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  Pharmaceutical Patents  

 

Title:  Diagnosis and monitoring of systemic lupus erythematosus and of scleroderma
United States Patent: 
7,390,631
Issued: 
June 24, 2008

Inventors:
 Ahearn; Joseph M. (Sewickley, PA), Manzi; Susan M. (Wexford, PA)
Assignee:
  University of Pittsburgh - Of the Commonwealth System of Higher Education (Pittsburgh, PA)
Appl. No.:
 10/489,219
Filed:
 September 9, 2002
PCT Filed:
 September 09, 2002
PCT No.:
 PCT/US02/28910
371(c)(1),(2),(4) Date:
 March 10, 2004
PCT Pub. No.:
 WO03/022223
PCT Pub. Date:
 March 20, 2003


 

Woodbury College's Master of Science in Law


Abstract

Methods for diagnosing and monitoring systemic lupus erythematosus (SLE) or scleroderma by determining, in a blood sample from the individual being diagnosed or monitored, complement component C4d deposited on surfaces of red blood cells in the sample, and optionally also determining complement receptor CR1 deposited on the red blood cell surfaces. For diagnosis this is compared with the quantity of C4d (and optionally CR1) present on red blood cells of normal individuals. For monitoring it is compared with a value in a sample or samples previously obtained from the individual patient. The comparison may be made with individual values for C4d and CR1 and/or with a ratio of the two found in normal individuals.

Description of the Invention

BRIEF SUMMARY OF THE INVENTION

The invention involves the use of determinations of complement component C4d, and of complement receptor CR1, a receptor present on the surfaces of erythrocytes that acts as a receptor for proteolytic fragments of C4 and C3.

In one aspect, this invention comprises a method of diagnosing systemic lupus erythematosus in an individual, comprising (a) determining, in a blood sample from the individual containing red blood cells, complement component C4d deposited on surfaces of red blood cells in the sample, and (b) comparing said determination with the quantity of component C4d deposited on the surface of red blood cells of individuals not having systemic lupus erythematosus.

In a second aspect, this invention comprises a method of monitoring systemic lupus erythematosus in an individual, comprising (a) determining, in a blood sample from the individual containing red blood cells, complement component C4d deposited on surfaces of red blood cells in the sample, and (b) comparing said determination with the quantity of component C4d deposited on the surface of red blood cells previously obtained from the individual.

In another aspect, this invention comprises a method of diagnosing scleroderma in an individual comprising (a) determining, in a blood sample containing red blood cells from the individual, complement component C4d deposited on surfaces of red blood cells in the sample, and (b) comparing the determination of said component with the quantity of said component known to be present on the surface of red blood cells of individuals not having scleroderma.

The invention further comprises a method of monitoring scleroderma in an individual, comprising (a) determining, in a blood sample from the individual containing red blood cells, complement component C4d deposited on surfaces of red blood cells in the sample, and (b) comparing said determination with the quantity of component C4d deposited on the surfaces of red blood cells previously obtained from the individual.

In preferred aspects of this invention, the method also includes determining complement receptor CR1 on the surfaces of red blood cells in the sample and comparing that determination with the quantity of CR1 present on the surfaces of red blood cells of patients not having SLE or scleroderma, respectively.

In other preferred aspects of this invention, the ratio of C4d to CR1 on surfaces of red blood cells is determined and compared with that of patients not having SLE or scleroderma, respectively, or if monitoring of a patient is being conducted, that ratio is compared with such a ratio previously determined for the patient (or for cells previously obtained from the patient).

In another preferred aspect of this invention, the method also includes determining complement receptor CR1 on the surfaces of red blood cells in the sample, and comparing said determinations with the quantity of CR1 deposited on the surfaces of red blood cells previously obtained from individuals with systemic lupus erythematosus and scleroderma respectively as a method of monitoring disease activity.

The invention also comprises automated methods of the above types, computer software for performing such automated methods, and kits for performing the methods described herein.

DETAILED DESCRIPTION OF THE INVENTION

General Discussion

The methods of this invention enable the diagnosis and/or monitoring of SLE and scleroderma. Because these two conditions are serious health problems, there is a need for relatively accurate and early diagnosis of these conditions. Likewise, the ability to monitor the activity of these diseases is of great importance.

The invention involves the use of determinations of complement component C4d and/or complement receptor CR1, a receptor present on the surfaces of erythrocytes that acts as a receptor for proteolytic fragments of C3 and C4.

In the most general sense, the methods of this invention are based on the discovery by the inventors that a determination of C4d deposited on surfaces of red blood cells of a patient can serve as a diagnostic marker for either SLE or scleroderma. As will be discussed below, a combination of this determination with a determination of CR1 expressed on surfaces of red blood cells of the same patient can aid in distinguishing between SLE and scleroderma as well as helping doctors distinguish between the two diseases and other diseases having similar manifestations.

In diagnosing the occurrence, or previous occurrence, of either disease, complement component C4d deposited on surfaces of red blood cells in a sample is determined. Complement receptor CR1 on the surfaces of the same red blood cells is preferably also determined. One or both of these determinations is then compared with the quantities of C4d and CR1, respectively, usually found on the surfaces of red blood cells of individuals not having SLE or scleroderma.

In monitoring disease activity of a patient with either disorder, the same determinations are made in the patient's blood sample, and are then compared with determinations of the quantities of C4d and CR1 present on surfaces of red blood cells in a sample obtained from the same patient in the past.

In both instances, when speaking of "determination" and "quantity," we mean to include both an absolute amount or quantity of material, as well as (in addition, or alternatively), a ratio of C4d to CR1. As will be discussed below, either or both of these measurements can be used, particularly in diagnosing either SLE or scleroderma in a patient.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

General Procedures

The invention involves conducting assays on blood samples obtained from patients to determine C4d and preferably also CR1.

Samples of blood are obtained from the patient and are treated with EDTA (ethylenediaminetetraacetate) to inhibit complement activation. The samples are maintained at room temperature or under cold conditions. Assays are run preferably within 48 hours.

The determination of C4d and CR1 may be done by a number of methods including flow cytometry, ELISA using red blood cell lysates, and radioimmunoassay. In one embodiment of this invention, the determination of the level of C4d and CR1 is made using flow cytometric methods, with measurements taken by direct or indirect immunofluorescence using polyclonal or monoclonal antibodies specific for each of the two molecules. Each of these two molecules can be measured with a separate sample or using a single sample. The mean fluorescence channel (MFC) for erythrocyte CR1 and C4d is determined individually. The same type of assay may be used for diagnosis or for monitoring disease activity in patients known to have SLE or scleroderma.

Development of an assay of this type for CR1 and for C4d is known in the art and described in Freysdottir, et al., J. Immunol. Meth. vol. 135, 2005 (1991). That assay was a flow cytometric assay for CR1 and for protein fragments C4d and C3d on erythrocytes, and was described as enabling the identification of individuals having comparatively high or comparatively low levels of CR1. However, erythrocyte C4d and C3d were low and often not detectable above background (limits of detection). These authors suggest a possible use of their assay in providing general information regarding immune complex load or clearing in patients. However, their work was limited to developing the assay for general use. They did not investigate the use or application of their work for diagnosing or monitoring the activity of particular diseases.

Kits

Kits for conducting the assays for both the diagnosing of disease and monitoring of disease activity are a part of this invention. Said kits will use any of the various reagents needed to perform the methods described herein. For example using the immunofluorescence assays, the kits will generally comprise a conjugate of a monoclonal antibody specific for complement component C4d with a fluorescent moiety, and preferably also a conjugate of a monoclonal antibody specific for complement receptor CR1 with a different fluorescent moiety. Additionally, the kits will comprise such other material as may be needed in carrying out assays of this type, for example, buffers, radiolabelled antibodies, colorimeter reagents etc.

The antibodies for use in these methods and kits are known. Hybridomas secreting Anti-CR1 antibodies are available from the American Type Culture Collection in Maryland (ATCC #HB 8592). A general reference is U.S. Pat. No. 4,672,044. Scripps Clinic and Research Foundation, La Jolla, Calif. Anti-C4d antibodies are available from Quidel Corp. in San Diego, Calif. (#A213) and are generally described in Rogers, J., N. Cooper, et al. Complement activation by beta-amyloid in Alzheimer disease. PNAS 89:10016-10020, 1992; Schwab, C. et al. Neurofibrillary tangles of Guam Parkinson-dementia are associated with reactive microglia and complement proteins. Brain Res 707(2):196 1996; Gemmell, C. A flow cytometric immunoassay to quantify adsorption of complement activation products on artificial surfaces. J Biomed Mater Res 37:474-480, 1997; and, Stoltzner, S. E., et al. Temporal accrual of complement proteins in amyloid plaques in patients with Down's syndrome with Alzheimer's disease. Am J Path 156:489-499, 2000.

The determination of the C4d and CR1 values may alternatively be conducted using a number of standard measurement techniques such as ELISA. Instead of fluorescent labels, there may be used labels of other types, such as radioactive and colorimetric labels. If such other types of assays are to be used, the kits will comprise monoclonal antibodies specific for C4d and CR1 conjugated with appropriate labels such as radioactive iodine, avidin, biotin or enzymes such as peroxidase.

Diagnostic Methods

Diagnosis of a patient with SLE or scleroderma is carried out by comparing the determination of C4d and preferably also of CR1 with a base value or range of values for the quantities of these entities typically present on the surfaces of red blood cells in normal individuals. In normal individuals, C4d is present in relatively low levels on surfaces of red blood cells. When using flow cytometric measurement with indirect immunofluorescence, the MFC of C4d on red blood cells in healthy individuals ranged from 1.06 to 16.12 (mean 5.7).  The MFC of erythrocyte C4d in patients having SLE was higher than that of healthy individuals and ranged from 2.66 to 155.03 (mean 23.9). (Table III and Table VII, see Original Patent). Patients with scleroderma also had elevated levels of C4d as compared to healthy individuals. In patients with scleroderma, the MFC of erythrocyte C4d ranged from 2.86 to 28.89 (mean 11.6). (Table VI and Table V, see Original Patent).

Conversely, as is generally known in the art, the level of CR1 on surfaces of erythrocytes of individuals having SLE was usually lower than in healthy individuals. In the latter, the value of the MFC for erythrocyte CR1 ranged from 10.53 to 50.83 (mean 25.4) (Table II and Table VII, see Original Patent), whereas the MFC for erythrocyte CR1 from patients having SLE ranged from 1.41 to 40.89 (mean 12.4). (Table III and Table VII, see Original Patent). Patients with scleroderma had MFC values for erythrocyte CR1 (range 4.69 to 38.26, mean 18.4) (Table VI and Table VII, see Original Patent) that were lower than healthy individuals but higher than those with SLE.

A further indication of a diagnosis of SLE is based on the ratio of erythrocyte CR1 to erythrocyte C4d in these assays. More than 93% of patients with SLE had a CR1:C4d ratio less than 3.00, whereas more than 77% of healthy individuals had a ratio of greater than 3.00. Thus, a ratio of CR1:C4d less than 3.00 in an individual is an indication of SLE.

A further method to distinguish between a diagnosis of SLE and a diagnosis of scleroderma is to compare the erythrocyte CR1:C4d ratios. Greater than 47% of patients with SLE had a CR1:C4d ratio less than 0.69, whereas only one of 30 patients with scleroderma had a CR1:C4d ratio less than 0.69. Thus, a ratio of CR1:C4d less than 0.69 distinguishes SLE from scleroderma.

Monitoring of Patients

A particular feature of the methods of this invention is the ability to monitor the activity of a patient's disease. The life span of a red blood cell is approximately 120 days. Therefore, a particular feature of this assay or method is to indicate or reflect SLE or scleroderma activity that has occurred in the patient during the preceding several weeks or even several months. It is possible, using this procedure, to identify the occurrence of a flare-up of SLE or scleroderma during the previous few weeks or possibly even the previous several months due to persistence of C4d deposited on the surface of red blood cells. The timing of a previous occurrence may be approximated by separating from the sample the C4d-bearing erythrocytes and ascertaining their age by conventional techniques such as density gradient centrifugation (Rennie, C. M., S. Thompson, et al. (1979). Human erythrocyte fractionation in Percoll density gradients. Clinica Chimica Acta 98: 119-125).

Also of importance is that the method of this invention is capable of detecting evidence of complement activation in scleroderma, which is a relatively non-inflammatory disease. At the present time, there are no useful circulating markers, or, for that matter, markers of any kind, for measuring disease activity of scleroderma.

Automation and Computer Software

The determinations of C4d and CR1 and the diagnostic and disease activity monitoring methods described above can be carried out manually, but often are conveniently carried out using an automated system and/or equipment, in which the blood sample is analyzed automatically to make the necessary determination or determinations, and the comparison with the base or reference value is carried out automatically, using computer software appropriate to that purpose.

Thus, in one aspect, the invention comprises a method for diagnosing or monitoring systemic lupus erythematosus in an individual comprising (a) automatically determining, in a blood sample from the individual containing red blood cells, complement component C4d and complement receptor CR1 deposited on surfaces of red blood cells in the sample, and (b) automatically comparing said determinations with reference values for component C4d and receptor CR1, respectively, on surfaces of red blood cells. In another aspect this automated method includes one in which the reference values comprise a ratio of C4d:CR1.

The invention also comprises a method for diagnosing or monitoring scleroderma in an individual comprising (a) automatically determining, in a blood sample from the individual containing red blood cells, complement component C4d and complement receptor CR1 deposited on surfaces of red blood cells in the sample, and (b) automatically comparing said determinations with reference values for component C4d and receptor CR1, respectively, on surfaces of red blood cells. In another aspect this automated method also includes one in which the reference values comprise a ratio of C4d:CR1.

Another aspect of the invention comprises a method for diagnosing or monitoring systemic lupus erythematosus in an individual comprising (a) automatically determining, in a blood sample from the individual containing red blood cells, complement component C4d deposited on surfaces of the blood cells in the sample, and (b) automatically comparing said determination with a reference value for component C4d deposited on surfaces of red blood cells.

A further aspect of the invention comprises a method for diagnosing or monitoring scleroderma in an individual comprising (a) automatically determining, in a blood sample from the individual containing red blood cells, complement component C4d deposited on surfaces of red blood cells in the sample, and (b) automatically comparing said determination with a reference value for component C4d deposited on surfaces of red blood cells.

Computer software, or computer-readable media for use in the methods of this invention include: (1): a computer readable medium, comprising: (a) code for receiving data corresponding to a determination of complement component C4d deposited on surfaces of red blood cells; (b) code for retrieving a reference value for complement component C4d deposited on surfaces of red blood cells of individuals; and (c) code for comparing the data in (a) with the reference value of (b); and (2): a computer readable medium as just described, further comprising: (d) code for receiving data corresponding to a determination of complement receptor CR1 deposited on surfaces of red blood cells; (e) code for retrieving a range of reference values for complement receptor CR1 deposited on surfaces of red blood cells of individuals; and f) code for comparing the data in (d) with the reference values of (e).

In embodiments of the invention, one or more reference values may be stored in a memory associated with a digital computer. After data corresponding to a determination of complement C4d is obtained (e.g., from an appropriate analytical instrument), the digital computer may compare the C4d data with one or more appropriate reference values. After this comparison takes place, the digital computer can automatically determine if the data corresponding to the determination of complement C4d is associated with SLE.

Accordingly, some embodiments of the invention may be embodied by computer code that is executed by a digital computer. The digital computer may be a micro, mini or large frame computer using any standard or specialized operating system such as a Windows.TM. based operating system. The code may be stored on any suitable computer readable media. Examples of computer readable media include magnetic, electronic, or optical disks, tapes, sticks, chips, etc. The code may also be written by those of ordinary skill in the art and in any suitable computer programming language including, C, C++, etc.
 

Claim 1 of 21 Claims

1. A method for diagnosing systemic lupus erythematosus in an individual, comprising: (a) detecting, in a blood sample from the individual containing red blood cells, quantities of complement component C4d and complement receptor CR1 both of which are on surfaces of red blood cells in the sample, (b) comparing said quantities with, respectively, the quantities of component C4d and receptor CR1 both on surfaces of red blood cells in samples from individuals not having systemic lupus erythematosus, and (c) diagnosing systemic lupus erythematosus in the individual by observing a significantly increased quantity in complement component C4d on surfaces of red blood cells and a significantly decreased quantity of receptor CR1 on surfaces of red blood cells in said sample from the individual compared to said samples from said individuals not having systemic lupus erythematosus.

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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