Method for Removal of Human Antibodies to Native DNA From Serum

In one preferred mode, the monoclonal, anti-idiotypic antibody is coupled to sepharose to produce a separation medium which could be disposed in a column. Serum samples are applied to the column and the anti-nDNA antibody in the serum binds to the antiidiotypic antibody coupled to the sepharose in the column. The filtrate, which is depleted of anti-nDNA antibodies, is removed from the column. The serum sample can also be depleted of circulating immune complexes of anti-nDNA antibody and DNA if one uses the antibody of the present invention which is capable of reacting with a shared idiotypic determinant on human anti-nDNA antibody which is located outside the DNA binding region. Finally, the anti-nDNA antibody may be eluted from the column to permit reuse of the column.

Ten mL of serum from an SLE patient (A.W.) containing antiDNA antibodies was mixed with 10 mL of 0.01M Na phosphate buffer, pH 6.6, containing calf thymus DNA (Type 1, Sigma Chemical Co., St. Louis, Mo.) at a concentration of 1.5 mg/mL. This mixture was incubated for 1 hour at room temperature followed by 16 hours at 4.degree. C. to allow DNA/anti-DNA antibody complexes to form and was then applied to a 300 mL DE-52 column (Whatman Ltd., Clifton, N.J.) equilibrated with 0.01M Na phosphate (pH 6.6) containing 0.1M NaC1. The complexes were eluted with 0.5M NaC1, dissociated with 8M urea and rechromatographed on a DE-52 column to yield an enriched preparation of human anti-DNA antibodies of the IgG class which were free of native DNA.

Five BALB/c mice were immunized by intraperitoneal in jection with 100 .mu.g of an immunogen which is the enriched anti-DNA antibody emulsified in complete Freund's adjuvant. The mice were boosted weekly for 6 to 10 weeks with the immunogen without adjuvant. Three of the five mice were shown, using a solid phase radioimmunoassay (see below), to have produced high levels of antibodies against the immunogen. Spleen cells from these three immunized mice were fused to thioguanine-resistant non immunoglobulin secreting, mouse myeloma cells (line X63Ag8.653) and plated in 96 well microtiter plates. Supernatant fluids from those cultures containing viable hybridomas were screened to detect clones producing antibodies reactive with purified immunogen using a solid phase radioimmunoassay. Samples of each culture supernatant fluid were incubated in wells of Immulon-II (Dynatech Labs., Inc., Alexandria, Va.) microtiter plates which had been pre-coated by overnight incubation with either purified anti-DNA antibodies from an SLE patient or with purified normal human immunoglobulin. After extensive washing of the coated plates, bound mouse immunoglobulin was detected by addition of a Sulfur-35-methionine labeled monoclonal rat anti-mouse kappa light chain antibody. Differential binding of radiolabel to the anti-DNA plate relative to the normal human immunoglobulin plate was taken as evidence for the presence of a putative anti-idiotypic antibody specific for anti-DNA in the culture supernatant fluid of a given hybridoma. Five of such hybridomas were identified as shown in Table I.

Either enriched AW anti-DNA antibodies or control serum was absorbed to polystyrene wells. Mouse serum or hybridoma supernatant was added, followed by radiolabeled rat anti-mouse light chain. Duplicate values are shown.

Cells from these hybridomas were further cloned in agarose on a feeder layer of rat embryo fibroblasts. Clones were picked, grown to mass culture and retested for anti-idiotypic activity. Pristane-primed BALB/c mice were injected intraperitoneally with 1 .times.10.sup.7 hybridoma cells, and the ascites fluid was harvested 10-14 days later.

The monoclonal antibody hereinafter designated 3I which was produced by one of these clones was further characterized as follows. Ouchterlony analysis indicated 3I to be a moust IgG.sub. 1 immunoglobulin with kappa light chains. Activity of 3I against antiDNA antibodies was not inhibited by a fifty-fold excess of normal human serum. Furthermore, its binding to anti-DNA antibody was not blocked by the addition of a large excess of free DNA. 3I did not contain anti-DNA acitivity as measured in a conventional Millipore filter assay. (Iodine-125 labeled DNA is reacted with a dilution of serum suspected of containing anti-DNA antibody. Immune complex is trapped on a 0.45um filter which is then counted for radioactivity.) Furthermore, 3I antibody when combined with a rabbit anti-mouse antibody was capable of precipitating radiolabeled DNA only if the DNA had been preincubated with anti-DNA antibodies from patient sera. Finally, 3I was found to bind to the Fab region of anti-DNA antibodies.

From these data, it was concluded that the 3I antibody was anti-idiotypic and was specific for an idiotype present on a species of anti-DNA antibodies from patient AW. Furthermore, the 3I antibody is not directed against the antigen combing site of anti-DNA antibody because binding to anti-DNA is not blocked by the addition of excess DNA, and because it binds to DNA/anti-DNA antibody complexes.

Sera obtained from active SLE patients and normal healthy donors were tested to determine their reactivity with 3I using a solid phase radioimmunoassay. In this assay, the sera, diluted 1/10 in phosphate buffered saline (PBS), were incubated overnight at 4.degree. C. in the wells of an Immulon-II microtiter plate. Plates were washed three times with PBS and then incubated for 1 hours with PBS containing 5% (w/v) bovine serum albumin (BSA). Culture supernatant fluid or a 1/50 dilution of ascites containing 3I was added to each well. After 90 minutes incubation at room temperature, the plates were washed with a washing solution containing 0.5% Tween 20, 0.15M NaC1 and 2% BSA (pH 8.3). Sulfur-35-methionine-labeled monoclonal rat anti-mouse kappa light chain antibody (clone 187.1, ATCC-HB 58) was added and the plates further incubated at room temperature for 90 minutes. After extensive washing with the washing solution, the extent of binding was determined in each well by scintillation counting.

Eight of nine active SLE patients previously shown to have high levels of anti-DNA antibodies showed significant reactivity with 3I. Furthermore, four of nine active patients without significant reactivity in the Millipore filter assay for anti-DNA antibodies also reacted with 3I. Many of these sera from patients with SLE reacted with 3I more strongly than did serum from patient AW, from which the anti-DNA antibody was purified. In contrast, none of the ten normal donors showed significant 3I-reactivity in their sera.

Serum samples were obtained from thirteen patients during active disease and during clinical remission. Remission sera from all patients showed a loss of anti-nDNA antibody in the Millipore filter assay. Six of these remission sera showed a concomitant loss of 3I reactivity in the solid phase radioimmunoassay. However, the remaining seven remission sera showed little or no change in 3I reactivity. The continued reactivity of these seven sera with 3I suggests that anti-nDNA antibodies were present, but were prevented from reacting with DNA in the Millipore filter assay. Therefore an assay for detecting anti-nDNA in serum samples using 3I as a reagent has greater diagnostic and prognostic utility than anti-nDNA assays of the prior art. The hybridoma secreting 3I has been deposited in accordance with the provisions of the Budapest Treaty at the American Type Culture Collection, Rockville, Md., where it is catalogued as ATCC HB8376.

The antibody of the present invention can be used to remove anti-nDNA antibody from biological fluids, including the serum of SLE patients, by contacting the serum to a medium containing the monoclonal, anti-idiotypic antibody of this invention. Specifically, cyanogen bromide was used to activate normally inert sepharose to permit the coupling of the antibody 3I to the sepharose. The sepharose was then placed in a suitable container to form a column. Serum samples from patients with SLE were then applied to this column so that the anti-nDNA in the serum binds to the 3I on the sepharose. The column was then washed with phosphate buffered saline to remove the serum from the column, this filtrate being depleted of anti-nDNA antibodies. The anti-nDNA antibody was then eluted from the column using 8M urea to dissociate the complex formed by the 3I and anti-nDNA antibodies, leaving the 3I in the column which may now be used again. This fraction containing the eluted anti-nDNA antibodies is then dialyzed against 6M urea, 4M urea, 2M urea and finally against phosphate buffered saline, leaving the fraction containing the eluted anti-nDNA antibody free of urea.

The filtrate (serum passed through the column) was then shown to be depleted of 3I reactive anti-nDNA antibodies by the radioimmunoassay technique described above for the detection of anti-nDNA antibodies in a sample. The fraction containing the eluted anti-nDNA antibody was also shown to be enriched with 3I reactive anti-nDNA antibodies by the same radioimmunoassay technique. In addition, the eluted fraction was shown to have increased anti-DNA activity and the filtrate was shown to have decreased anti-nDNA activity by both a crithidia assay and by an enzyme linked immunosorbent assay (ELISA). Table II shows the optical density of the serum, the eluted fraction and the filtrate, each of which was contacted with free DNA. The optical density was measured at 405 nm, so that the reading corresponds to the amount of anti-nDNA antibody in the sample. As seen in Table II, for each of the patients the amount of anti-nDNA antibody is the greatest in the eluted fraction which contains the 3I reactive anti-nDNA antibodies and the lowest in the filtrate which is depleted of the 3I reactive anti-nDNA antibodies. As such, the serum has been successfully depleted of anti-nDNA antibodies by passage through the column.

A column formed with the antibodies of the present invention can specifically remove only the anti-nDNA antibodies from the serum of an SLE patient and may therefore eliminate the generalized immunosuppression caused by current SLE therapies involving removal of all antibodies. In addition, the column can deplete the serum of circulating immunocomplexes of anti-nDNA antibody and DNA if one uses the antibody of the present invention which is capable of reacting with a shared idiotypic determinant on human anti-nDNA antibody which is located outside the DNA binding region. This is especially important as it is known that these immune complexes are a major pathogenic component of disease in SLE patients. A column formed with the antibodies of the present invention removes from the serum of SLE patients anti-nDNA present in immune complexes as well as monomeric anti-nDNA and as such is a desirable therapeutic modality for SLE patients.

As will be readily apparent to those skilled in the art, the invention may be used in other specific forms or for other purposes without departing from its spirit or central characteristics. The present embodiments are therefore to be considered as illustrative and not restrictive, the scope of the invention being indicated by the claims rather than by the foregoing description, and all embodiments which come within the range of equivalence of the claims are intended to be embraced.