METHODS OF USING sIP-10, CD26 INHIBITORS AND CXCR3 LEVELS IN A SAMPLE TO ASSESS CLEARANCE OF INFECTION, RESPONSE TO INTERFERON THERAPY, AND TREATING CHRONIC INFECTIONS
The present invention relates to methods for the treatment of infection, a disease, or a condition using CD26 (DPIV) inhibitors. The present invention also relates to an antibody that binds to the IP-10 protein and a method of monitoring the necessity for administering a CD26 inhibitor to a patient, comprising evaluating a level of sIP-10, a activity of CD26, and/or a level of CXCR3 cells in a sample.
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This application a Continuation-in-Part of U.S. application Ser. No. 11/855,238, filed Sep. 14, 2007, now U.S. Pat. No. 7,674,578, issued Mar. 9, 2010, which claims benefit of Provisional Application Ser. No. 60/844,375, filed Sep. 14, 2006, the entire contents of which are incorporated herein by reference. This application also claims benefit of Provisional Application Ser. No. 61/085,102, filed Jul. 31, 2008, the entire content of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION1. Field of the Invention
The present invention relates to methods for evaluating and predicting clinical outcomes in patients by measuring levels of protein expression as well as therapies using sIP-10 and its fragments. The present invention also relates to methods for the treatment of infections using CD26 (DPIV) inhibitors. The present invention also relates to methods of monitoring levels of CXCR3 cells in blood, a level of IP-10 and a CD26 activity for assessing patients as to clearing chronic infections and responding to IFN therapy and the necessity of administering CD26 inhibitors.
Description of the Background
Hepatitis C virus (HCV) is a significant health problem in that chronic hepatitis occurs in over 50% of infected individuals and may lead to the development of life threatening cirrhosis, hepatic cancer, as well as other sequellae. Chronic hepatitis is often treated with interferon α and/or β and other type I and III interferons that can also be administered in combination with ribavirin with varying levels of success depending on genotypes of the virus.
There are nearly 170 million HCV infected individuals worldwide (1). Many studies have explored the mechanisms by which the immune system is capable of mediating viral clearance (2). Although an HCV-specific humoral response is commonly seen, HCV-specific antibodies are not thought capable of conferring protection (3). Instead, protection in chimpanzees has been shown to correspond with HCV-reactive CD8+ T cell responses. Similarly in humans, a tetramer analysis of successful resolvers indicates the presence of a high frequency of functional HCV-specific CD8+ T cells (4). In addition, there is genetic data to support a role for NK cells based on the higher likelihood of. KIR2DL3 and HLA-Cl individuals to clear infection (5). One thing is clear—cure is possible—20-40% of patients spontaneously resolve infection and 40-80% of chronically infected patients (numbers vary depending on viral genotype) that receive peg-IFN-α2/RBV therapy clear the virus and are sustained virologic responders (SVR) (6). Still for many, the virus manages to circumvent natural immunity and our therapeutic strategies, thus resulting in significant morbidity and mortality including liver cirrhosis or hepatocellular carcinoma.
To better define the distinct clinical outcomes of HCV infection many investigators have performed candidate molecule screens or transcriptional profiling in order to identify correlates of viral clearance. One molecule that has gained significant attention is CXCL10 (also known as interferon-gamma induced protein-10 or IP-10), an interferon- and TNFα-inducible chemokine that can be highly expressed by endothelial cells, keratinocytes, fibroblast, mesangial cells, astrocytes, monocytes, neutrophils and hepatocytes (7). In addition to HCV disease, it has been shown to be expressed in many Th1-type inflammatory diseases, often correlating with the target organ infiltration by T cells (8, 9). CXCL10 is a part of a family of α-chemokines that bind CXCR3, which also includes CXCL9 (also known as monokine induced by IFNγ or MIG) and CXCL11 (also known as IFN-inducible T cell a chemoattractant or I-TAC). While all three ligands are induced by IFN and bind the same receptor, there now exists substantial data to support their unique roles in disease pathogenesis. This is clearly evident in chronic HCV, wherein elevated levels of all three have been demonstrated, but only CXCL10 is predictive of a response to the therapy (10).
Regarding published studies of chronic HCV patients, at least five independent cohorts have demonstrated that baseline levels of CXCL10 are predictive of the failure to respond to HCV treatment (10-13). Specifically, this has been demonstrated in patients with genotype 1 and 4 HCV and in the largest study a negative predictive value of 79% was reported for genotype 1 patients (12). In addition, elevated levels of CXCL10 have been reported in patients co-infected by HCV/HIV (14, 15); and with HCV-associated cryoglobulinemia (16). In several of these reports, CXCL10 levels correlate with necroinflammatory activity, fibrosis stage and/or HCV viral load. However, none of the reports have resolved the paradox of why a pro-inflammatory chemokine, responsible for recruiting activated lymphocytes to the liver, is a negative prognostic marker for response to therapy.
The inventors hypothesized that CXCL10 in chronic HCV is catabolized and exists in an antagonist form, thus offering a rationale for its negative impact on treatment responsiveness. Such a mechanism of regulating a chemokine activity has been proposed for several chemokines. Pertinent in vivo examples include the degradation of monocyte chemoattractant protein-3 (MCP-3) by matrix metalloproteinase-2 (MMP-2), reported to be relevant to the pathogenesis of spondyloarthritis (17); and the cleavage of stromal cell derived factor-1 (SDF-1 or CXCL12) in HIV-associated neurodegeneration, also mediated by MMP-2 (18). Herein, the inventors provide direct evidence that the NH2-terminal residues of plasma CXCL10 have been cleaved in HCV patients. Moreover, the inventors have found a correlation between the CXCL10 levels and the precursor frequency of CXCR3+ cells in circulation, indicating in vivo that the chemokine gradient is non-functional. The inventors have investigated matrix metalloprotease (MMP) and dipeptidylpeptidase (DPP) family members as mediators of the truncation and evaluated a role of the enzyme dipeptidylpeptidase IV (DPP4 or CD26). Following from the observations, the inventors have found an important link between the metabolic complications and the chronic inflammation seen in HCV patients. The inventors have determined that DPP4 is a novel therapeutic target, inhibition of which results in the restoration of the CXCL10 gradient and allows for increased responsiveness to peg-IFN-α2/RBV therapy.
Even with interferon therapies, there are substantial side effects, and the costs of therapy are very high. Accordingly, there is a need to develop a way to assess when successful results can be expected in an individual patient with a particular IFN therapy.
Thus, an object of the invention is to provide a way in which to predict whether such therapies would be useful for treating certain patients and also a treatment of infections using CD26 (DPIV) inhibitors.
SUMMARY OF THE INVENTIONThere exists a need to determine and predict clinical outcomes in patients with HCV. It is therefore an object of the invention to provide a means for evaluating (e.g., determining and/or predicting) clinical outcome for a patient suffering from a clinical condition or syndrome.
One aspect of the invention is using the short form of IP-10 (sIP-10) as a predictive marker for failure to clear HCV and failure to respond to IFN based therapy.
Another aspect is determining the total, long and short forms of IP-10 using antibodies that are specific for the long and/or short forms of IP-10.
Another aspect is to provide a treatment for infections, diseases, or conditions. In one embodiment a treatment of chronic Hepatitis C by administering a patient in need of the treatment an effective amount of a CD26 inhibitor to restore the chemokine gradient is provided. In a further aspect, predicting which patients will respond to therapy and improve upon the existing treatment of chronic HCV patients using combination therapy of interferon, ribavirin and CD26 inhibitors. In other aspect, CD26 inhibitors may be used as monotherapy to address some of the sequellae of chronic HCV, e.g., type II diabetes.
In another embodiment, a method of monitoring the necessity for administering CD26 inhibitors to a patient, comprising evaluating chemokine antagonism, is provided.
In a different embodiment, a level of CXCR3 in blood may be monitored.
In other aspects, the antibodies that distinguish between the short and long forms of IP-10 can be used to evaluate patients with chronic inflammation (e.g., infection, cancer, obesity, autoimmunity, graft vs. host disease).
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skill artisan in chemistry, biochemistry, cellular biology, molecular biology, and medical sciences.
All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.
The phrase “pre-selected gene(s)” refers to proteins that have been determined to be suitable in practice of the invention. Preferably, in accordance with practice of the invention, such proteins are selected where there is a correlation between the level of protein expression and the nature and extent of a disease state or other undesired condition.
Methods are provided for quantitating protein expression levels, and the measured levels are compared against reference populations. Deviations from the reference levels can be correlated with clinical outcomes. For example, the type and extent of a patient's response to a therapeutic intervention can be determined, or the prognosis for a patient's survival can be estimated. The protein levels can be measured in essentially any chosen body tissue or fluid.
IP-10 is CXC chemokine (NP 001556) and functions to recruit activated and memory lymphocytes to sites of inflammation. The secreted bioactive form (after cleavage of the signal peptide) is composed of a protein having 77 amino acids (positions 22-98). In situations where CD26 is expressed in an active state, IP-10 may be cleaved with the removal of its two N-terminal amino acids to a short form (termed sIP-10, amino acids 24-98). Importantly, sIP-10 has antagonist activities and repels activated and memory lymphocytes from sites of inflammation. IP-10 has previously been identified as a negative indicator for chronic HCV patients receiving the IFN/ribavirin treatment. This finding is somewhat counterintuitive as IP-10 is a pro-inflammatory molecule and should facilitate the priming of T cells by attracting CXCR3− cells (TH1 cells) to the infected organ.
The inventors have identified IP-10 as a negative prognostic indicator for chronic HCV patients receiving treatment. The observation that IP-10 correlates with failure to spontaneously clear the virus as well as with failure of chronic HCV patients to respond to therapy suggests a common mechanism for non-responsiveness (and possibly, a common mechanism by which patients successfully resolve infection, whether it be natural or therapeutic).
The inventors have identified a positive correlation between the IP-10 levels in patients and the number of CXCR3 cells in circulation. This too was counter intuitive as IP-10 in the tissue should result in the homing of CXCR3 ((the sole receptor for IP-10) and a depletion of CXCR3 cells from the blood. The resolution of these paradoxical observations comes from the prediction that IP-10 is acting as an antagonist. Indeed, the inventors have confirmed that the two N-terminal amino acids of IP-10 are cleaved by CD26 and, in turn the short form inhibits CXCR3 by acting as a dominant negative.
The inventors have analyzed the relationships of IP-10 and HCV and have discovered that IP-10 is a negative predictive marker for clearance of hepatitis C virus (HCV). This has been demonstrated, for example, in a cohort of chronically infected HCV patients with genotype 1 virus (Paris cohort). Notably, the study compared chronically infected patients and sustained virologic responders, but data from other groups (Butera et al, Blood, 106(4):1175 (2005)) support the inventors' findings. This observation has been confusing for the field as it is counter-intuitive because IP-10 is considered a pro-inflammatory molecule and should facilitate the efficient priming of anti-HCV specific T cells.
Thus, the invention and the solution to this problem are based on the identifying by the inventors a positive correlation between the IP-10 levels in patients and the absolute number of CXCR3 positive cells in circulation. It has been suggested in the literature that IP-10 may be cleaved by CD26 (also called DPPIV, dipeptidyl peptidase IV), resulting in the generation of a short form of IP-10 (sIP-10) that acts as an antagonist of CXCR3 (De Meester et al, Immunology Today, 20(8):367 (1999)). sIP-10 is implicated in the failure of chronically infected HCV patients to respond to interferon therapy. Additional applications may include evaluation of patients with chronic inflammation (e.g. cancer, obesity, autoimmunity, graft vs. host disease). Specific applications should be focused on diseases in which IP-10 and/or CD26 have been shown to be elevated (e.g., melanoma, type II diabetes, autoimmune vasculitis). IP-10 is implicated in every chronic infection, autoimmunity andncer. Also,there are related enzymes such as FAP that cleaves IP-and is upregulated in many cancers such as melanoma. Neville et al. (Cytokine and Growth Factor Review, 8(3):207-219 (1997)) describe the immunobiology of IP-10. This cytokine is implicated in various biological systems, e.g., cell development, cell division, cell differentiation, chemotaxis, cell adhesion, cell cytolysis, angiogenesis, anti-tumor mechanisms, and regulation of feeding (see, e.g., Table 3 of Neville et al.).
Thus, as used herein, it is understood that sIP-10 means a short form of IP-10 that has been cleaved by CD 26 and portions thereof. sIP-10 lacks some N-terminal residues but always conserves the C-terminal residues which are useful to the binding of the truncated cytokine to its CXCR3 receptor. For example, sIP-10 has an amino acid sequence shown as SEQ ID NO:3. In another embodiment, the sIP-10 has the sequence of RTVRC TCISISNQPV NPRSLEKLEI IPASQFCPRV EIIATMKKKG EKRCLNPESK AIKNLLKAVS KEMSKRSP (SEQ ID NO:4).
These sequences can be used as well as fragments thereof having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 and 75 amino acids thereof, as well as all values and ranges there between.
Therefore, sIP-10 is a clinically important predictive marker for failure to clear HCV and failure to respond to the IFN therapy. This represents an important and medically useful discovery as we have the ability to determine prior to treatment, which patients will fail therapeutic treatment, thus saving them from up to a year of an expensive treatment with significant side effects (e.g., major depression). Of note, ˜50% of genotype 1 patients fail to clear virus in response to IFN therapy. Furthermore, the introduction of such a diagnostic tool may assist physicians in determining responsive patients, thus pushing them to treat earlier with potentially more impact in avoiding the long-term sequella of HCV (e.g. cirrhosis, hepatocellular carcinoma).
Thus, the short (truncated) form of IP-10 (sIP-10) can be used as an improved predictive marker for failure to clear HCV and failure to respond to IFN therapy. It also permits optimal use of resources, an important factor in the treatment of HCV in regions such as Egypt and other developing countries. Further, CD26 can also be used as a therapeutic target. The CD26 inhibitors can be used to treat HCV patients with a combination therapy with Peg-IFNα2A/Ribavirin, Peg-IFNα2B/Ribavirin or a combination thereof. Other type I and III IFNs can be also used.
While there are many approaches aimed at increasing the numbers of antigen-specific T cells, we believe that this is not the problem in HCV disease pathogenesis. The real issue is getting the T cells to the right place—the liver. There are no current therapies aimed at reversing the effects of chemokine antagonism. Moreover, there are few approaches for HCV that are aimed at immune modulation: instead. most efforts are focused on inhibiting the vital protease and/or other viral proteins. While effective, such approaches are not yet available and seem to early carry serious side effects.
In one embodiment, a method of treating infections, diseases, or conditions composing administering to a patient in need thereof, an effective amount of a CD26 inhibitor to restore the chemokine gradient, thereby treating the infection, disease, or condition, is provided. The patient can be infected with hepatitis C virus. The patient can also be chronically infected with HCV.
Total, long and short (truncated) IP-10 can be determined utilizing polyclonal (rabbit) and monoclonal (mouse) antibodies that are specific for the long form of IP-10. In an added embodiment, in vivo IP-10 activity can be assessed by using CXCR3 cells as a surrogate for IP-10 activity. Flow cytometry can be used to measure the relative amount of CXCR3 cells in the peripheral blood of patients throughout their treatment. This will serve as a reflection of the ability of activated T cells and B cells to respond to a gradient of IP-10 (keeping in mind that there exist other CXCR3 agonists). In another added embodiment, a dipeptidyl peptidase IV activity can be measured in patient plasma, which can correlate to the IP-10 cleavage.
In these analysis, it is preferred to inactivate the plasma DPPIV activity, thus preventing an extra-corporeal cleavage of IP-10. Collections are thus preferably performed with protease inhibitors. For example, one such commercially available sample collection tube is BDTM P700 but other such BD™ 100 are also sufficient to preserve IP-10 in its circulating form.
In one embodiment, antibodies specific for the long form of IP-10 to evaluate the amount of long vs. short form of IP-10 are generated. Such antibodies that distinguish full-length IP-10 from short (truncated) form of IP-10, were generated by immunization of rabbits with a synthetic peptide coupled to KLH; (VPLSRTVRC (SEQ ID NO:5) corresponding of the NH2 terminal of the full-length IP-10 protein). Immunoglobulines G (IgG) can then be purified in two steps by chromatography affinity: the first step is a positive selection of IgG that recognize the long peptide. The second step is depletion of IgG that recognize the shorter peptide LSRTVRC (SEQ ID NO:6) common to short and full-length form of IP-10. Purified antibodies are specific to full-length IP-10. The same strategy can be used to obtain polyclonal antibodies from different species (e.g., goat, chicken, and others.). Monoclonal antibodies may also be derived from mice after immunization with the long peptide. After verifying the presence of antibody production in the serum, hybridomas are produced and cloned. The hybridoma screening can be made by direct ELISA with both, the long and short peptides, and the full-length and short form of recombinant IP-10 coated directly in ELISA plate. Then antibodies from hybridomas specific of the full-length can be purified. Validation of polyclonal antibodies can be performed by direct and by sandwiches Elisa assays against short and full-length IP-10 form. SIP-10 can be generated by following in vitro digestion of recombinant full-length IP-10 by recombinant CD26. The two NH2 amino-acids, Valine and Proline are truncated from the full-length.
Using these distinguishing antibodies, IP-10 can be detected in biological fluids, for example, using an ELISA assay to specifically detect the full-length IP-10. As W-10 binds to heparin, heparin crosslinked to agarose beads via amide bonds can be used to concentrate IP-10 from plasma. IP-10 can be eluted with NaC1 1M. To avoid or at least minimize ex vivo truncation of IP-10 in biologic samples, inhibitors of CD26 can be added into collection tubes. The HuCAl (Human Combinatorial Antibody library) technology allows to generate in vitro a 1.5 billion human antibodies candidates library. An automated panning process is used: the peptides specific of long and short forms of IP-10 are immobilized in 384-well microplate for screening against antibody-displaying phage library. Specificity of antibodies is then verified by ELISA.
To generate the short (truncated) IP-10 form, the full length IP-10 sequence (vplsrtvracisisnqpvnprslekleiipasqfcprveiiatmkkkgekrelnpeskaiknllkayskemskrsp, SEQ ID NO:2) can be incubated with CD26 at 25-37° C. to generate the short form (lsrtvretcisisnqpvnprslekleiipasqfcprveiiatmkkkgekrclnpeskaiknllkayskemskrsp, SEQ ID NO:3). CD26 is a known protein, with a cloned sequence (e.g., from mammals such as human and mouse). Resolution of IP-10 and sIP-10 can be accomplished by Western blotting or by MALDI-TOF-MS as is known in the art.
Chemokine antagonism is responsible for resistance to IFNαa2/Ribavirin therapy in patients with chronic HCV. Determining the levels of the antagonist form of IP-10 will serve as a predictive marker for responsiveness to therapy: and treatment with an inhibitor of CD26 (also known as dipeptityl peptidase IV) can be used to decrease the antagonist form of IP-10 and help re-direct HCV-specific T cells to the liver. Thus, sIP-10 can be used for studying the failure of chronically infected HCV patients to respond to interferon therapy. Thus, the invention provides means to improve treatments of Hepatitis C in patients (mammalian, generally with humans, preferred) and, in one embodiment, such is accomplished by predicting which patients will respond to therapy: and improve upon the existing treatment of chronic HCV patients using combination therapy. Such combination therapy can employ existing HCV treatments with interferons, e.g., interferon a (preferably IFN α2A and IFN α2B) and ribavirin with CD26 (DPIV) inhibitors as discussed herein. Other type I and III interferons can be used. One, two or more interferons can be used. The various inhibitors currently undergoing animal and human testing vary in both potency and length of action. For example, Novartis, Merck and Prosidion (PSN9301) compounds are currently in phase III trials and clinical data are likely to become available in the near future. Various CD26 (DPIV) inhibitors are also described in McIntosh et al., Applications of dipeptidyl peptidase IV inhibitors in diabetes mellitus, International Journal of Biochemistry & Cell Biology, 38(5-6): 860-872 (May-June 2006), e.g., a large number of DPIV inhibitors have been described and their structures and characteristics have been succinctly reviewed ([Augustyns et al., 1999], [Evans, 2002] and [Weber, 2004]; Wiedeman and J. M. Trevillyan, Dipeptidyl peptidase IV inhibitors for type 2 diabetes and metabolic syndrome, Drug Discovery Today Therapeutic Strategies, 2(2): 143-149 (2005)). The majority of inhibitors fall into one of three main classes (
In other embodiments, the antibodies that distinguish between the short and long forms of IP-10 can be used to evaluate patients with chronic inflammation (e.g., cancer, obesity, autoimmunity, graft vs. host disease). Specific applications can be focused on diseases in which IP-10 and/or CD26 have been shown to be elevated (e.g., melanoma, type II diabetes, autoimmune vasculitis).
CD26/dipeptidyl-peptidase IV (DPP-IV) inhibitors include, amino boronic dipeptide talabostat (PT-100), Ile-Pro-Ile (Diprotin A), alanyl pyrrolidine, isoleucyl thiazolidine, and N-valyl prolyl, and O-benzoyl hydroxylamine. One such inhibitor sold as a treatment for type 2 diabetes is JANUVIA TM (sitagliptin phosphate). One, two or more CD26/dipeptidyl-peptidase IV (DPP-IV) inhibitors can be used.
Other such inhibitors are known and described in U.S. Pat. No. 4,935,493, “Protease Inhibitors”,; U.S. Pat. No. 5,462,928, “Inhibitors of Dipeptidyl-aminopeptidase Type IV”; U.S. Pat. No. 5,543,396, “Proline Phosphonate Derivatives”; U.S. Pat. No. 5,296,604, “Proline Derivatives and Compositions for Their Use as Inhibitors of HIV Protease”; PCT/US92/09845, “Method for Making a Prolineboronate Ester”. Representative structures of the transition-state analog-based inhibitors Xaa-Boro-Pro, include Lys-boroPro, Pro-boroPro, Val-boroPro and Ala-boroPro in which “boroPro” refers to the analog of proline in which the carboxylate group (COOH) is replaced with a boronyl group (B) (OH)2. Compounds as described in U.S. Pat. No. 6,100,234. One, two or more inhibitors can be used. All of the preceding patents and patent applications are incorporated herein by reference.
One embodiment of the invention is to assess the ability of a patient to clear HCV from circulation and/or the body. In another embodiment, the effectiveness of interferon therapy can be assessed.
According to these embodiments, it is possible to assess these indices by determining the expression levels of sIP-10. In these embodiments, one can assess the expression and effects by comparing the expression level of sIP-10 to a set of reference expression levels predictive of a patient's ability to clear HCV. Likewise, an increased expression of sIP-10 relative to a reference expression standard is indicative of a decreased ability to clear HCV.
In another embodiment, a method of monitoring the necessity for administering a CD26 inhibitor to a patient, comprising evaluating chemokine antagonism in a sample is provided. In a different embodiment, a level of CXCR3 in blood of the patient and/or a level of NH2-terminal truncation of IP-10 protein can be also monitored/evaluated for assessing the necessity for administering a CD26 inhibitor to a patient.
Since the effectiveness of IFN therapy is not limited to an HCV infected patients, the methods can be carried out in patients not infected or not known to be infected.
A patient not infected with HCV who may benefit from this methodology may be a patient with a disease in which IFN is a normal course of therapy as known in the art.
An “individual” or “patient” which may be subjected to the methodology described herein may be any of mammalian animals including human, dog, cat, cattle, goat, pig, swine, sheep and monkey. Humans are preferable.
A “sample” is a biological sample such as blood, serum, plasma, lymph, and tissue. The “sample” may also be pretreated, for example, by homogenization, extraction, enzymatic and/or chemical treatments as commonly used in the field.
The preparation and/or isolation of protenacious material from a sample for analysis are also well known in this field.
Measuring the levels of sIP-10 expression in a sample is easily undertaken by one knowledgeable in this field. Non-limiting examples include, western-blot analysis, ELISA assays, immuno-reactivity assays, column assays using fluorescence, antibodies and radiolabeled markers to detect. Automated manners of performing the analysis, for example, using a computer processor can also be used.
The inventors developed the first method of detecting long form of IP-10. No detection system to date has been described for the detection of long form IP-10 and the determination of the amount of sIP-10 in a sample. This novel tool supports a method for predicting responsiveness to therapy in chronically infected HCV patients. Other applications may exist. This new tool solves the problem of determining the ratio of long to short form of IP-10. It has immediate use in a prospective observational study for developing predictive markers in HCV patients.
Inhibiting CD26 and/or providing a short form of IP-10 as an agonist of full length IP10 is part of the invention. The polypeptides can be naturally obtained, obtained from recombinant sources and/or synthesized chemically, the procedures for which are known in the field. Administration can be any suitable route, for example, intravenously, intranasally, peritoneally, intramuscularly, orally and other conventional methods. An active compound can be mixed and/or carried with one or more liquid and/or solid pharmaceutically acceptable carriers, ingredients and/or excipients.
High Levels of Plasma CXCL10 Correlates with Failure to Respond to Peg-IFN-α2/RBV Therapy
In order to identify biomarkers for response to therapy, the inventors have analyzed chronic HCV patients with plasma samples taken prior to, during and following their 48-week course of peg-IFN-α2/RBV. Multi-analyte profiling was performed on 84 selected molecules involved in inflammation, liver function and metabolism. The inventors performed a Kruskal-Wallis analysis of variance, comparing patients who failed to respond to therapy (non-responders, N−n=9), those who cleared the virus (sustained virologic responders, S−n=13) and healthy individuals (H−n=7) (
NH-2-Truncated CXCL10 is the Dominant Form Circulating in Chronic HCV Patients
Several independent studies have previously identified CXCL10 as a biomarker for chronic HCV patients (10-13), however, none of the published reports account for why a pro-inflammatory Thi-type chemokine might be a negative predictor for response to therapy. The inventors hypothesized that CXCL10 is present in an antagonist form in chronic HCV patients, thus accounting for it correlating with treatment failure. Briefly, chemokines act by binding to G-protein coupled receptors and mobilization of intracellular Ca++, which resulting in receptor internalization and the initiation of signaling pathways that facilitate chemotaxis. For the CXC class of chemokines, binding to, and activation of the receptor is thought to be a two-step process. First, the core of the ligand binds the outer surface of the receptor; a second step involving the re-orientation of the flexible N-terminal tail of the protein allows binding to a distinct domain within the receptor (24). Post-secretion modification of CXCL10 has been described, including C-terminal cleavage by metal metalloproteinase 9 (MMP9 or gelatinase B) and citruillination by peptidylarginine deiminase (PAD), both of which leave the protein in an agonist state (25, 26), Also reported is the N-terminal cleavage of two amino acids by members of the X-prolyl dipeptidylpeptidase (DPP) family, the most characterized being dipeptidylpeptidase IV (DPP4 or CD26)(27). DPP4 has been shown to cleave several chemokines, including members of the α-chemokine family (28, 29).
In order to test the hypothesis, it was necessary to generate novel reagents. First, recombinant full length CXCL10 (1-77) was exposed to recombinant DPP4 and cleavage was monitored by SELDI mass spectroscopy (
To overcome this hurdle, the inventors have generated antibodies capable of distinguishing the agonist form of CXCL10 (1-77) (see the Examples for details on the generation of the pAb specific for the NH2-terminal tail of the protein, termed antisera L3) (
CXCL10 Levels in Chronic HCV Patients Correlate with High Frequency of Circulating CXCR3+ Cells
As a second test for the inventors' hypothesis, the inventors have developed an ‘in vivo-bioassay’ to evaluate the fate of CXCR3+ cells in individuals who have high levels of plasma CXCL10. To the inventors' knowledge, there is no report that evaluated in vivo migration as a function of plasma chemokine expression while taking care to determine that the chemokine is in a bioactive form. To establish a positive control for the assay, blood samples were collected from chronic HCV patients during the initiation of their treatment with peg-IFN-α2/RBV. 6 hr following the initial dose of peg-IFN-α2, the inventors observed high levels of plasma CXCL10, all of which existed in the agonist form, based on detection with anti-CXCL10, antisera L3 (
Utilizing this in vivo-bioassay, the inventors next evaluated the frequency of circulating CXCR3+ cells in chronic HCV patients (not receiving therapy). Strikingly, the inventors have found a higher frequency of CXCR3+ cells in HCV non-responders, the same population with high plasma CXCL10 (
To provide direct evidence for in situ CXCL10 cleavage, the inventors have generated antibodies specific for the NH2-terminus of CXCL10 (3-77) (see the Examples for details on the generation of mAb clone 52.1) (
Elevated Dipeptidyl Peptidase Activity in Chronic HCV Patients is Due to DPP4
There exist five enzymes with X-prolyl DPP activity, including DPP2, DPP4, DPP8, DPP9 and FAP (27). CXCL10 is a known substrate of DPP4, DPP8 and FAP. DPP4 is a membrane protein that may be shed upon inflammation (referred to as sDPP4) (31). In addition, several chemokines have been shown to be substrates for MMPs. CXCL10 has been reported to be cleaved at its C-terminus by MMP9 and MMP12 (25, 32); and it has been demonstrated that the cleavage product produced by MMP9 acting on CXCL10 remains an agonist for CXCR3 (25). One important and biologically relevant unknown is the action of MMP2 on CXCL10. Indeed, MMP2 has been shown to cleave the N-terminal amino acids of MCP-1 and SDF-1, thus generating an antagonist form of these molecules.
To evaluate the potential enzymes implicated in cleavage of CXCL10, the inventors exposed full length recombinant CXCL10 to active MMP2, MMP9 and DPP4. As predicted, MMP9 cleaved 9 amino acids from CXCL10 (
To further investigate the mechanism of CXCL10 cleavage, the plasma DPP activity in patients with HCV infection has been assayed. Indeed, the plasma DPP activity was higher in chronic non-responders as compared to SVR and healthy individuals (
Interestingly, when the plasma concentration of CXCL10 and sDPP4 against each other was plotted, there was a positive correlation (rs=0.6,
Discussion
Chemokine Antagonism in Chronic HCV Patients
In a screen to identify correlates for treatment induced clearance, the inventors have re-discovered plasma CXCL10 as a pre-treatment negative prognostic marker in chronic HCV (
The described studies offer the first evidence for chemokine antagonism as a possible mechanism in HCV disease pathogenesis. Although some pieces to this puzzle have been known, however, a synthetic evaluation of the problem has not been previously reported. Several independent studies have shown CXCL10 to be a negative predictive marker for response to therapy in chronic HCV patients (10-13). This observation has held up for genotype 1 and 4 patients (12), including difficult to treat patients with high levels of liver fibrosis, co-infected HIV/HCV infected patients and individuals with autoimmune sequella (e.g. mixed cryoglobulinemia and thyroiditis). Regarding CXCL10 being a substrate for DPP4, the seminal in vitro studies were performed by Proost and Van Damme, who established that CXCL10 (3-77) is capable of acting as a dominant negative, binding CXCR3 without signaling and competitively inhibiting binding by the agonist form of CXCL10 (1-77) (29). DPP4 is constitutively expressed by a wide range of cell types including hepatocytes, fibroblasts, epithelial and endothelial cells; and it is an activation marker for lymphocytes, in particular Th1 and effector/memory CDS+ T cells. In addition to the cell associated DPP4, the enzyme is shed via the action of a still unidentified sheddase, and healthy donors harbor a physiologic plasma activity in a range of 12-25 U/L. While published kinetic studies suggest that this should be sufficient to cleave even high concentrations of CXCL10, no prior in vivo studies have confirmed CXCL10 to be present in an NH2-terminus cleaved form. Finally, there have been two clinical reports that have made a link between dipeptidylpeptidases and chronic HCV. Firneisz et al. noted the link between liver injury and DPP activity and in a large 144 person cohort reported higher plasma X-prolyl dipeptidylpeptidase activity in patients with chronic HCV infection than in healthy controls (33). This study, however, did not define the enzyme responsible for the enzymatic activity, nor did it make a link to any of the immunologic or metabolic findings in chronic HCV patients. Yang evaluated sDPP4 as a marker of Thl cell activation and followed 33 chronic HCV patients, reporting lower sDPP4 plasma concentrations as compared to healthy donors (34). Their study, however, did not take precautions to control for DPP4 shedding, nor did it evaluate enzymatic activity. Thus, it is believe that the study described herein, through the application of novel tools, has offered a synthetic picture and an important perspective regarding the inflammatory state of chronic HCV patients.
DPP4 Provides a Link Between Metabolic and Immunologic Dysregulation in Chronic HCV Patients
Insulin resistance and type II diabetes are common co-morbidities in chronic hepatitis C patients, with recent estimates indicating that 30-50% of patients have some degree of metabolic glucose abnormality. The incretin hormone, glucagon-like peptide-1 (GLP-1) enhances the release of insulin from islet g cells in a glucose-dependant manner. It also facilitates uptake of glucose by myocytes and hepatocytes while simultaneously suppressing glucagon secretion. GLP-1 has come under close scrutiny as a contributor to insulin resistance, and the mechanism is linked to cleavage by DPP4 (35). In fact, GLP-1 is the most characterized substrate of DPP4, and as the inventors have observed for agonist CXCL10, removal of the two N-terminal amino acids of GLP-1 converts it from an agonist to an antagonist.
It is worthy of mention that insulin resistance is a negative predictor for response to IFN/ribavirin therapy (36, 37). There is also a case report of a diabetic chronic HCV patients who received a 48 wk course of IFN/ribavirin and was cured of their type II diabetes (37). One possible explanation that might account for such a finding is that HCV-driven liver inflammation results in increase DPP4 activity that in turn revealed a predilection for type II diabetes. While not currently focused on DPP4, recent clinical studies have aimed at stabilizing patients' glucose control as a means of improving their response to therapy. These initial trials have failed, but perhaps they were utilizing the wrong anti-hyperglycemic agent(s).
DPP4 as a Novel Drug Target for the Treatment of Chronic HCV
Following from the clinical use of recombinant GLP-1 as a means of enhancing insulin production and more recently from the biochemical evidence that GLP-1 is cleaved by DPP4 into an antagonist form that results in insulin resistance, many biotechnology groups and pharmaceutical companies have made an effort to develop DPP4-specific inhibitors as a novel treatment for type-2 diabetes. DPP4 inhibition prevents the inactivation of glucagon-like peptide 1 (GLP-1), resulting in increased endogenous levels of agonist GLP-1. Sitagliptin (Merck; approved by the FDA in 2007) and Vildagliptin (Novartis; approved by the FDA in 2009) are orally active compounds with a long duration, and both have been used as monotherapy with excellent safety profiles and seem to be well tolerated with minimal risk of hypoglycaemia or weight gain. DPP4 inhibitors are quickly being phased in as first-line treatment of type II diabetes.
The identification of agonist CXCL10 as an in vivo target for DPP4 and the possibility that this cleavage event is a mechanism of treatment failure in chronic HCV patients are attractive. As such, DPP4 inhibition are beneficial for HCV patients, with the objective being restoration of the chemokine gradient and the ability of CXCR3+ cells to properly traffic to sites of inflammation. Not only can we envision DPP4-inhibition enhancing clearance rates achieved by peg-IFN-α2/ribavirin therapy, but it offers the added benefit of addressing underlying metabolic problems present within the same patient population.
Pharmaceutical Applications
As used herein, “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. If necessary, the chimeric compounds can be co-administered with a solubilizing agent, such as cyclodextran.
A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., chimericcompounds) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
Nucleic acid molecules encoding the chimeric compounds of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) PNAS 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
The dosages can be assessed by the physician administrating the therapy taking into consideration factors such as weight, age, sex, race, severity of the infection and other prior, concurrent and/or future therapies.
EXAMPLESMaterial and Methods
Reagents:
Human recombinant IP10 was provided by Abcam and Peprotech
Recombinant CD26 was provided by Alexis or Sigma
Monoclonal (clone 33036) and Goat polyclonal anti-IP10 antibodies were obtained from R&D Systems.
Luminex reagents were provided by Biosource.
Luminex technology. The luminex technology allows us to follow up to 25 analytes in a single plasma sample. It is a bead based sandwich ELISA that utilizes cytometric principles and permits the simultaneous analysis of up to 100 analytes while using a small amount of patient plasma or cell supernatent.
Flow cytometry. Thawed PBMCs from patients were stained with CXCR3-PE and CD19-fitc (BD) for 20 min. Cells were washed, fixed with 1% PFA and read on FACSCALIBUR (BD).
IP-10 AA sequence
Pro-IP-10: mnqtailicc lifltlsgiq gvplsrtvrc tcisisnqpv nprsleklei ipasqfcpry eiiatmkkkg ekrclnpesk aiknllkays kemskrsp (SEQ ID NO:1).
Secreted full length protein: vplsrtvrc tcisisnqpv nprsleklei ipasqfcpry eiiatmkkkg ekrclnpesk aiknllkays kemskrsp (SEQ ID NO:2).
Cleavage product after CD26 treatment, as reported in literature:
In vitro cleavage of IP10:
100 ug of IP10 were incubated with 0.25U of recombinant CD26 in a 100 mM Tris-HCL pH 8 solution, for 2 h at 37° C., in 100 ul final volume. The cleavage was controlled by spotting 1 ul of the digested product in a H4 protein chip array and analyzed by SELDI-TOF (Ciphergen mass spectrometry technology).
Western blot:
Recombinant IP10 samples, treated or not with CD26 were run in a 15% bis-acryalmide gel. We used 48 cm plates and 1 mm spacer, and run the gel in tris-tricine buffer. Proteins were transferred to Hybond-P membrane (Amersham Biosciences) by a semi-dry western blot transfer procedure (EBU-4000 CBS-scientific Co). The membrane was saturated with TBS-1% tween-20-1% BSA solution. Revelation was done with the monoclonal antibody 33036 incubated O/N at 4° C. in the saturation buffer. 3 Washes step were performed with TBS −1% Tween at RT. Detection was done with anti-Mouse IgG (H+L) coupled to HRP (Vector laboratories). After 3 washes step, the ECL plus detection system (Amersham) was used for blot revelation.
We used SeeBlue Plus™ prestained standard (Invitrogen) as molecular weight markers.
Mass spectrometry conditions: 3 μl of 0.5 mg/ml monoclonal antibody (33036 clone) were added to the spots of a preactivated ProteinChip array (PS10; Ciphergen Biosystems). The PS10 array consists of a surface with carbonyldiimidazole moieties that dock proteins by covalently reacting with their amine groups. The arrays were incubated overnight at 4° C. in a humidified chamber. After blocking residual active sites (with 2×5 μl of ethanolamine 1M pH 8 for 15 min at RT). The arrays were assembled into a deep-well-type Bioprocessor assembly, then washed once, 10 min, with 200 μl PBS-0.5% triton-X100 and twice, 3 min, with PBS. The arrays were then incubated with 100 ul of PBS-0,1%Triton X-100 containing the recombinant IP10 protein at RT during for 1 h30 on a shaker, then washed 3-times with 150 μl of PBS. After rinsing with 1 mM HEPES, the arrays were removed from the Bioprocessor assembly and air-dried. The following energy-absorbing molecules (EAMs) was used: sinapinic acid saturated in 1:1 acetonitrile/0.5% aqueous TFA (SPA). Two 0.5-μl aliquots of EAM solution were applied to the spot surface, and the sample was allowed to dry.
The ProteinChip arrays were analyzed in the ProteinChip System Series 4000 (Ciphergen). The range of molecular weights (MWs) investigated was from 0 to 20,000. The focus mass was set at 8.5 kDa. The data were analyzed using CiphergenExpress Software.
Generation of rabbit polyclonal anti-LIP10 antibodies: Production of polyclonal anti-long form IP10 antibodies was generated by Proteogenix company (Alsace, France): the VPLSRTVRC (SEC ID NO:5) peptide (corresponding of the NH2 terminal of the full-length IP-10 protein of SEQ ID NO:2), corresponding to the NH2 peptidic sequence of Human IP-10, was synthesized and coupled to KLH. 5 New Zealand White rabbits were bled, and pre-immune serum was collected before the immunization. After immunization, the specific immunoglobulins production was tested bleeding rabbits; sera titrations were done by direct Elisa: the plate was coated with the same peptide as used for the immunization. After a 90 days immunization protocol, Rabbit IgG were purified: first by an affinity column containing the VPLSRTVRC (SEC ID NO:5) peptide, then a depletion step was achieved by a second affinity column containing the shorter peptide LSRTVRC (SEQ ID NO:6) (corresponding to the IP-10 shared peptidic sequence and the NH2 terminal of the full-length IP-10 protein of SEQ ID NO:2) to remove the non specific IgG.
Sandwich ELISA to quantify TOTAL AND L-IP10:
Maxisorp plate (Nunc) were coated with 2 ug/ml of Monoclonal antibody (33036) in PBS O/N at 4° C. The wells were saturated with 200 ul PBS-0.05% Tween-20—1% BSA1 to 2 h at RT. Samples were diluted ¼ or 1/10 in saturation buffer and incubated at RT for 2 h. A standard curve was obtained by diluting recombinant IP10, truncated or not by CD26 before use, in the same solution used for saturation. Each experiment was duplicated in the same plate. For secondary revelation we used the biotinylated goat polyclonal antibody (Total IP10, 0.25 ug/ml final concentration) and Rabbit polyclonal anti-VPL (long IP10, 2 ug/ml final concentration) (incubation 1H30 at RT) in the same plate.
For the last step revelation, we used HRP-streptavidin (BD Pharmingen) for the goat polyclonal and HRP-goat anti-rabbit IgG (H+L), mouse and human adsorbed (Southern Biotech) for the rabbit polyclonal. After last wash step, 100 ul of ‘TMB substrat (Sigma) were added to each well and 100 ul of HCl 1N to stop the reaction. Plates were read in a Labsystems Multiskan MS (Thermo) device.
For the ELISA our limit of detection is 50 pg/ml for total IP10 and 250 pg/ml for L-IP10.
Plasma Samples:
Healthy donors whole blood (EFS, Necker Paris France) was stimulated with Intron A, human recombinant interferon alpha 2 (Schering Plough): 1.5 ml whole blood was diluted with 1.5 ml of RPMI. Diluted blood was stimulated with: 3000 to 10000 U/ml of IFNa during 20 h to 24 h at 37° C. Plasma were collected and stored between −20° and −80° C. until analysis by Elisa.
HCV plasma from Paris: Blood samples were collected from 21 HCV chronic patients (initially defined by a persistence of HCV in the plasma and elevated ALT) who previously received IFNa therapy. 13 patients responded to therapy and controlled the infection (“Responder”) whereas 8 patients failed to respond to therapy (“non responder”).
For each cohort, plasma were isolated, frozen and analyzed by Luminex technology.
For the ELISA discriminating total and long form IP10 in HCV patients we concentrated 10× the plasma by a vivaspin-2 system with a 3000 Da MW cut off membrane. Samples were diluted twice for the ELISA analysis.
Surface CXCR3 expression: Blood samples were collected from 12 HCV chronic patients (initially defined by a persistence of HCV in the plasma and elevated ALT) who previously received IFNa therapy. 6 patients responded to therapy and controlled the infection (“SVR”) whereas 6 patients failed to respond to therapy (“Chronic”). PBMCs were isolated and frozen till further analysis. Thawed PBMCs were analyzed by flow cytometry to compare CXCR3 expression on different cell subsets. B cells were identified in PBMCs as CD19+ cells and CXCR3 expression was analyzed on their subset population.
sCD26 ELISA: human DPPIV/cd26 quantification in the plasma was performed with a Quantikine Immunoasay kit (R &D Systems). Samples were diluted 1/100 and tested in duplicate as manufactor instructions.
DPPIV activity in plasma sample: DPPIV activity was performed with DPPIV-Glo protease Assay (Promega). Briefly, in a white plate (Greiner bio-one), 50 ul of kit reagent (with luciferase substrate) were added to 50 μl plasma sample diluted in 10 mM Tris-HC1 pH8-0.1% prionex (Calbiochem). Samples were tested in duplicate at different dilutions. Plates were read in a Microluminat LB (EG&G Berthold) device.
Additional Patient Samples
For multi-analyte profiling, MMP2, MMP9 and DPP assays, banked patient samples were obtained from prior collections performed by S. Pol. during the treatment and management of chronic HCV patients receiving peg-IFN-α2/ribivirin therapy. All patients had genotype 1 infection. Plasma samples were obtained pre-, during and post-treatment. Samples were stored at -80 prior to use. For analysis of agonist and NH,-truncated forms of CXCL10, new collections were required so that blood samples could be collected in BD700 tubes (which contain a DPP4 inhibitor), thus preserving the circulating form of CXCL10. These samples were obtained as part of the study protocol RBM 03-59, approved by the INSERM clinical investigation department with ethical approval from the committee for the protection of persons (CPP) of Necker Hospital. HCV patients with chronic infection were defined by anti-HCV Abs and HCV RNA positivity for a period of time greater than six months. All patients had genotype 1 infection and were treated by a combination of peg-IFN-α2 and ribivirin for at least three month. Sustained virological responders (SVR) were defined as individuals absent of HCV RNA for longer than 6 months after termination of therapy. Protocols were reviewed and approved by local ethical committees and patients provided informed consent. The study protocols conformed to the ethical guidelines of the Declaration of Helsinki.
Multi-Analyte Profiling
Plasma were clarified by a high-speed centrifugation and analyzed using the Luminex xMAP technology. Samples were measured by a diagnostic lab, Rules Based Medicine for measurement of 83 molecules with all assays being CLIA certified (validated using guidelines set forth by the Clinical and Laboratory Standards Institute). The cut-off value was derived by determining the average of >200 normals and adding 3 standard deviations to the mean. The Least Detectable Dose (LDD) is the lower limit at which the system can accurately calculate the concentration of an experimental sample, having confidence that the concentration is higher than a blank sample. The LDD is determined by analyzing 20 blank samples, calculating the mean background, and adding 3 standard deviations to this mean. The Lower Assay Limit (LAL) is each assay's working sensitivity as defined by the lowest concentration calibrator found on the standard curve that provides a quantifiable measurement above background. The LAL is typically lower than the LDD. Analyte measurements reported below the LDD and above the LAL may be real values whose precision was examined closely. Values above the LDD possess excellent precision with coefficients of variation (CV) less than 10%. For data mining, values below the LAL were replaced with a value that is 50% of the lowest value measured in the data set. For all analytes used in this study, the LDD can be found in Table S3. In parallel, analytes not part of this multiplex screen (IFNα and CXCL10) were analyzed by Luminex, using commercially available kits (Invitrogen).
Generation of Agonist CXCL10 and NH2-Truncated CXCL10 Specific pAbs
Production of polyclonal agonist CXCL10 specific antibodies were generated by Proteogenix company (Alsace, France). The NH2-terminal 9 amino acids of the secreted form of CXCL10 (VPLSRTVRC) were synthesized and coupled to KLH. Eight New Zealand White rabbits were bled, and pre-immune serum was collected before the immunization. After immunization, anti-sera were tested by direct ELISA (plates were coated with the same peptide used for immunization). After a ninety days immunization protocol consisting of five injections, serum IgGs were subjected to an affinity column containing the VPLSRTVRC peptide, then a depletion column containing the short peptide (LSRTVRC, corresponding to NH2-terminal 7 amino acids of DPP4 cleaved CXCL10). Two of the eight rabbits tested had high affinity and specific reactivity for agonist CXCL10 (1-77). IgG purification was performed and further tested. For these studies, we utilized the pAbs isolated from rabbit L3 (and called antisera L3), which after immunodepletion showed no binding to CXCL10 (3-77) (up to concentrations of 2 μg/ml) and has a limit of detection of 125 pg/ml for CXCL10 (1-77) in a sandwich ELISA. Concentration of patient plasma provided an assay with an effective limit of detection of 50 pg/ml. Additionally, we demonstrated lack of cross-reactivity with other α-chemokines (data not shown). Production of monoclonal single chain antibodies specific for the neo-epitope created by DPP4 cleavage of CXCL10 were generated by AbD Serotec (Dusseldorf, Germany). Briefly, peptide corresponding to the NH2-terminal 7 amino acids of DPP4 cleaved CXCL10 (LSRTVRC) and peptide corresponding to the native protein (VPLSRTVRC) were conjugated to BSA and TRF. The HuCal library (Human Combinatorial Antibody Library) was screened first with the conjugated peptides coated in the ELISA plate, clones reactive for the neo-epitope and not the native peptide were then screened with the recombinant proteins (CXCL10 (3-77) and (1-77)). The clone 52.1 is specific of the neo-epitope and was used as the coating antibody for the sandwich ELISA specific of the antagonist form.
Assays for Discriminating Agonist CXCL10 and NH2-Truncated CXCL10
To generate CXCL10 (3-77), recombinant human CXCL10 (Peprotech, Rocky Hill, N.Y. USA) was incubated with recombinant DPP4 (Sigma) in a 100 mM Tris-HCL pH 8 solution, for 2 h at 37° C. SELDI-TOF Mass spectrometry: 1 μl of the digested product was spotted onto an H4 protein chip. Arrays were incubated and washed as per manufacturers instructions and analyzed using the ProteinChip System Series 4000 (Ciphergen). The range of molecular weights (MWs) investigated was from 0 to 20,000 Da. The focus mass was set at 8.5 kDa. Data were analyzed using CiphergenExpress Software. Chemotaxis assays: Peripheral blood mononuclear cells were purified from healthy volunteers and treated with 1 mg/ml phytohemagglutinin for 3 days then 40 U/ml of recombinant hIL-2 (R&D Systems) was added for and additional 2 days prior to use of the cells. Lymphocyte chemotaxis was performed using 5 μm pore size Multiscreen—MIC plates (Milliopore). To monitor CXCL10 (1-77) or CXCL10 (3-77) directed migration, lymphocytes were added to the top chamber and chemokine was added to the bottom. Lymphocytes were allowed to migrate for 2 hours at 37° C. and cells in the bottom chamber were counted using a Guava cytometer (Guava Technologies, CA, USA). Calcium-flux assays: Increased intracellular [Ca2+] in response to CXCR3 signaling were monitored by FACS. Briefly, CHO-K1 cells were transfected with a plasmid encoding the full-length open reading frame of CXCR3 (generously provided by Dr. C P Tensen, Leiden, The Netherlands). Stably transfected cells were screened for CXCR3 expression (anti-human CXCR3 clone 106, Becton Dickinson) and loaded with Fura-3-AM. Ca++ release was measured using a Canto II Cytometer. Baseline signal was collect prior to addition of chemokine. For competitive antagonist assessment, cells were first incubated for 5 min with CXCL10 (3-77) then exposed to CXCL10 (1-77). Sandwich ELISA. Maxisorp plates (Nunc) were coated with 2 □g/ml of the capture antibody—mAb anti-CXCL10 (clone 33036) in PBS overnight at 4° C. The wells were saturated with 200 μl 0.05% Tween-20, 1% BSA in PBS for 1-2 h at room temperature. Samples were diluted in saturation buffer and incubated at room temperature for 2 h. A standard curve was obtained by diluting recombinant CXCL10 (1-77) or CXCL10 (3-77). Each experiment was run in duplicate with two different detection antibodies—a commercially available goat pAb that recognized both forms of CXCL10 or a rabbit pAb (antisera L3) that is specific for the agonist form of CXCL10. For detection of the two amino acid NH2-truncated form of CXCL10, we utilized the scAb (clone 52.1) as the capture reagent and goat pAb anti-CXCL10 for detection. Plates were read in a Labsystems Multiskan MS (Thermo) device.
Concentration of Patient Plasma
The ProteoMiner kit (Bio-Rad) was used as per manufacture's instructions. Briefly, the kit contains 100 μ of ProteoMiner beads in minispin columns. Human serum samples (1 ml) were applied to the columns. To ensure effective binding, columns were rotated for 2 hr at room temperature prior to washing away unbound proteins with PBS. To elute bound proteins, the columns were treated 2 times with 100 μl of an acidic urea/CHAPS buffer. Collected fractions were neutralized with 15 μl of 2M sodium carbonate and desalted used a Zeba spin column (Pierce).
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Claims
1. A method of treating infection, a disease, or a condition, comprising administering to a patient in need thereof, an effective amount of a CD26 inhibitor, thereby restoring the chemokine gradient and treating the infection.
2. The method of claim 1, wherein the patient is infected with hepatitis C virus.
3. The method of claim 1, wherein the CD26 inhibitor is at least one selected from the group consisting of amino boronic dipeptide talabostat (PT-100), Ile-Pro-Ile (Diprotin A), alanyl pyrrolidine, isoleucyl thiazolidine, N-valyl prolyl, O-benzoyl hydroxylamine and sitagliptin phosphate (JANUVIATM)
4. The method of claim 1, wherein the patient is chronically infected with hepatitis C virus.
5. The method of claim 1, which further comprises administering interferon.
6. The method of claim 5, where the interferon is interferon α2A.
7. The method of claim 5, where the interferon is interferon α2B.
8. The method of claim 5, where interferon α2A is administered in combination with ribavirin.
9. The method of claim 5, where interferon α2B is administered in combination with a viral protease inhibitor.
Type: Application
Filed: Jan 20, 2012
Publication Date: Jul 5, 2012
Applicant: INSTITUT PASTEUR (PARIS CEDEX)
Inventors: Matthew Albert (Paris), Armanda Casrouge (Draveil), Jeremie Decalf (Paris), Stanislas Pol (Juvisy-sur-Orge), Arnaud Fontanet (Paris), Mostafa Mohamed (Heliopolis Cairo)
Application Number: 13/354,458
International Classification: A61K 38/21 (20060101); A61K 31/135 (20060101); A61K 31/426 (20060101); A61P 31/00 (20060101); A61P 31/14 (20060101); A61K 31/40 (20060101); A61K 31/4985 (20060101); A61K 38/55 (20060101);
