Patent Application
20150299252
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 2,126 byte ASCII (text) file named “Seq_List” created on Dec. 9, 2013.
The present invention is directed to therapeutic peptides and their uses in modulating the innate and adaptive immune systems in a subject.
Viruses such as HIV-1 enter into cells by first attaching to one or more receptors on the surface of a cell, thereby inducing conformational and/or structural changes that allow insertion of the viral genome into the cell [1]. Once inside the cell, the viral RNA genome is transcribed into DNA, is integrated into the host cell's genome, and is then free to replicate. The primary therapy against HIV infections is daily administration of a combination of anti-retroviral drugs that inhibit viral replication after entry into the cell and subsequent maturation. The most commonly used are nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, integrase inhibitors, and protease inhibitors that block enzymatic processing of viral products. These drugs effectively inhibit replication of the virus inside an infected cell and reduce viral load in the blood to undetectable levels [2].
Another therapeutic approach uses fusion inhibitors, including proteins (e.g. monoclonal antibodies), peptides and small molecule agents (e.g. drugs), some of which act on the outside of the cell to prevent HIV from binding to receptors and fusing with the cell membrane [3]. If HIV-1 cannot penetrate the host cell membrane and infect the cell, the virus cannot replicate. Fusion inhibitors effectively block infection by HIV-1 and significantly reduce the systemic viral load. However, sufficient quantities of these drugs must be present in patients continuously to compete with the virus for the receptors. Vaccines that elicit antibodies that inhibit such fusion are of interest in this regard, and great effort is being directed towards achieving this goal [4].
Combinations of small molecular weight drugs achieve undetectable levels of HIV virus in only about 50 to 60% of treated patients. Significant toxicity develops in some patients, which limits the long-term, continuous use of these drugs. In addition, the development of treatments that involve exogenous antibodies is generally costly and requires considerable medical infrastructure for production and treatment. Furthermore, although the development of prophylactic treatments such as vaccines is an important effort, particularly for susceptible target populations, this approach has thus far been unsuccessful. Therefore, protocols must also be developed for those already infected.
A particularly confounding aspect of HIV-1 infections is the establishment of latent reservoirs, in which the integrated provirus stage can remain dormant for long periods of time. Consequently, the virus cannot be completely cleared from an infected individual by current treatments. Upon discontinuation of anti-retroviral treatment, these reservoirs are activated and the virus “rebounds” to pretreatment levels within a few weeks [5]. The question of whether the provirus is indeed dormant or simply replicates at a very low level has not been completely resolved.
Recent advances in the treatment of the RNA hepatitis C virus (HCV) involve development of protease inhibitors that act in a similar manner as those used to treat HIV-1 infections. For HCV, the protease inhibitors are added to the currently accepted drug regime of pegylated interferon-alpha and ribavirin.
An antiviral drug, ganciclovir, a precursor of a nucleotide analog that inhibits the viral DNA polymerase, is the commonly used treatment for acute infections of CMV.
In contrast to therapeutic approaches aimed at prevention or control of the disease by directly inhibiting a step in the viral replication cycle, as described above, reactivation of patients' immune system is an alternative therapy that holds promise for restoring health and productivity to an infected patient in a practical, cost-effective manner. This approach provides a general defense against diseases rather than a pathogen-specific treatment. As a result, an intense interest in immunotherapy, as indicated by the development of cytokine treatments, is leading to products that can stimulate or inhibit the immune system. One developmental cytokine/immunomodulator project for the treatment of HIV/AIDS, for example, identified two key peptides derived from Thymus Nuclear Protein (TNP) technology (Viral Genetics, Inc., Azusa, Calif.), These peptides occur naturally in a variety of mammals, including humans.
The role of cytokines in the inhibition of HIV infectivity, particularly interleukin-16 (IL-16), interleukin-8 (IL-8) and RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted; also known as CCL5), is very important. HIV-1 enters cells by first binding to two key molecular components on the cell surface, the protein CD4 and co-receptor CCR5 or CXCR4. CD4− cells are therefore insensitive to HIV, and a genetic mutation in CCR5 correlates strongly with resistance to HIV-1 infection. Cytokines such as IL-16, IL-8 and RANTES, which have overlapping and complementary functions, can act to attenuate viral infection by competing with viral binding with the receptors and by interfering with viral entry into cells by down-regulating the receptors required for entry. Other cytokines such as interferons (e.g., IFN-α. and IFN-γ) act to reduce viral load by activating intracellular anti-viral enzymes and also by stimulating antibody-mediated phagocytosis.
Interleukins (IL's) and interferons (IFN's) are potent cellular stimulants that are released from a variety of cells in response to insult or injury. Consequently, these proteins have attracted intense interest as therapeutic agents. However, similar to general stimulants such as lipopolysaccharide (LPS), IL's and IFN's induce release of inflammatory cytokines and thus, when given at higher than normal endogenous concentrations during therapy, have substantial adverse effects, which can be life-threatening and may require inpatient treatment facilities. Similarly, levels of TNF-α, IL-1β and IL-6 are directly correlated with the probability of death in humans. Moreover, production of recombinant IL's and IFN's and their application are very costly, and even lower-dosage immunostimulant treatments developed for out-patient use have lower success rates and are not suitable in some situations such as, for example, to extend remission from cancer therapy or control a disease such as HIV at a chronic level.
In general, a stimulant of IL-8, IL-16 or IL-21 release appears to be particularly suited for a role in enhancing host defense. Selective release of IL-8 by monocytes is possible without the release of inflammatory cytokines such as IL-1β and IL-6 [7]. However, a potentially adverse effect of IL-8 production is the enhanced recruitment of neutrophils to inflamed endothelial cells and subsequent release of cytotoxic factors which cause cell/tissue damage, in addition to the continued production of IL-8 by adjacent (non-inflamed) endothelial cells. The consequence is a vicious cycle of recruitment of neutrophils in response to IL-8, damage to tissues, and more production of IL-8, although higher concentrations of IL-8 can be beneficial when they lead to internalization of receptors and de-sensitization of the cells. Therefore, exogenous therapeutic agents such as large, intact cytokine molecules are not well suited for general therapeutic use. IL-16 is a natural ligand of CD4 and should compete with virus for binding to T cells. IL-21 is required to avoid depletion of CD8+ T cells and also essential to maintain immunity and resolve persistent viral infections [8,9].
Usually, viral infections are cleared by the immune system through (i) generation of antibodies by B cells, (ii) lysis of pathogen-infected cells by NK cells and CD8+ cytotoxic T lymphocytes (CTL), and (iii) destruction of the virus by antibody-mediated phagocytosis. While neutralizing antibody responses are subject to viral escape, many non-neutralizating antibodies that nevertheless bind HIV-1 are present in infected patients. Restoration of immune effector cell functions, in particular phagocytic activity, which can recognize the resulting antigen-antibody complexes and destroy the complexes by antibody (Fc)-mediated phagocytosis, may be applicable to the clearance of HIV-1 and viral infections in general. The cell types that have significant involvement in HIV-1 infections in addition to phagocytic cells are in particular, two subsets of the T cell population, NK cells (CD56+) and CTL. These cells are able to kill virus-infected cells by antibody-dependent cellular cytotoxicity (ADCC) in addition to an ability to directly lyse infected cells. NK cells are an integral component of the innate immune system and are primarily responsible for killing virus-infected and cancer cells. NK cells and CTL kill their targets mainly by releasing cytotoxic molecules such as perforin, granzymes and granlysin, which are contained in intracellular granules. These molecules are released when these cells make contact with target cells that contain antigens on the surface of viral infected or cancer cells to which antibodies bind. Activated NK cells also release cytokines and chemokines such as IFN-γ that activates macrophages and drives differentiation of CD4+ T cells into type 1 (Th1) cells [10, 11].
Information relevant to attempts to address one or more of these problems can be found in the following references: U.S. Patent Publication No. 2007/0003542; U.S. Patent Publication No. 2006/0269519; U.S. Patent Publication No. 2004/0248192; P. W. Latham, 1999; Fatkenheuer et al., 2005; Stover et al., 2006; Cohen, 2007; GlaxoSmithKline, 2005a and GlaxoSmithKline, 2005b. However, each one of these references suffers from one or more of the following disadvantages:
1. the size or composition of the agent provides significant challenges to cost-effective synthesis and purification;
2. the agent is specific for particular pathogen and/or cell type, rendering them unsuitable for general therapeutic use;
3. treatment with the agent induces clinically deleterious side effects that can be life-threatening, such as inflammation or hepatotoxicity, and require inpatient treatment facilities;
4. termination of treatment is followed soon thereafter by an increased systemic viral load;
5. long term exposure to agent often leads to treatment-resistant pathogens;
6. lower-dosage treatments developed for out-patient use have lower success rates and are not suitable in some situations;
7. treatment is ineffective, impractical, or cost-prohibitive for a large proportion of patients;
8. development of therapeutic antibodies require considerable medical infrastructure;
9. treatment such as vaccines may be appropriate to prevent infection but not to treat those already infected and who have a suppressed immune system;
10. no beneficial synergy between the immunogenic response induced and the effects of other endogenous immunoregulators;
11. agent inhibits the release of inhibitory cytokines that suppress release of beneficial cytokines, an indirect treatment; and
12. agent acts to restore baseline cytokine levels to balance responses of the immune system rather than promoting activation of phagocytes.
Many of these therapeutic protocols also become ineffective with time because mutation of the pathogen allows it to escape the treatment. Moreover, any immunosuppression that accompanies the disease attenuates the ability of the innate and adaptive immune systems to respond to antigenic changes and thereby keep the infection under control.
The immune system in individuals infected with a pathogenic agent such as HIV-1 initiates a defense response by production of antibodies. Even though the virus may mutate at one or a few sites and thereby escape the neutralizing activity of antibodies, endogenously produced non-neutralizing antibodies are usually polyclonal and may still bind the virus. The presence of anti-HIV-1 antibodies is often used as a diagnostic test for infection. During the course of the disease, the antibody level remains high whereas the ability to maintain a minimal viral load gradually weakens as the population of CD4+ T cells declines. The cellular components of the innate and adaptive immune response then become absent or quiescent. When the immune defense mechanisms reach a sufficiently low level, viral replication is not held in check and rapidly leads to a final stage of the disease, designated AIDS. However, even at this late stage, patients can be rescued from death by aggressive anti-retroviral therapy. Therefore, an agent that reactivates cells of the immune system, in particular phagocytes and NK cells, will likely also restore an immune defense against progression of the disease.
In light of the available treatments for infections such as HIV-induced AIDS, there are large numbers of people worldwide that need alternative, practical, cost-effective, non-specific therapies that directly bolster a patient's immune system during the course of the disease without causing deleterious side effects. Ideally, such therapies should also be effective against other types of pathogens. Work described in this document is focused on HIV-1 infections as a model disease, although it is expected that the technology should be effective against other pathogens such as HCV, CMV and cancer cells.
Therapeutic agents that activate/reactivate the immune system show particular promise in this regard, including cytokines and immunomodulators, although therapies based on exogenous agents such as large, intact cytokine molecules are not generally well suited for therapeutic use. Peptides, however, are often much more suitable therapeutic agents than large polypeptides or proteins. Peptides can, for example, be designed to induce one or more particular desired effects in vitro or in vivo, often without concomitantly inducing deleterious effects, and can usually be synthesized in a cost effective manner.
Chronic infections by viruses such as HIV-1, Hepatitis C virus (HCV) and Cytomegalovirus (CMV), with about 33M, 200M and 4B infected individuals world-wide, respectively, exact a heavy toll on humans and world economies. Each of these viruses develops latency, which can persist for extended periods of time, and causes life-threatening disease when reactivated in immuno-compromised individuals. Several innate and adaptive immune functions are required for long-term defense against these infections such as active phagocytic cells for antigen presentation and subsequent generation of antibodies against these viruses for destruction of antibody-virion immune complexes. However, most of the antibodies do not prevent infection, i.e., are non-neutralizing. Also of importance is the activity of natural killer (NK) cells and cytotoxic T cells, which are components of the innate defense system and have the ability to destroy infected and cancerous cells directly as well as by antibody-dependent cellular cytotoxicity (ADCC). Potential benefit could be achieved by activation of these cells as an immuno-therapeutic protocol. The implications of this approach are enormous and form the basis of the technology described herein.
Approximately 10% of the 33M individuals infected with HIV-1 will die each year from AIDS. In addition, the annual number of new infections is estimated to be 5 million and rising. The cost of treating this disease with anti-retroviral drugs is enormous, and varies from $2,500 per patient in Brazil to over $15,000 per patient per year in developed countries, which continues for each year of an individual's life. Cost of prevention is estimated at more than $120 billion over the next 10 years, although the long-term benefit from prevention would dramatically reduce future costs for treatment and care. The bulk of the cost of current treatment is for anti-retroviral drugs, which are remarkably effective but often lead to resistance. Furthermore, life-long control of the infection, most likely by management as a low-grade, chronic disease, increases the cost burden beyond that which can be afforded in low- and middle-income countries.
Of additional concern is the number of people who are infected with HCV, which world-wide is 5- to 8-fold greater than those infected with HIV-1, with about 4 million in the US. HCV infects liver cells and as the disease progresses causes cirrhosis of the liver and eventually failure of the organ. The current treatment for HCV infections, to which most people will respond, includes anti-retroviral drugs and pegylated interferon-alpha.
As people age, the incidence of infection by CMV increases from about 50% in young children to about 80% in the elderly. A significant percentage of infants that become infected with CMV will develop disabilities that include blindness and loss of hearing. CMV, as a member of the Herpes virus family, lies latent until the immune system is suppressed. Consequently, CMV infections are opportunistic and often accompany infections by HIV-1 and other pathogens.
The development of this technology has been focused on HIV-1 as a pathogen, although the technology should also be applicable to diseases caused by other viruses, bacteria, fungi and cancers.
The present invention is directed to compositions and methods of modulating the innate and adaptive immune systems in a subject, preferably including activation of natural killer (NK) cells and/or CD8+ cytotoxic T lymphocytes. The method comprises: administering to the subject a composition comprising a therapeutic peptide or a multivalent structured polypeptide comprising multiple copies of the therapeutic peptide, the therapeutic peptide consisting of 5 to 8 amino acids, the peptide being selected from the group consisting of:
In one embodiment, the method preferably, further comprises determining the level of NK cells and/or CD8+ cytotoxic T cells in the subject's blood. In this embodiment, it is advantageous to further establish a ratio of NK cells and/or CD8+ cytotoxic T cells compared to monocytes in the subject's blood.
The method also preferably further comprises administering at least one agent selected from the group consisting of: a cytotoxic T cell proliferation agent, NK cell proliferation agent, or an anti-inflammatory agent.
The therapeutic peptide used is preferably selected from the group consisting of: VGGGS (SEQ ID NO:1), PSSNA (SEQ ID NO:2), HPSLK (SEQ ID NO:3), HPSLG (SEQ ID NO:4), HPSLL (SEQ ID NO:5), HPSLA (SEQ ID NO:6), NPSHPLSG (SEQ ID NO:7), and NPSHPSLG (SEQ ID NO:8). A branched multivalent structured polypeptide comprising multiple copies of the therapeutic peptide is preferred.
The composition advantageously comprises the therapeutic peptide in an amount sufficient to induce activation of NK cells in the subject and the subject is a human. The therapeutic peptide preferably functionally mimics a terminal sequence 5-acetylneuraminic acid-galactose on complex glycans, the terminal sequence being linked α(2-3) or α(2-6).
The therapeutic peptides are advantageously configured to bind to the receptor NKG2D and/or sialic acid-binding immunoglobulin-like lectins and function as modulators of the immune system by binding to receptors on natural killer cells, cytotoxic T cells and/or phagocytic cells.
In one embodiment, the therapeutic peptide or multivalent structured polypeptide is administered in an amount sufficient to induce antibody-mediated cellular cytotoxicity in the subject, preferably to increase the expression of at least one endogenous cytokine from lymphocytes elected from the group consisting of: IL-2, IL-4, IL-16, IL-17, IL-21, TNF-β, IFN-γ and RANTES and/or decreases at least one endogenous cytokines elected from the group consisting of: IL-1α, IL-1β, IL-13, IL-12p40, and IL-12p70, TNF-α.
The method may advantageously further comprise the step of administering an antibody preparation admixed in an amount sufficient to enhance antibody-mediated cellular cytotoxicity.
The present invention is also directed to a therapeutic composition. The therapeutic composition preferably comprises a carrier; at least one agent selected from the group consisting of: an anti-inflammatory agent, a cytotoxic T cell proliferation agent, or a NK cell proliferation agent; and a therapeutic peptide or a multivalent structured polypeptide as described above. In certain embodiments, the composition further comprises an antibody preparation admixed in an amount sufficient to enhance antibody-mediated cellular cytotoxicity in a subject; or further comprises an immunoglobulin admixed with the polypeptide composition in an amount sufficient to enhance passive immunoprotection.
The present invention also provides a method of treating rheumatoid arthritis in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition or therapeutic peptide disclosed herein.
The present invention is directed to compositions and methods of activating of NK cells and/or CD8+ cytotoxic T lymphocytes.
An important component of immune system stimulation by the peptides is activation of NK (natural killer) cells and CTL (cytotoxic T lymphocytes) in addition to activation of phagocytic cells. To this end, peptidic mimetics of the glycan 5-acetyl-neuraminic acid-galactose [Neu5Ac(α2-3)Gal and Neu5Ac(α2-6)Gal] were designed. These glycans bind to NKG2D, an important activating receptor on NK cells, γδ T cells and CD8+ cytotoxic T cells [11,12], and to the family of siglecs (sialic acid-binding Ig-like lectin) receptors that occur on most cells of the immune system [13]. Whereas identified endogenous ligands of NKG2D are several protein-based activating ligands [10], binding of glycans should also activate these cells [14]. Activation of phagocytes occurs by binding of peptides to siglecs or other receptors on these cells. The therapeutic peptides consist of a multivalent structure in which the arms consist of sequences only 9 to 12 amino acids long (including a linker sequence). The active sequences of the relevant peptides that were described previously [U.S. Pat. No. 7,811,995 incorporated by reference thereto] are VGGGS (SEQ ID NO:1), HPLSK (SEQ ID NO:3), NPSHPLSG (SEQ ID NO:7) and NPSHPSLG (SEQ ID NO:8).
Preferably, the peptides are in substantially pure form. Typically it is desired that the peptides be at least 70%, more preferably at least 80%, and most preferably at least 95% pure by weight. In one embodiment the N-terminus may also be acetylated.
In a preferred embodiment, the peptides of the invention comprise a peptide construct with at least two arms. The construct typically has a central framework and each arm comprises a therapeutic sequence linked to the central framework via a linker. Each therapeutic sequence of the peptide construct can be the same or different. In a preferred embodiment, the therapeutic sequence is the same for each arm of peptide construct. The therapeutic sequence is preferably selected from the group of therapeutic peptides described above.
The present invention also provides therapeutic compositions comprising at least one peptide of the invention and a pharmaceutically acceptable carrier. In a preferred embodiment, the composition is an immunostimulatory composition, preferably further comprising an antigen and/or an antibody preparation admixed therewith in an amount sufficient to enhance antibody-mediated cytotoxicity or phagocytosis. Alternatively, the composition may comprise an immunoglobulin admixed with the therapeutic peptide in an amount sufficient to substantially enhance passive immune protection, e.g., at least 30% increase compared to the control.
In another embodiment, the therapeutic compositions comprises a carrier; at least one agent selected from the group consisting of: an anti-inflammatory agent, a cytotoxic T cell proliferation agent, or a NK cell proliferation agent; and a therapeutic peptide or a multivalent structured polypeptide as described above. In certain embodiments, the composition further comprises an antibody preparation admixed in an amount sufficient to enhance antibody-mediated cellular cytotoxicity in a subject; or further comprises an immunoglobulin admixed with the polypeptide composition in an amount sufficient to enhance passive immunoprotection.
Preferred anti-inflammatory agents include anti-inflammatory peptides, antibodies, and small molecules, e.g., anti-TNF-α antibody; non-steroidal anti-inflammatory molecules, etc.
Preferred cytotoxic T cell proliferation agents and/or NK cell proliferation agents include molecules that increase IL-2, IL-15 and IL-21 expression. Alternatively, molecules that induce IL-12 and IL-18 expression are included.
The peptides of the invention are useful in treating the subject having a disease, especially those diseases treatable by endogenous induction of antibodies against invading pathogens or endogenous antigens of harmful cells. The peptides of the invention can specifically be used to treat such diseases as viral infections, cancer, bacterial and yeast infections, and/or other autoimmune diseases which require treatment through stimulation of the immune system. Such autoimmune diseases include rheumatoid arthritis, psoriasis; dermatitis; systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease; Crohn's disease; ulcerative colitis; respiratory distress syndrome; adult respiratory distress syndrome (ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions; eczema; asthma; conditions involving infiltration of T cells and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; systemic lupus erythematosus (SLE); diabetes mellitus; multiple sclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjorgen's syndrome; juvenile onset diabetes; immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes; tuberculosis; sarcoidosis; polymyositis; granulomatosis; vasculitis; pernicious anemia (Addison's disease); diseases involving leukocyte diapedesis; central nervous system (CNS) inflammatory disorder; multiple organ injury syndrome; hemolytic anemia; myasthenia gravis; antigen-antibody complex mediated diseases; anti-glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous; pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-man syndrome; Behcet disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathies; idiopathic thrombocytopenic purpura (ITP) and autoimmune thrombocytopenia.
The invention encompasses methods of substantially activating subsets of lymphocytes in a subject, in particular NK cells that attack diseased cells directly or by antibody-dependent cellular cytotoxicity, which complements activation of Fc-mediated phagocytosis, to treat a subject. In a preferred embodiment, HIV-1 replication is inhibited in the subject by at least 50%, more preferably by at least 90% as compared to a control and/or levels prior to administration of the peptide in the subject. In the presence of antibodies, inhibition may reach 100%.
In a preferred embodiment, to provide a non-specific therapeutic agent with a relatively broad front, an agent that activates NK and cytotoxic T cells preferably works in concert with the phagocytic cells of the immune system. The peptides of the present invention can accomplish this goal by concomitantly stimulating the immune cells, including NK cells and phagocytes, and to respond in particular to the presence of pathogen-directed antibodies. Treatment with the peptides of the present invention therefore preferably induce activation of cells of the immune system in vivo and provide a sustained endogenous defense against the pathogen.
In the following description, and for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the various aspects of the invention. It will be understood, however, by those skilled in the art, that the structures, compositions, and methods are sometimes shown or discussed generally in order to avoid obscuring the invention. In many cases, a description of the material and operation is sufficient to enable one to implement the various forms of the invention. It should be noted that there are many different and alternative technologies and treatments to which the disclosed inventions may be applied, and the full scope of the inventions is not limited to the examples that are described below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term “pharmaceutically acceptable” as used herein means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which an active ingredient is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, other excipients can be used.
Preferably, the subject being treated by the methods described herein is a mammal, e.g., monkey, dog, cat, horse, cow, sheep, pig, and more preferably the subject is human.
“Effective amount” or “therapeutically effective amount” is meant to describe an amount of therapeutic peptide or composition of the present invention effective to modulate the innate and adaptive immune systems and/or treat or prevent a disease (e.g., rheumatoid arthritis) in a subject and thus produce the desired therapeutic effect in the subject.
Typical compositions and dosage forms may comprise one or more excipients. Suitable excipients are well known to those skilled in the art of pharmacy, and non-limiting examples of suitable excipients are provided herein. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).
The present invention comprises therapeutic peptides, compositions of those therapeutic peptides for administration to a subject in need, and methods to stimulate the immune system of a subject through the administration of compositions containing those therapeutic peptides. In general, the advantage of this invention is the modulated release of specific cytokines and the stimulation of immune cells, including but not limited to NK cells, CD8+ T cells and phagocytes, to respond to the presence of pathogen-directed antibodies. Non-limiting examples of cytokines include immunoregulatory proteins such as interleukins and interferons, which are secreted by cells of the immune system and can affect the immune response. A non-limiting example of the stimulation of immune cells is the induction of Fc-mediated phagocytosis. An additional example is direct activation of NK cells for antibody-dependent cellular cytotoxicity. A further example is activation of NK cells and CTL to lyse infected or cancer cells by direct cellular cytotoxicity.
The single letter designation for amino acids is used predominately herein. As is well known by one of skill in the art, the single letter designations are as follows: A is alanine; C is cysteine; D is aspartic acid; E is glutamic acid; F is phenylalanine; G is glycine; H is histidine; I is isoleucine; K is lysine; L is leucine; M is methionine; N is asparagine; P is proline; Q is glutamine; R is arginine; S is serine; T is threonine; V is valine; W is tryptophan; Y is tyrosine.
The therapeutic peptide is preferably 5 to 8 amino acids. Preferred therapeutic peptides are selected from the group consisting of:
In a most preferred embodiment, the therapeutic peptide is selected from the group consisting of: VGGGS (SEQ ID NO:1), PSSNA (SEQ ID NO:2), HPSLK (SEQ ID NO:3), HPSLG (SEQ ID NO:4), HPSLL (SEQ ID NO:5), HPSLA (SEQ ID NO:6), NPSHPLSG (SEQ ID NO:7), and NPSHPSLG (SEQ ID NO:8).
Multivalent structured polypeptides comprising multiple copies of the therapeutic peptide are preferred. In one embodiment, the multivalent structured polypeptide comprises a construct and at least two arms, the construct having a central framework and each arm comprising a therapeutic peptide sequence linked to the central framework via a linker, wherein each therapeutic sequence is preferably the same.
As used herein, “construct” is defined as the entire molecule and comprises the central framework linked with at least two arms. In a preferred embodiment, the construct comprises the central framework linked to 2 or more arms, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 arms, preferably 2 to 8 arms. In a further preferred embodiment, the construct comprises the central framework linked to 4 arms. Each arm within the construct may consist of the same or different therapeutic sequence and/or linker. In one preferred embodiment, the therapeutic sequence is the same between arms.
The “central framework” provides a structure for attaching the arms. The central framework is based on a core molecule which has at least two functional groups to which molecular branches having terminal functional groups are bonded, e.g., a tri-lysine to which the peptide arms are added. Such molecules may be developed or created to present a varying number of branches, depending on the number of monomers branched from the core molecule. Each terminal functional group on each branch provides a means of attachment to an arm. Non-limiting examples of preferred central framework include: ethylenediamine(1,2-ethanediamine), ethylene glycol (1,2-dihydroxyethane), polyols such as glycerol, 3,5-diaminobenzoic acid, 1,3,5-triaminobenzene, and monocarboxylic-diamino compounds of intermediate length. Preferably, the monocarboxylic-diamino compounds are within the range of 2 to 10 carbons in length. Non-limiting examples of such compounds are 2,3-diaminopropionic acid and 2,6-diaminocaproic acid. In a more preferred embodiment, the monocarboxylic-diamino compound is 6 carbons in length. Compounds that provide an aromatic central framework which absorbs light may be beneficial for determining peptide concentration as well. The carboxyl group of the monocarboxylic-diamino compounds allows the addition of C-terminal tags including biotin derivatives. In a preferred embodiment, the central framework comprises a tri-lysine core (a lysine residue as the central molecule bonded to two lysine residues, each through its carboxyl group, to one of the amino groups of the central lysine residue), providing a central framework for four arms.
The “arm” comprises the therapeutic sequence, plus the linker. The “linker” comprises a peptide chain or other molecule that connects the central framework to the core sequence. In a preferred embodiment, the linker comprises, but is not limited to, certain linker peptide sequences, polyethylene glycol, 6-aminocaproic acid (6-aminohexanoic acid), 8-aminooctanoic acid, and dextran. In a most preferred embodiment, the linker is GGGS (SEQ ID NO:9), GGGSGGGS (SEQ ID NO:10), SSSS (SEQ ID NO:11), SSSSSSSS (SEQ ID NO:12), or variations thereof. The length of the linker can be adjusted, for example, the linker GGGS (SEQ ID NO:9) can be repeated to provide variable lengths, e.g., repeated twice (GGGSGGGS (SEQ ID NO:10)), or even three or more times; additional serine residues could be added to SSSS (SEQ ID NO:11) to also produce varying lengths of the linker. The therapeutic peptide preferably functionally mimics a terminal sequence 5-acetylneuraminic acid-galactose on complex glycans, the terminal sequence being linked α(2-3) or α(2-6). The therapeutic peptides are advantageously configured to bind to the receptor NKG2D and/or sialic acid-binding immunoglobulin-like lectins and function as modulators of the immune system by binding to receptors on natural killer cells, cytotoxic T cells and/or phagocytic cells.
The therapeutic peptide is preferably administered in an amount sufficient to induce activation of NK cells in the subject and the subject is a human. In one embodiment, the therapeutic peptide or multivalent structured polypeptide is administered in an amount sufficient to induce antibody-mediated cellular cytotoxicity in the subject, preferably to increase the expression of at least one endogenous cytokine from lymphocytes elected from the group consisting of: IL-2, IL-4, IL-16, IL-17, IL-21, TNF-β, IFN-γ and RANTES and/or decreases at least one endogenous cytokines elected from the group consisting of: IL-la, IL-1β, IL-13, IL-12p40, and IL-12p70, TNF-α.
The method may advantageously further comprise the step of administering an antibody preparation admixed in an amount sufficient to enhance antibody-mediated cellular cytotoxicity.
The step of determining the level of NK cells and/or CD8+ cytotoxic T cells in the subject's blood is done using well known methods in the art, e.g., flow cytometric analysis of peripheral blood mononuclear cells with use of antibodies against cell-specific surface markers. It is advantageous to further establish a ratio of NK cells and/or CD8+ cytotoxic T cells compared to monocytes in the subject's blood. In a preferred embodiment, the ratio of NK cells or CD8+ cytotoxic T cells to monocytes is 3:1 or more preferably 4:1. The present invention is most effective with a higher ratio NK cells and/or CD8+ cytotoxic T cells compared to monocytes.
In another aspect, the present invention provides a method of treating rheumatoid arthritis in a subject, the method comprising administering to the subject a therapeutically effective amount of a therapeutic peptide or composition disclosed herein. The therapeutic peptide may be selected from the group consisting of: VGGGS (SEQ ID NO:1), PSSNA (SEQ ID NO:2), HPSLK (SEQ ID NO:3), HPSLG (SEQ ID NO:4), HPSLL (SEQ ID NO:5), HPSLA (SEQ ID NO:6), NPSHPLSG (SEQ ID NO:7), and NPSHPSLG (SEQ ID NO:8).
The present invention identifies a series of peptides that stimulate immune response and modulate the release of specific cytokines Thus, in a first aspect, the present invention provides a therapeutic peptide consisting of 9 to 12 amino acids in length (including a spacer sequence). In a preferred embodiment, the therapeutic peptide is in a substantially purified form. As used herein, the term “substantially purified” refers to material which is substantially or essentially free from components which normally accompany it as found in its synthesized state. When the material is synthesized, the material is substantially or essentially free of cellular material, gel materials, culture medium, chemical precursors, contaminating polypeptides, nucleic acids, and other organic chemicals. Preferably, the peptide is purified to represent greater than 90% (dry weight) of all organic molecular species present. More preferably the peptide is purified to greater than 95% (dry weight), and most preferably the peptide is purified to essential homogeneity, wherein other organic molecular species are not detected by conventional techniques. Advantageously, the therapeutic peptide is reacted with acetic anhydride to acetylate the N-terminus of the therapeutic peptide. Acetylation protects the peptide from N-terminal degradation and therefore is preferred.
Peptide sequences were identified by computer-aided molecular modeling of docking to the sugar-binding site of lectins, which served as receptor analogs. The concept underlying the design of Susavion's peptides had several components. From knowledge that a number of receptors on cells of the immune system bind carbohydrate ligands [15,16], we focused on developing peptidic mimetics of these glycan ligands. Peptides of 5 to 8 amino acids in length fill the glycan binding site of lectins and receptors and are sufficiently short to be invisible to the immune system. An important aspect of the final peptide is a multivalent structure that is capable of cross-linking receptors, an event that is critical to initiation of a signal transduction pathway within the cell [17,18]. To determine the most effective amino acid sequence of a peptide, molecular modeling was performed of docking of a single (monovalent) sequence into the glycan-binding site of well-characterized lectins, which were selected as analogs of cell-surface receptors. The crystal structures of the lectins were down-loaded from the Protein Data Bank (PDB). ArgusLab 4.0.1 software (Mark A. Thompson, Planaria Software LLC, Seattle, Wash.) was used for modeling. Amino acid residues that comprise the binding site of a lectin or receptor were selected from the literature that describes each protein. Through this approach, unique peptide sequences were evaluated by predicted binding energy. These in silico experiments predicted that some peptides would bind to a variety of lectins with sufficiently high affinities to encourage further characterization.
A model for interaction of the peptide designated SVH1C (SEQ ID NO:7) with the glycan-binding site of the lectin MAA from Maackia amurensis (accession no. 1DBN), which binds with high specificity to trisaccharides containing a terminal Neu5Ac(α2-3)Gal linkage, is shown in
The short peptide sequence was then incorporated into multivalent structures (
The concept of the importance of valency for high affinity binding [21-23] was tested directly by synthesizing a monovalent peptide in which the sequence was extended from the α-amino group of ε-biotinyl-lysine amide. The bivalent molecule contained the peptide sequence extended from the α and ε amino groups of a second lysine residue linked to ε-biotinyl-lysine amide. The quadravalent molecule contained the peptide sequence extended from the four amino groups of a tri-lysine scaffold in which the C-terminal amide group (as in
The lectins, their specificities, and PDB accession numbers were the GalNAc/Gal-specific lectin from Helix pomatia (2CE6), GalNAc-specific lectin from Vicia villosa (1N47), GalNAc-specific lectin from Dolichos biflorus (1LU2), the lectin from Triticus vulgaris (1WGT), which binds GlcNAc and Neu5Ac but also clusters of GalNAc, and the lectin from Canavalia ensiformis (3CNA), which binds Man. The lectins MAA and SNA1, described below, were also included in the study.
As shown in
Binding of SVH1C to MAA and SNA1 was examined further as a function of the amount of peptide added to assay wells containing bound lectin. As shown in
To confirm that the peptides interacted with carbohydrate-binding sites on the lectins, competition binding assays were performed with the glycoprotein fetuin. Each fetuin molecule contains collectively 12 to 15 oligosaccharides that terminate predominantly as Neu5Ac-Gal, with nearly equal α(2-3) and α(2-6) linkages, on three N-linked and three O-linked glycans [27,28]. If indeed SVH1C mimics Neu5Ac-Gal termini, fetuin should compete effectively with the peptides and inhibit their binding to MAA and SNA1. As shown in
To further examine the ability of fetuin to inhibit binding of peptides to MAA and SNA1, concentrations were chosen that were one-third, equal or 5-fold the molar concentration of peptide in the assay. Similar to the data shown in
The binding of SVH1C to lectins such as MAA and SNA1 suggests that the peptide mimics Neu5Ac-Gal sequences on the termini of complex glycans. This sequence is a ligand for the receptor NKG2D on NK cells and δγ T cells and CD8+ cytotoxic T cells [12]. Also, a family of approximately 15 lectin-type receptors, the siglecs (sialic acid-binding Ig-like lectin) receptors, binds Neu5Ac-Gal- sequences (reviewed in ref 13). The siglecs are thought to promote cell-cell interactions and regulate the functions of cells in the innate and adaptive immune systems through glycan recognition. These receptors are possible targets of the peptide, as predicted by molecular modeling (
Binding of SVH1C to NKG2D was assayed by isothermal microcalorimetry. SVH1C (100 μM) was titrated into a solution of NKG2D (11 μM, monomer concentration) and changes in heat content of the system was measured.
The thermodynamics of the binding reaction, with a positive AH, suggests that the entropy was a major factor in the strong binding. Similar characteristics of ligand binding to NKG2D, with positive enthalpy and a large entropy contribution, were reported in the literature [19,20,30,31]. Furthermore, NKG2D occurs as a homodimer but the concentration was introduced into the analysis as the monomeric number of binding sites. In the analysis, the stoichiometry, n, yielded a value of 0.71. This number suggests that approximately half of the bound peptide cross-linked the two binding sites of the dimeric protein or that only half of the protein had both binding sites filled.
NKG2D is not known to function as a glycan receptor in vivo, although the Neu5Ac(α2-3)Gal structure binds to the C-type lectin domain of the receptor [12]. On the other hand, the siglecs have been characterized as receptors that bind Neu5Ac(α2-3) or (α2-6)Gal [13,32]. These receptors function as either inhibitory or activating when bound with a ligand. Siglec-1 is expressed on monocytes and macrophages and is involved in cellular adhesion but also enhances endocytosis. As such, it enhances infection of these cells by HIV-1 by binding to glycans on the envelop of the virus [33,34]. Because siglecs are known to bind to the same glycans as the lectins MAA and SNA1 (see
Direct binding of SVH1C to siglecs was demonstrated by a solid-phase assay in which recombinant chimeric siglecs were bound in microtiter wells coated with protein A/G. The chimeric siglecs contained an N-terminal glycan-binding domain and a C-terminal Fcγ domain, which bound strongly to protein A. Biotinylated peptides were then incubated with the siglecs, the wells were stringently washed and the bound peptide was detected by binding of streptavidin conjugated with peroxidase.
The solid-phase assay was also used to demonstrate binding of SVH1C to NKG2D (
Among the siglec receptors, most are inhibitory receptors and contain an ITIM (immunoreceptor tyrosine-based inhibitory motif) within their cytosolic domain, whereas a few, in particular Siglec-14 and Siglec-15, function with an activating adaptor protein [13,32,36]. NKG2D is also an activating receptor and contains an ITAM (immunoreceptor tyrosine-based activation motif) within its cytosolic, C-terminal domain. The function of these receptors is regulated by phosphorylation of the tyrosine residue within the regulatory motif. As illustrated in
To determine whether activation of cells by the peptides could be detected by induction of release of cytokines, cultured peripheral blood mononuclear cells (PBMCs) were treated with one peptide embodiment of the present invention and, after 4 h incubation, the medium was collected and assayed for changes in the amounts of 40 different cytokines. A therapeutic peptide construct containing four copies of the therapeutic sequence VGGGS (SEQ ID NO:1), HPSLK (SEQ ID NO:3) or NSPHPLSG (SEQ ID NO:7) was added at a concentration of 100 nM in each of the assays. Approximately 3 million cells of frozen human PBMCs were thawed at 37° C. and transferred to a 50 mL conical tube where 8 mL of wash medium were added slowly. Then an additional 8 mI of wash medium were added and swirled to mix. The cells were then centrifuged at 330 g for 10 min, the supernatant was removed and the pellet was resuspended in 10 mL wash medium and centrifuged as above. The washed cells were then resuspended in RPMI-1640 medium containing 10% FBS to about 6 million cells per mL and 100 mL of the suspension were added into each well of a 96-well microtiter plate and incubated overnight at 37° C. in humidified 5% CO2. After 24 h the medium was replaced with 200 μL fresh RPMI-1640 medium containing 10% FBS and incubated at 37° C. in humidified 5% CO2 for 2 days. The peptide aliquot was then added to samples in duplicate at a final concentration of 100 nM and incubated at 37° C. in humidifed 5% CO2 for 4 h. The medium was then removed and stored at −80° C. The samples were analyzed for production of cytokines One set of control cells was not treated with an experimental agent. A second set of control cells was treated with lipopolysaccharide, an agent commonly used to induce production of a variety of inflammatory cytokines. The positive control for inflammation was essential to ensure that the peptides did not produce an unregulated inflammatory response.
Culture medium was removed for assay of cytokine levels with methods developed by RayBiotech, Inc. (Norcross, Ga.). In this technology, membrane arrays of antibodies against cytokines are incubated with samples of media. After washing, the array was incubated with a cocktail of all antibodies tagged with biotin. The membrane was then washed free of unbound antibodies and incubated with streptavidin, labeled with a fluorescent dye, which binds to biotin. After a final wash, the membrane arrays were read in a fluorescence detector.
The peptides stimulated release of several important cytokines. In particular, IL-21, a cytokine produced by CD4+ T cells that is required for proliferation and differentiation of natural killer cells and CD8+ cytotoxic lymphocytes. Additional cytokines released by the general population of T cells in response to treatment with the peptides of this invention were IFN-γ, IL-4, IL-8, IL-16, IL-17, TNF-β, and RANTES. Of importance, release of the inflammatory cytokines IL-1α, IL-1β, IL-6, IL-10, and TNF-α were not induced. Release of other important cytokines, notably Eotaxin-2, IL-10, IL-13, IL-12p40, and IL-12p70, was reduced (Table 1).
The mixture of cytokines released from PBMCs, in particular T cells, in response to the peptides described herein should provide, either in isolation or in combination with other treatments, an effective modulation of the immune system. Treatment with the peptides of the present invention should induce activation of cells of the immune system in vivo and provide a sustained endogenous elevation of beneficial cytokines, in contrast to the rapid disappearance of these proteins when given exogenously. These cytokine responses are presumably in addition to direct activation of the immune cells engaged in fighting a disease.
The embodiments and examples set forth herein were presented in order to best explain the present invention and its practical application and to thereby enable those of ordinary skill in the art to make and use the invention. However, those of ordinary skill in the art will recognize that the foregoing description and examples have been presented for the purposes of illustration and example only. The description as set forth is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the teachings above without departing from the spirit and scope of the forthcoming claims. Although the examples herein disclose the therapeutic efficacy of the peptides of the present invention, with respect to neutralizing replication of the HIV virus, for example, the peptides should be useful to treat a wide variety of infections or disorders, including prophylactic treatments for prevention of such maladies, and for enhancing or stabilizing the well-being of healthy subjects.
Human PBMCs were incubated 3 days with peptides and then assay plates were stained with the soluble tetrazolium-based dye MTS to determine cell viability. The mitochondrial enzymes of metabolically active cells metabolize MTS to yield a colored formazan product. After an incubation period of 4 to 6 h at 37° C., the plates were read spectrophotometrically. Cells treated with peptide SVH1C alone or with diluted anti-HIV antiserum were 100±2% viable at peptide concentrations of 1 nM to 1 μM. In another assay to determine cytotoxicity, cells were doubly stained with acridine orange and ethidium bromide. In this assay, viable cells fluoresce green while dead cells fluoresce red. SVH1C did not exhibit cytotoxicity at a concentration of 1 mM, a concentration 106-fold greater than an effective bioactive concentration of 1 nM [37].
Toxicity of the peptide in vivo was tested by injection of a peptide into animals. In preliminary studies on rats, intravenous injections of peptides that provided 1000-fold greater concentrations than an expected therapeutic dose was well tolerated by the animals and no adverse effects of the peptide were been observed. The peptides can be administered in a number of ways, including without limitation by injection (intravenously, subcutaneously, intramuscularly or intraperitoneally, topically (transmucosally, transbuccally, sublingually, or transdermally) and/or orally (liquid, tablet or capsule).
The ability of the peptides to inhibit replication of HIV-1, both alone and in combination with antibodies, was tested as follows. Approximately 3 million cells of frozen human PBMCs, obtained from the California Blood Bank system, were thawed at 37° C. and transferred to a 50 mL conical tube where 8 mL of wash medium were added slowly. Then an additional 8 mL of wash medium were added and swirled to mix. The cells were then centrifuged at 330 g for 10 min, the supernatant was removed and the pellet was resuspended in 10 mL wash medium and centrifuged as above. The washed cells were then resuspended in RPMI-B medium containing 10% FBS, phytohemagglutinin was added to 5 μg/mL, and cells were incubated at 37° C. for 24 or 72 h in humidified 5% CO2. Cells were washed, suspended to about 6 million cells per ml, and 50 μL (about 250,000 viable cells) were added to each test well. Then 100 μL of the test peptide were added at a concentration sufficient to provide the desired final concentration, followed by 100 μL of virus suspension [25 to 100 median tissue culture infective doses (TCID50)]. The assay plate was incubated 3 days at 37° C., then washed 3 times to remove unbound virus, and the cells were again suspended to 250 μL of medium. After an additional 24 h of incubation, cells were lysed with Triton X-100 and each sample was assayed by an enzyme-linked immunoassay for virus protein p24 to quantify neutralization of virus. In another set of samples, antibody preparations were also added at a desired concentration (i.e., in addition to peptide).
Complete inhibition of HIV-1 replication (neutralization) in PBMCs by several peptide constructs containing four copies of a therapeutic sequence linked to a branched central framework structure was demonstrated previously [37]. This peptide construct was assayed for activity with two subtypes of HIV, clade B (Strain SF162) and clade C (Strain 97ZA009), both of which were provided by the California Department of Public Health (Richmond, Calif.). HIV-1 clade B is the major subtype in North America and Europe and HIV-1 clade C is the major subtype in central and southern Africa, India and China. The peptide was assayed either alone or in combination with serum from HIV-infected individuals. In the absence of peptide, the antibody preparation (serum) provided only 25 to 40% neutralization at the same dilution (data not shown).
To explore this phenomenon further, PBMCs were depleted of monocytes by positive selection with magnetic beads to which anti-CD14 antibodies were attached. Depletion of CD14+ monocytes was verified by flow cytometry (
It was found that when the cultures were overwhelmed with a high input of the virus, the percent of neutralization was reduced. Thus, in subsequent experiments the viral input was reduced to about 30 TCID50. An assay was performed in which peptides were added to the culture without antiserum. For the results shown in
The data shown in
Siglecs occur on the surface of most of the cells of the immune system. In general, siglecs show low affinity (a KD of 0.1-3 mM) for the sialic acid Neu5Ac (α2-3) and (α2-6) linkages to galactose [Neu5Acα(2-3)Gal and Neu5Acα(2-6)Gal]. It is assumed that this recognition is important for modulating the functions of siglecs as regulators of adhesion, cell signaling and endocytosis [13,32].
The fact that the peptides act alone as well as in concert with antibodies against a virus suggest that they may be effective as immune system modulators for use in therapy for a variety of infections against which an individual develops antibodies. The data shown herein demonstrate that the peptides SVH1C (SEQ ID NO:7), SVH1D (SEQ ID NO:8) and SV6B (SEQ ID NO:3), as well as SVC2 SEQ ID NO:1) which is not shown, functionally mimic glycans with terminal Neu5Ac-Gal sequences. Receptors such as NKG2D and siglecs bind these glycans. Siglec-1 is expressed on monocytes and macrophages and is involved in cellular adhesion but also enhances endocytosis. As such, it enhances infection of these cells by HIV-1 by binding to glycans on the envelop of the virus [33,34]. Because siglecs are known to bind to the same glycans as the lectins MAA and SNA1 (see
Unless defined otherwise, all technical and scientific terms 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 be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patents, and patent publications cited are incorporated by reference herein in their entirety for all purposes.
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.
It is understood that the disclosed invention is not limited to the particular methodology, protocols and materials described as these can vary. It is also understood that the terminology used herein is for the purposes of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
This application is a continuation-in-part of U.S. patent application Ser. No. 13/287,102, filed on Nov. 1, 2011, which claims the benefit of U.S. Provisional Application No. 61/409,044, filed Nov. 1, 2010, the contents of each of which are incorporated herein by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 61409044 | Nov 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13287102 | Nov 2011 | US |
| Child | 14101334 | US |
