that allows detection of insulin granules. Granulation of the tissue was scored as follows: 4=completely granulated; 3=75% of islet granulated; 2=50% of islet granulated, and peri-insulitis; 1=25% of islet granulated; 0=no insulin granules detected. The results of this analysis are shown in
This analysis demonstrates that the 15-mer peptide preserved insulin granules in the majority of the mice and was significantly improved in peptide-reversed diabetic mice compared to diabetic mice that received an irrelevant peptide.
This example demonstrates the effect of mutations in the 15-mer peptide on its ability to prevent the onset of diabetes.
Peptide were designed and produced as described in Example 1. Variant peptides were produced so that in each variant, a glycine was substituted for an amino acid corresponding to an amino acid in positions 1−9 of SEQ ID NO:9, as follows:
NOD mice were placed in groups of 10, and the mice in each group injected IV weekly with 50 ug of either wild-type (WT; Legend) peptide or a variant peptide (in PBD, ph 7.2) listed above. The development of diabetes was monitored by measuring blood glucose levels on a weekly basis. Mice were considered “diabetic” when blood glucose was 250 mg/dl or greater for 2 consecutive readings. Injections began at 6 weeks of age=pre-diabetes.
This example demonstrates that substitution of a glycine at any of positions 1−7, or 9-12, reduces the ability of the 15-mer peptide to inhibit the development of diabetes. It also shows that such mutations do not completely abolish the ability of the mutated 15-mer peptide to inhibit the development of diabetes.
This application is a continuation of U.S. application Ser. No. 15/423,822, filed Feb. 3, 2017, which is a continuation of U.S. application Ser. No. 13/880,387, filed May 23, 2013, now U.S. Pat. No. 9,562,088, which is a national stage filing of International Application No.: PCT/US11/56860, filed Oct. 19, 2011, which claims the benefit of U.S. Provisional Application No.: 61/394,699, filed Oct. 19, 2010, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61394699 | Oct 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15423822 | Feb 2017 | US |
| Child | 17400880 | US | |
| Parent | 13880387 | May 2013 | US |
| Child | 15423822 | US |
