WO1998039658A1 - Methods for diagnosing and treating autoimmune disease - Google Patents

Abstract

Methods and compositions for diagnosing autoimmune diseases are disclosed. The methods include determining the presence in a biological sample of the individual of an antibody specific for a phosphoprotein, an active protein kinase, or a protein complex containing a phosphoprotein or an active protein kinase which is present in a human apoptotic cell extract but which is not present in a human non-apoptotic cell extract. Also disclosed are methods of treating or preventing autoimmune diseases in an individual diagnosed in accordance with the aforementioned method.

Description

METHODS FOR DIAGNOSING AND TREATING AUTOIMMUNE DISEASE

Background of the Invention A common feature of autoimmune diseases such as systemic lupus erythematosus (SLE), systemic sclerosis (SS), Sjδgren's disease (SD), rheumatoid arthritis (RA), and mixed connective tissue disease (MCTD) is the breakdown of tolerance to self antigens. A consequence of this immune dysfunction is the production of antibodies reactive with multiple self proteins (von Muhlen et al. (1995) Sem. in. Arthritis Rheum. 24: 323-258.). Remarkably, the self proteins recognized by these antibodies are culled from a relatively small subset of total cellular proteins. Protein targets for autoantibody production can be grouped into distinct classes sharing structural and/or functional properties. One such class is the ribonucleoprotein (RNP) particles involved in the regulation of RNA metabolism. Autoantigens belonging to this class include heterogeneous nuclear RNPs (hnRNPs), small nuclear RNPs (snRNPs), the Th/To RNP complex, and the Ro complex (Astaldi-Ricotti et al. (1989) J.Cell.Biochem. 40: 43-47; Gold et al. (1989) Science 245:1377-1380; Montecucco et al. (1990) Arthritis Rheum. 33:180-186; Van Veenrooij et al. (1989) Clin. Exp. Rheum. 7: 635- 639). The U-snRNPs are a group of related nuclear particles containing a unique, uridine-rich, structural RNA (termed the U-snRNA) and a core of six or more polypeptides, Craft, J. Rheum Dis Clin North Amer 18:311-335. The most abundant of these, the U1-, U2-, U5- and U4/U6- snRNP complexes are known autoantigens (Craft et al. (1988) J. Clin. Invest. 81:1716-1724; Craft et al. (1992) J., Rheum. Dis. Clin. North Amer. 18:311-335; Lerner et al. (1979) Proc. Natl. Acad. Sci. USA 76:5495-5499; Okano et al. (1991) Journal of Immunology 146:535-542; Okano et al. (1991) Arthritis and Rheumatism 34:S103), and play critical roles in the splicing of pre-mRNA molecules. During splicing, U-snRNPs assemble into a macromolecular structure termed a spliceosome whose function is to efficiently and precisely process introns from pre-mRNA prior to export of the mature mRNA from the nucleus. The fidelity of this complex process is facilitated by other splicing factors which transiently associate with the U-snRNP complexes, particularly the UI- and U2 -snRNPs (Horowitz et al. (1994) Trends in Genetics 100:100-106; Hodges et al. (1994) Current Biology 4:264-267). Splicing factors belonging to the SR family are highly conserved proteins containing one or more RNA recognition motifs (RRMs) at their amino termini and a serine/arginine (SR) repeat of varying length in their carboxyl termini (Screaton et al. (1995) EMBO Journal 14:4336-4349). Structural analysis of the SR protein ASF/SF2 demonstrates that the SR domains are required for protein phosphorylation and constitutive RNA splicing but are dispensable for alternative splicing. Targeted disruption of the RRM domains blocks RNA binding and constitutive splicing activity (Caceres et al. (1993) E 5OJ L2.4715-4726; Zuo et al. (1993) EMBOJ 12:4727-4737). At least eight SR domain containing proteins have been identified in humans, including the Ul-70 kD protein, SRp75, SRp54, SRp40, ASF/SF2, SC35, U2AF35, and SRp20. It has been postulated, but never shown, that SR proteins enhance splicing by binding to the Ul-snRNP during the formation of a commitment complex, thus stabilizing the spliceosome assembly (Fu et al. (1994) Nature 365:82-85; Kohtz et al. (1994) Nature 368:119-124; Wu et al. (1993) Cell 75:1061-1070). Individual SR proteins can substitute for the Ul-snRNP in in vitro splicing assays (Crispino et al. (1994) Science 265:1866-1869), and SR proteins have been implicated in regulation of both constitutive and alternative splicing of several mRNAs (Caceres et al. (1994) Science 265:1706; Ge et al. (1991) Cell 66:373; Krainer et al. (1991) Cell 66:383). Reversible protein phosphorylation is also thought to regulate both constitutive and alternative mRNA splicing. Experiments utilizing phosphatase inhibitors, nonhydrolyzable ATP analogues, or purified phosphatases in in vitro splicing reactions demonstrates a requirement for reversible protein phosphorylation for mRNA splicing (Mermoud et al. (1992) Nucleic Acids Res. 20:5263; Mermoud et al. (1994) EMBOJ. H:5679; Tazi et al. (1992) J. Biol. Chem. 267:4322), and several kinases capable of phosphorylating SR proteins have been identified. The Ul-70 kD snRNP protein is an in vivo and in vitro substrate for an unidentified serine kinase that copurifies with the Ul-snRNP complex (Woppmann et al. (1993) Nucleic Acids Res. 21:2815). A second kinase, SR protein kinase- 1 (SRPK-1), capable of phosphorylating multiple different SR proteins has also been identified (Gui et al. (1994) Nature 169:678; Gui et al. (1994) PNAS 91:10824; Colwill et al. (1996) J. Biol. Chem. 271:24569). Interestingly, this kinase is active during mitosis, phosphorylates substrates exclusively on serine residues, copurifies with snRNP complexes, and disrupts both nuclear speckles and in vitro pre-rnRNA splicing (Gui et al. (1994) Nature 369:678). SRPK-1 has five known in vitro substrates, including SRp55, SRp40, SC35, ASF/SF2 and SRp20 (Gui et al. (1994) PNAS 91:10824). A related kinase, Clk/Sty, has also been shown to phosphorylate SR proteins in vitro (Colwill et al. (1996) J Biol. Chem. 271:24569; Melcher et al. (1996) J. Biol. Chem. 271:29958). Recently, two other kinases have been shown to phosphorylate SR proteins, the nuclear envelope (lamin B receptor) associated kinase (Nikolakaki et al. (1996) J. Biol. Chem. 271:8365) and mammalian DNA topoisomerase I (Rossi et al. (1996) Nαtwre 381:80-82). Currently, patients exhibiting symptoms of autoimmune disease, such as fever, weight loss, hair loss, oral ulcers, rash, photosensitivity, chest and/or abdominal pain, and arthritis, are serologically diagnosed using an antinuclear antibody (ANA) test. This test detects the presence of antibodies directed against the nucleus of cells where autoantigens commonly reside. ANA titers of greater than about 1 : 80 are generally considered positive for an autoimmune disease.

The ANA serological test for diagnosing autoimmune diseases has the drawback of being cumbersome, technician dependent and poorly reproducible. In addition, the test lacks specificity; providing no information with respect to the specific autoantigens involved in a disease phenotype. Accordingly, more practical, reproducible and specific methods of diagnosing autoimmune diseases would be beneficial to the art.

Summary of the Invention

The present invention provides advanced methods for diagnosing, treating and preventing autoimmune diseases. The methods are advanced in that, among other advantages, they are more sensitive, accurate, reproducible and informative than currently used methods. The invention further features an assay kit for diagnosing an autoimmune disease in an individual, methods for screening a chemical libraries for therapeutic compounds capable of treating autoimmune diseases. Accordingly, in one embodiment, the invention provides a method for diagnosing an autoimmune disease in an individual by determining in a biological sample of the individual the presence of antibodies specific for one or more phosphoproteins or protein kinases which are found in human apoptotic cells but are not found in human non- apoptotic cells, or the presence of antibodies specific for a protein complex containing one or more phosphoproteins or active protein kinases which are found in human apoptotic cells but are not found in human non-apoptotic cells. In a particular embodiment, antibodies are detected which are specific for one or more of eight different phosphoproteins (phosphorylated autoantigens) referred to as ppl7, pp23, pp34, pp42, pp46, pp54, pp90 and pp200 (named according to their molecular weights as measured by PAGE). In another particular embodiment, antibodies are detected which are specific for one or more phosphorylated serine/arginine (SR) splicing factors, such as SRp54, SRp40, ASF/SF2, SC35, U2AF35, and SRp20. In another particular embodiment, antibodies are detected which are specific for one or more active protein kinases, such as SRPK-1, Clk/Sty, DNA topoisomerase I, nuclear envelope-associated kinase, and Ul-70 kD-associated kinase. In yet another particular embodiment, antibodies are detected which are specific for a protein complex containing one or a combination of the aforementioned phosphoproteins or active protein kinases. For example, antibodies can be detected which are specific for a complex containing one of the aforementioned phosphoproteins or active protein kinases in association with a Ul- or U2-snRNP. In such cases, the antibody being detected (i.e., the autoantibody) may recognize either the phosphoprotein (e.g., phosphoepitope) within the complex or a component of the UI- or U2-snRNP within the complex (e.g., a UI A or U2B" protein). Various assays can be employed to detect autoantibodies of the present invention. In one embodiment, a sandwich ELISA assay is used to detect in a biological sample (e.g., serum) of an individual suspected of having or being at risk for an autoimmune disease the presence of antibodies directed against phosphoproteins, such as ppl7, pp23, pp34, pp42, pp46, pp54, pp90, pp200, SRp54, SRp40, ASF/SF2, SC35, U2AF35, SRp20, or protein complexes containing these phosphoproteins. Accordingly, phosphoproteins implicated by way of the present invention in autioimmune diseases can be recognized by autoantibodies either directly or indirectly in the form of a complex (i.e., associated with one or more other proteins, such as UI- or U2-snRNPs, which are recognized directly by autoantibodies).

Methods of the invention which employ a sandwich ELISA to detect the presence of one or more of the above-described autoantibodies involve collecting a blood sample from an individual which is then contacted separately (e.g., divided into two or more portions which are compared) with an extract from human apoptotic cells and an extract from human non-apoptotic cells under conditions which allow binding of proteins in the extracts by antibodies in the sample. Unbound proteins are then removed, and the sample is tested for bound phosphoproteins by contacting the sample with a labeled anti-phosphoprotein antibody or antibody fragment (e.g., one directed against a phosphoserine residue), removing unbound antibody, and detecting the presence of the label. The labelling pattern for the portion of the sample contacted with the apoptotic cell extract can then be compared to the pattern observed for the portion contacted with the non-apoptotic cell extract to determine the presence of an antibody in the individual's biological sample which recognizes a phosphoprotein or phosphoprotein complex which is present in human apoptotic cell extracts, but not in human non-apoptotic cell extracts. In accordance with the present invention, the presence of such an antibody is indicative of an autoimmune disease.

Preferred anti-phosphoprotein antibodies for use in the methods of the present invention are highly specific, including monospecific and, more preferably, monoclonal antibodies or fragments thereof, particularly those directed against phosphoserine residues. In one embodiment, the method employs a monoclonal antibody directed against a phosphoserine residue, preferably an antibody which recognizes and binds to phosphoserine residues in ppl7, pp23, pp34, pp42, pp46, pp54, pp90, pp200, and SR proteins including, but not limited to, SRp54, SRp40, ASF/SF2, SC35, U2AF35 and SRp20. Other antibodies which can be used in the methods of the invention include those directed against proteins, such as core components of the UI- and U2-snRNPs (e.g., U1A or U2B"), which are found (in apoptotic cell extracts) in association (e.g., in a complex) with the aforementioned phosphoproteins. Exemplary antibodies for use in the invention include the anti-Ul A/U2B" mAb 9A9, the anti-Sm mAb Y16, the anti-SR mAbl04, anti-SC35 mAb, and variable domain antibody fragments directed against Ul- snRNP (e.g., U1A protein component of Ul-snRNP).

In another embodiment of the invention, an in vitro kinase activity assay is used to detect in a biological sample from a patient suspected of having or being at risk for an autoimmune disease the presence of an antibody specific for one or more protein kinases, preferably an active protein kinase or a protein complex containing an active protein kinase, which is present in apoptotic cells but not in non-apoptotic cells. Exemplary protein kinases include SRPK-1, Clk/Sty, DNA topoisomerase I, nuclear envelope-associated kinase, and Ul-70 kD-associated kinase.

Methods of the invention which employ an in vitro kinase assay involve collecting a biological sample (e.g., serum) from the individual and contacting the sample separately (e.g., dividing the sample into two or more portions which are contacted) with an extract from human apoptotic cells and an extract from human non- apoptotic cells under conditions which promote binding of proteins in the extracts by antibodies in the samples. Unbound proteins are then removed, and the sample is tested for protein kinase activity using any standard in vitro assay known in the art (e.g., a colorimetric, radioactive, or fluorometric assay). The kinase activity of the portion of the sample contacted with the apoptotic cell extract can then be compared to the portion contacted with the apoptotic cell extract to determine the presence in the individual's biological sample of an antibody specific for one or more active protein kinases, or protein complexes containing active protein kinases, which are found in apoptotic cell extracts but not in non-apoptotic cell extracts. In accordance with the present invention, the presence of such an antibody is indicative of an autoimmune disease. In yet another aspect, the present invention provides an assay kit for diagnosing an autoimmune disease in an individual. The kit contains a solid support (e.g., an ELISA plate) capable of adsorbing immunoglobulin (e.g., IgG, IgM and IgA) from a biological sample (preferably a human biological sample, such as serum), a first extract from an apoptotic cell culture, a second extract from a non-apoptotic cell culture, and a monoclonal antibody or fragment thereof specific for a phosphoprotein. Preferred monoclonal antibodies for inclusion in the assay kit are specific for phosphoserine residues, such as those present on phosphoproteins selected from the group consisting of ppl7, pp23, pp34, pp42, pp46, pp54, pp90, pp200, SRp54, SRp40, ASF/SF2, SC35, U2AF35, SRp20, and phosphoproteins associated with UI- and U2-snRNPs. The assay kit can optionally include additional reagents such as a solution for washing unbound proteins from the solid support, and materials needed for performing an in vitro kinase assay, such as a kinase buffer and labeled (e.g., radioactively or fluorescently) ATP. In still another aspect, the present invention provides a method of treating or preventing an autoimmune disease in an individual. The method involves first detecting in a biological sample from the individual the presence of an antibody specific for one or more phosphoproteins (e.g., ppl7, pp23, pp34, pp42, pp46, pp54, pp90, pp200, SRp54, SRp40, ASF/SF2, SC35, U2AF35, SRp20) or activated protein kinases which are present in extracts from human apoptotic cells but not in extracts from human non- apoptotic cells (e.g., SRPK-1, Ok/Sty, DNA topoisomerase I, nuclear envelope- associated kinase, and Ul-70 kD-associated kinase). Once the presence of such an autoantibody is determined using, for example, the sandwich ELISA assay described above, then the individual can be administered a protein kinase inhibitor to reduce or prevent phosphorylation of autoantigens involved in the individual's disease phenotype. The protein kinase inhibitor can be any of a variety of kinase inhibitors known in the art and is preferably administered as a composition along with a pharmaceutically acceptable carrier. In yet another aspect of the invention, a method is provided for screening a chemical or peptide library for a compound which inhibits a protein kinase (e.g., SRPK- 1, Clk/Sty, DNA topoisomerase I, nuclear envelope-associated kinase, and Ul-70 kD- associated kinase) involved in phosphorylation of autoantigens during apoptosis. Such a kinase inhibitor can be administered to patients to treat or to prevent autoimmune disease.

These and other embodiments of the invention will be apparent from the following detailed description and working examples.

Brief Description of the Figures Figure 1 shows phosphoproteins precipitated from apoptotic Jurkat cell lysates using human autoimmune sera. In Panel A, Jurkat cells were labeled with 3 p. orthophosphate, treated with the anti-Fas monoclonal antibody 7C11, and lysed either before (odd numbered lanes) or 2.5 hours after (even numbered lanes) the addition of antibodies. Proteins were then precipitated using the indicated autoimmune serum, separated on a 12% SDS-polyacrylamide gel, transferred to nitrocellulose, and exposed for autoradiography. Arrows point to new phosphoproteins in the anti-Fas treatment lanes. In Panel B, the identical experiment with 35g labeled cells was performed. Patient numbers are located above each figure and correspond to those in Table 1 shown below. Lane numbers appear beneath the corresponding lane. The relative migration of molecular size markers in kilodaltons are indicated on the left side of the gel.

Figure 2 shows phosphoproteins precipitated using human autoimmune sera from Jurkat cells subjected to various apoptotic or mitogenic stimuli. Jurkat cells were labeled with 32p orthophosphate, triggered with apoptotic or mitogenic stimuli, and solubilized using NP40 lysis buffer at the indicated times prior to immuno-precipitation using sera derived from the indicated patient. Immunoprecipitates were separated on a 12% SDS polyacrylamide gel, transferred to nitrocellulose, and subjected to autoradiographic analysis. Panel A shows immunoprecipitates from cells treated with anti-Fas; Panel B shows immunoprecipitates from cells exposed to gamma irradiation; Panel C shows immunoprecipitates from cells exposed to UV irradiation; and Panel D shows immunoprecipitates from cells subjected to CD3 cross-linking. Arrows point to new phosphoproteins. The patient number is indicated above each time course. The time, in hours, appear beneath the corresponding lane. The relative migration of molecular size markers in kilodaltons is indicated on the left side of each panel.

Figure 3 shows that autoantigen phosphorylation coincides with or precedes the onset of DNA fragmentation in apoptotic Jurkat cells. Jurkat cells were triggered to undergo apoptosis by anti-Fas treatment (Panel A); gamma irradiation (Panel B); UV irradiation (Panel C); and anti-CD3 treatment (Panel D) and harvested at the indicated times. Each time point represents a total of 1 million cells. The DNA was prepared as described in the Materials and Methods section of the Exemplification below, separated on a 0.8% agarose gel and visualized by staining with ethidium bromide prior to ultraviolet exposure. The time, in hours, from initial exposure to each stimulus is indicated at the top of each lane. The relative migration of molecular size markers in kilobases is indicated on the right side of each panel.

Figure 4 shows a PVDF autoradiograph demonstrating that autoantigens are phosphorylated exclusively on serine residues during Fas-mediated apoptosis. Jurkat cells were labeled with 32p orthophosphate, treated with the anti-Fas monoclonal antibody 7C11 , and solubilized using NP 40 lysis buffer after 2.5 hours. Proteins were then precipitated with autoimmune serum, separated on a 12% SDS-polyacrylamide gel, transferred to polyvinylidene difluoride (PVDF), and exposed for autoradiography. Individual phosphoproteins were localized on the membrane, excised, and subjected to acid hydrolysis. Phosphoaminoacids were separated by two-dimensional electrophoresis in pH 1.9 buffer in the horizontal dimension, followed by pH 3.5 buffer in the vertical dimension prior to autoradiographic analysis. Individual proteins correspond to those described in Table 1 as follows: Panel A - Patient 1, pp200; Panel B - Patient 1, pp54; Panel C - Patient 7, pp46; Panel D - Patient 11 , pp42; Panel E - Patient 3, pp34; Panel F - Patient 8, pp23; and Panel G - Patient 11, ppl7. Migration of phosphoaminoacid standards are labeled with circles as follows: phosphoserine (pS), phosphothreonine (pT), phosphotyrosine (pY). Figure 5 shows serine kinase activity precipitated from apoptotic Jurkat lysates using autoimmune serum. In Panel A, Jurkat cells cultured in the absence (odd numbered lanes) or presence (even numbered lanes) of anti-Fas were solubilized in NP40 lysis buffer after 2.5 hours, and precipitated using 3.5 μl of serum derived from the indicated patient. Individual precipitates were subjected to an in vitro kinase reaction. Serum derived from the patient number indicated at the top of the Figure corresponds to patients described in Table 1 shown below. The relative migration of molecular size markers in kilodaltons is indicated on the right side of the panel. In Panel B, the kinetics of kinase activation following Fas ligation was measured using an in vitro kinase reaction performed on immunoprecipates using serum derived from patient 7. The time, in minutes, from initial exposure to anti-Fas is indicated at the top of each lane. The position of pp46 is indicated with an arrow on the left side of the panel. In Panel C, phosphoaminoacid analysis of the in vitro phosphorylated 46 kDa protein was performed. Migration of phosphoaminoacid standards are labeled with circles as follows: phosphoserine (pS), phosphothreonine (pT), phosphotyrosine (pY). Figure 6 shows that in vivo phosphorylation of pp46 correlates with the induction of apoptosis and is inhibited in Jurkat cells overexpressing bcl-2. Jurkat (bcl-2) transformants (first six lanes) or Jurkat (neo) control transformants (last six lanes) were labeled with 32p orthophosphate, subjected to apoptotic stimulus as follows: Panel A: anti-Fas treatment; Panel B: gamma irradiation; Panel C: UV irradiation, solubilized in NP40 lysis buffer, and precipitated using serum derived from patient 7 prior to electrophoretic separation. The relative migration of molecular size markers in kilodaltons is indicated on the right side of each panel. The time, in hours, from initial exposure to each stimulus is indicated at the top of each lane.

Figure 7 shows that human autoimmune monospecific sera specific for U-snRNP complexes precipitate phosphoproteins from apoptotic Jurkat cell lysates. Panel A: Jurkat cells were labeled with 3 p orthophosphate and lysed either before (-) or 3 hours after (+) the addition of anti-Fas 7C11. Proteins were then precipitated using the following indicated autoimmune serum, separated on a 12% SDS-polyacrylamide gel, transferred to nitrocellulose, and exposed for autoradiography: U-serum 1, Immuno vision anti-histone/RNP; U-serum 2, CDC/AF reference serum 4 (anti-Ul RNP); U-serum 3, serum Ga; U-serum 4, Serum Ya; U-serum 5, CDC/AF reference serum 5 (anti-Sm). The relative migration of molecular size markers in kilodaltons is indicated on the left side of the gel. Panel B: Immunoprecipitation from 35s-labeled Jurkat cells. Jurkat cells were labeled with 35s methionine and cysteine and lysed either before (-) or 3 hours after (+) the addition of anti-Fas 7C11 prior to immunoprecipitation using sera derived from the indicated patient. Immunoprecipitates were separated on a 12%) SDS polyacrylamide gel, transferred to nitrocellulose, and subjected to autoradiographic analysis. The relative migration of molecular size markers in kilodaltons is indicated on the left side of each panel. Bands corresponding to the U- snRNP proteins, A, B, B', and C are indicated on the right side of the panel.

Figure 8 shows coprecipitation of Ul-snRNA using selected autoantisera. Jurkat cells were labeled with 3 p orthophosphate and solubilized in NP40 lysis buffer. Following immunoprecipitation with the indicated serum, RNA was extracted and separated on 6% sequencing gels prior to drying and autoradiographic exposure. The relative migration of known RNA moieties is depicted on the right side of the figure. The serum specificity is indicated above each sample. Lanes are numbered at the bottom of the figure. Lanes 1-4 correspond respectively to patients 1, 8, 11 and 12 identified in Table 1 (see Exemplification). Lanes 5-10 correspond to U-serum 1, Immunovision anti-histone/RNP (lane 5); U-serum 2, CDC/AF reference serum 4 (anti Ul-RNP) (lane 6); U-serum 3, serum Ga (lane 7); U-serum 4, serum Ya (lane 8); U-serum 5, CDC/AF reference serum 5 (anti-Sm) (lane 9); and U-serum 6, anti-Ul-70 kD serum (lane 10). Figure 9 shows that U 1 -monospecific autoantisera coprecipitate the U 1 -snRNA molecule and phosphoproteins pp54, pp42, pp34, and pp23 from apoptotic extracts. Panel A: Jurkat cells were labeled with 3 p orthophosphate and lysed in NP40 lysis buffer. Following immunoprecipitation with the indicated serum, RNA was extracted and separated on 6% sequencing gels prior to drying and autoradiographic exposure. Patient sera specific for the Ul-snRNP complex were used in lanes 1-7. A patient serum (V26, lane 8) capable of precipitating both the UI- and U2-snRNPs is shown for comparison. The relative migration of the UI- and U2- snRNAs is depicted on the right side of the figure. Panel B: Jurkat cells were labeled with 32p orthophosphate and lysed either before (-) or 3 hours after (+) the addition of anti-Fas (7C11) prior to immunoprecipitation using sera derived from the indicated patient. Immunoprecipitates were separated on a 12% SDS polyacrylamide gel, transferred to nitrocellulose, and subjected to autoradiographic analysis. Sera correspond to the seven Ul-specific autoantisera shown in Figure 9 A. The relative migration of molecular size markers in kilodaltons is indicated on the right side of the panel. A high molecular weight complex is indicated with a large arrowhead. Lanes are numbered at the bottom of the figure. Figure 10 shows that monoclonal antibodies directed against Ul-snRNP components precipitate phosphoproteins pp54, pp42, pp34, and pp23 from extracts prepared from apoptotic Jurkat cells. Panel A: Jurkat cells were labeled with 32p orthophosphate and lysed either before (-) or 3 hours after (+) the addition of anti-Fas 7C11. Proteins were then precipitated using the indicated autoimmune serum, separated on a 12%) SDS-polyacrylamide gel, transferred to nitrocellulose, and exposed for autoradiography. The relative migration of molecular size markers in kilodaltons is indicated on the left side of the gel. Bands corresponding to pp90, pp54, pp42, pp34, and pp23 are shown on the right side of the panel. Lanes are numbered at the bottom of the figure. Panel B: shows the identical experiment shown in Panel A in 35s-labeled Jurkat cells. The relative migration of molecular size markers in kilodaltons is indicated on the left side of the gel. Lanes are numbered at the bottom figure. Panel C: shows phosphoaminoacid analysis of pp54, pp42, pp34, and pp23. Jurkat cells were labeled with 3 p orthophosphate, treated with the anti-Fas monoclonal antibody 7C11, and solubilized using NP40 lysis buffer after 3 hours. Proteins were then precipitated with the anti-Ul A/U2B" monoclonal antibody 9A9, separated on a 12%) SDS-polyacrylamide gel, transferred to PVDF, and exposed for autoradiography. Individual phosphoproteins were localized on the membrane, excised, and subjected to acid hydrolysis. Phosphoaminoacids were separated by two-dimensional electrophoresis in pH 1.9 buffer in the horizontal dimension, followed by pH 3.5 buffer in the vertical dimension prior to autoradiographic analysis. Individual proteins are labeled on the side of each panel. Migration of phosphoaminoacid standards are labeled with circles as follows: phosphoserine (pS), phosphothreonine (pT), phosphotyrosine (pY). Panel D: shows that anti-Ul A antibody fragments coprecipitate pp54, pp42, pp34, and pp23 from apoptotic Jurkat cell lysates. Labeled Jurkat cell extracts were prepared as described above. Proteins were precipitated using the indicated anti-Ul A antibody fragments, separated on a 12% SDS-polyacrylamide gel, transferred to nitrocellulose, and exposed for autoradiography. The relative migration of molecular size markers in kilodaltons is indicated on the right side of the gel. Bands corresponding to pp54, pp42, pp34, and pp23 are shown on the left side of the panel.

Figure 11 shows that phosphoprotein components of the Ul-snRNP complex are precipitated following multiple apoptotic stimuli but not an activation stimulus. Jurkat cells were labeled with 32p orthophosphate, treated with the indicated stimulus, and solubilized using NP40 lysis buffer at the indicated times. Proteins were then precipitated with anti-Ul A/U2B, separated on a 12% SDS-polyacrylamide gel, transferred to nitrocellulose, and exposed for autoradiography. The time in hours following each stimulus is indicated above each lane. The relative migration of molecular size markers in kilodaltons is indicated on the left side of the panel. Bands corresponding to pp54, pp42, pp34, and pp23 are shown on the right side of the panel. Lanes are numbered at the bottom of each panel.

Figure 12 shows that in vivo phosphorylation of Ul-snRNP components is inhibited in gamma-irradiated Jurkat cells overexpressing bcl-2. Jurkat (bcl-2) transformants (lanes 1-4) or Jurkat (neo) control transformants (lanes 5-8) were labeled with 32p orthophosphate, subjected to gamma irradiation, solubilized in NP40 lysis buffer, precipitated using anti-Ul A/U2B antibodies, separated on a 12% SDS- polyacrylamide gel, transferred to nitrocellulose, and exposed for autoradiography. Bands corresponding to pp54, pp42, pp34, and pp23 are shown on the right side of the figure. The time, in hours, from initial exposure to gamma irradiation, is indicated at the top of each lane. Lane numbers appear at the bottom of the figure.

Figure 13 : shows that monoclonal antibodies specific for SR proteins precipitate pp54, pp42, pp34, and pp23 from apoptotic Jurkat cell extracts. Panel A: Jurkat cells were labeled with 32p orthophosphate and lysed either before (-) or 3 hours after (+) the addition of anti-Fas 7C 11. Proteins were then precipitated using the indicated monoclonal antibody, separated on a 12% SDS-polyacrylamide gel, transferred to nitrocellulose, and exposed for autoradiography. The relative migration of pp34 is indicated on the left side of the gel. Lanes are numbered at the bottom of the panel. Panel B: shows a two dimensional phosphotryptic map of pp34. The 34 kD band from the U1A/U2B immunoprecipitate (lane 2) and the anti-SC35 immunoprecipitate (lane 4) were excised and digested with trypsin. Phosphopeptides were separated electrophoretically at pH 1.9 in the first dimension, and by thin layer chromatography in the second dimension prior to autoradiographic exposure. Direction of separation is shown by arrows. E indicates electrophoresis. C indicates chromatography. O indicates Origin.

Detailed Description of the Invention

The present invention is based, at least in part, on the discovery that proteins phosphorylated in cells undergoing apoptosis may be preferred targets for autoantibody production in patients with autoimmune disease. Initially, as part of the present invention, it was found that sera from 10/12 patients containing antinuclear antibodies (ANA) meeting diagnostic criteria for systemic lupus erythematosus (SLE) and SLE in association with a second inflammatory condition (SLE overlap syndrome) precipitated new phosphoproteins from lysates derived from Jurkat T cells treated with apoptotic stimuli (e.g., Fas-ligation, gamma irradiation, UV irradiation), but not with an activation (e.g., CD3-ligation) stimulus. As a further step, it was found as part of the present invention that autoantigen phosphoproteins (e.g., phosphoproteins recognized by autoimmune sera) from apoptotic cell lysates could be detected in patients' sera using monoclonal antibodies and monospecific sera which recognize specific SR proteins and core components of snRNPs (e.g., UI- and U2-snRNPs) which associate with phosphoproteins. More particularly, it has been found as part of the present invention that autoimmune sera derived from individual patients precipitate from human apoptotic cell extracts but not from human non-apoptotic cell extracts different combinations of eight distinct serine-phosphorylated proteins having approximate molecular weights of 17 kDa, 23 kDA, 34 kD, 42 kDa, 46 kDa, 54 kDa, 90 kDa and 200 kDa, as measured by PAGE. These phosphoproteins are respectively referred to herein as ppl7, pp23, pp34, pp42, pp46, pp54, pp90 and pp200. None of these phosphoproteins have been identified in precipitates prepared using sera from patients with diseases that are not associated with autoantibody production or using serum from rheumatoid arthritis patients. It has also been found as part of the present invention that pp54, pp42, pp34 and pp23, in particular, are recognized and precipitated by monospecific autoimmune sera, monoclonal antibodies, and variable domain antibody fragments directed against proteins associated with the Ul-snRNP complex, including the SR splicing factors (phosphorylated proteins) SRp54, SRp40, ASF/SF2, SC35, U2AF35, and SRp20. It has further been found as part of the present invention that pp90, in particular, is recognized and precipitated by a monoclonal antibody which binds commonly to both the Ul- snRNP complex and the U2-snRNP complex, and a monoclonal antibody which binds only to the U2-snRNP complex (e.g., U2B").

In addition, it has been found as part of the present invention that serum from four autoimmune patients precipitates a serine/threonine kinase from apoptotic cell lysates that phosphorylates proteins of 23 kDa, 34 kDa and 46 kDa in in vitro kinase assays. Therefore, detection of such autoimmune disease-linked phosphorylated proteins and kinases in patients' sera by, for example, indirectly assaying for antibodies (e.g., IgG, IgM and IgA) specific for these proteins, or protein complexes containing these proteins, may be used to diagnose a variety of autoimmune diseases. Moreover, identification of such disease-linked phosphorylated proteins and kinases may be used to identify agents, such as kinase inhibitors, which can be administered therapeutically or prophylactically to patients to prevent phosphorylation of autoantigens during cell death.

Accordingly, in one embodiment, the present invention provides a method for diagnosing an autoimmune disease in an individual by determining in a biological sample from an individual suspected of having or being at risk for an autoimmune disease the presence of antibodies specific for one or more phosphoproteins or active protein kinases, or a complex containing one or more phosphoproteins or active protein kinases, which are found in human apoptotic cells but which are not found in human non-apoptotic cells. Such an individual may exhibit common symptoms of autoimmune disease such as fever, weight loss, hair loss, oral ulcers, rash, photosensitivity, chest and/or abdominal pain, or arthritis. Alternatively, such an individual may be at risk for developing such symptoms based on medical or genetic history (e.g., the individual may have a family history of autoimmune disease, may have had a transplantation or miscarriage, or may be taking an autoimmune disease inducing drug, such as procainamide which induces SLE). Such an individual may also have already been tested for autoimmune activity by, for example, an antinuclear antibody (ANA) test and shown to have a positive ANA titer of, for example, greater than about 1 :80. Methods for performing such serological ANA testing are described in U.S. 5,583,053, the contents of which is incorporated herein. However, the diagnostic method of the invention is intended to replace the current ANA test as an easier, more accurate diagnostic tool for autoimmune disease. For example, the present invention provides methods for detecting autoantibodies against particular phosphoproteins and protein kinases (or complexes thereof) which are found in serum from patients with SLE overlap syndrome. Thus, these methods can be used specifically to diagnose SLE, whereas ANA tests and other previously known diagnositic assays can be positive for multiple autoimmune diseases, such as RA, sclerodoma, etc. The term "phosphoprotein" as used herein means any phosphorylated protein and particularly encompasses a variety of autoantigens which are phosphorylated during apoptosis. The term "protein kinase" as used herein means an enzyme capable of catalyzing phosphorylation of proteins, particularly those which are activated during apoptosis and which are commonly known in the art as "stress activated protein kinases" (SAP kinases). As used herein the terms "phosphoprotein" and "protein kinase" also encompass protein complexes containing these proteins in association with other proteins (e.g., substrates).

The term "apoptotic" or "apoptosis" as used herein refers to the induction of cell death. Recent studies have established that inflammatory cytokines (e.g., TNF-α, Fas- ligand) and environmental stress (e.g., heat shock, UV light, and X-irradiation) are all potent triggers of apoptotic cell death (Alderson et al. (1995) J. Exp. Med. 181 :71-77; Chen et al. (1996)/. Biol. Chem. 271:631-634; Kyriakis et al. (1994) Nature 369:156- 160; Kyriakis et al. (1996) Bioassays 18: 567-577; Ju et al. (1995) N twre 373:444-448: Verheij et al. (1996) Nature 180:75-79; Mathias et al. (1991) Proc. Natl. Acad. Sci.. USA. 88: 10009-10013). All such triggers of apoptotic cell death, as well as many other triggers known in the art (e.g., chemotherapeutic agents etc.), can be employed to obtain aopototic human cells for use in the methods of the present invention. The term "biological sample" as used herein refers to a biological tissue or fluid of an individual which contains antibodies or fragments thereof capable of detection by the methods of the invention. Such biological samples include, for example, blood, serum, urine, joint fluid, cerebral spinal fluid, saliva, bronchial wash. A preferred biological sample is serum.

Anti-phosphoprotein antibodies can be serologically detected using a number of different screening assays known in the art, such as an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), or a Western Blot Assay, and their binding patterns compared for apoptotic verses non-apoptotic cell extracts. Each assay generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. Accordingly, in the present invention, these assays are used to detect protein-antibody complexes formed between immunoglobulins (e.g., human IgG, IgM and IgA) contained in a biological sample and phosphoproteins contained in apoptotic and non-apoptotic cell extracts. As will be described below, these protein-antibody complexes are preferably detected using an enzyme-linked antibody or antibody fragment (e.g., a monoclonal antibody or fragment thereof) which recognizes and specifically binds to the phosphoprotein portion of the protein-antibody complexes.

In a preferred embodiment, the present invention employs a sandwich ELISA assay to screen a biological sample of an individual suspected of having or being at risk for an autoimmune disease for the presence of antibodies specific for one or more phosphoproteins or protein kinases which are found in human apoptotic cells but which are not found in human non-apoptotic cells. The assay is so named because it involves the use of an antibody-antigen-antibody sandwich on a solid phase. To perform the assay, a biological sample is first collected from the individual and is concentrated (e.g., centrifuged) to collect the biological sample, e.g., serum. The serum is then adsorbed onto a solid support such as a microtiter plate (e.g., a 96 well ELISA plate) by incubating the serum and the plate for between 2-20 hours at between about 1-24°C. The unbound components of the serum sample are then removed in a manner which leaves intact the immunoglobulins (e.g., IgG, IgA, IgM) adsorbed onto the plate. The removal is preferably carried out by washing the solid support with an eluent to which the immunoglobulins are inert (e.g., PBS-Tween).

In a next step, a portion of the plate is treated (i.e., contacted, e.g., incubated) with an extract from an apoptotic cell culture and another portion of the plate is treated with an extract from a non-apoptotic cell culture (alternatively two separate plates can be used). Any human cells suitable for growth in culture can be used for this purpose, such a human Jurkat cells which can be cultured and subjected to various apoptotic stimuli (e.g., which include but are not limited to Fas ligand, UV irradiation or X-rays). Such cell cultures can be prepared as described in the examples below. The cell extracts are incubated on the serum coated plate(s) under conditions which permit antibody-protein binding between the immunoglobulins from the serum sample and proteins which they recognize from the apoptotic and non-apoptotic cell extracts. The incubation step is preferably carried out for a period of about 2-20 hours at a temperature of between 1- 24°C, higher temperatures being required for shorter incubation periods. This step results in the formation of active protein-antibody complexes which are bound to the surface of the plate. After the protein-antibody complexes have been allowed to form, unbound components (i.e., components which have not been recognized by antibodies in the patient's serum) from the apoptotic and non-apoptotic cell extracts are removed from the plate(s) in a manner which leaves the complexes intact. The removal is preferably carried out by washing the plate(s) with an eluent to which the complexes are inert (e.g., PBS-Tween). The plate(s) are then contacted with a solution containing at least one antibody or antibody fragment, preferably a monoclonal antibody or fragment thereof, specific for a phosphoprotein. As used herein, the terms "antibody specific for a phosphoprotein or a protein kinase" or "anti-phosphoprotein antibody" or "anti-kinase antibody," as used herein, encompass all forms of antibodies known in the art, such as chimeric, recombinatorial and humanized antibodies, as well as fragments thereof (e.g., F(ab')2 fragments), which specifically bind to a phosphoprotein or protein kinase, or to a protein complex containing a phosphoprotein or protein kinase and, therefore, can be used as screening tool in the assays described herein. The term "antibody fragment" as used herein means an antibody fragment which specifically binds to a phosphoprotein or protein kinase, or to a protein complex containing a phosphoprotein or protein kinase and, therefore, can be used as screening tool in the assays described herein (i.e., a functional antibody fragment).

In one embodiment, the invention employs a monoclonal antibody or fragment thereof which recognizes at least one, and preferably more than one, of the following phosphoproteins: ppl7, pp23, pp34, pp42, pp46, pp54, pp90, pp200, SRp54, SRp40, ASF/SF2, SC35, U2AF35, SRp20. For example, the monoclonal antibody can bind to a phosphorylated serine residue common to these phosphoproteins. Alternatively, the invention employs a monoclonal antibody or fragment thereof which recognizes a protein, such as UI- or U2-snRNP (e.g., a core component of UI- or U2-snRNP, such as UI A or U2B") associated with one or more of the aforementioned phosphoproteins (e.g., in a protein complex). In a particular embodiment, the invention employs one or more of the monoclonal antibodies identified in Figure 10, including but not limited to the anti- Ul A/U2B" mAb 9A9, the anti-Sm mAb Y16, the anti-U2B" mAb 4G3, the anti-SR mAb 104, anti-SC35 mAb, and variable domain antibody fragments directed against Ul- snRNP (the UI A protein). Other suitable monoclonal antibodies and fragments for use in the invention (e.g., those which bind phosphoproteins identified herein and/or components of UI- or U2 snRNPs) can be obtained by methods known in the art, such as those described below, or, alternatively, can be purchased in the form of commercially available cell lines (e.g., ATCC cell lines) which produce antibodies directed against phosphorylated proteins (e.g., phosphate groups) or components of snRNPs.

Alternatively, a combination of the aforementioned monoclonal antibodies or antibody fragments can be employed.

The anti-phosphoprotein antibody or fragment thereof of the present invention is preferably incubated on the plate(s) under conditions which allow the antibody to recognize and bind to phosphoproteins (or phosphoprotein complexes) which are themselves bound onto the plate(s) via antibodies from the patient's serum (e.g., immunoglobulins), thereby forming an antibody-protein-antibody sandwich. Again, the incubation step can be carried out for a period of about 2-20 hours at a temperature of between 1-24°C and any unbound anti-phosphoprotein antibody can be removed by washing with an appropriate eluent. After the anti-phosphoprotein antibody has been reacted with the plate(s), the presence of bound anti-phosphoprotein antibody is determined and thus the presence of antibodies in the patient's biological sample specific for a phosphoprotein can be both determined and quantified.

By comparing the portion of the plate (or separate plate) which was incubated with apoptotic cell extract with the portion of the plate which was incubated with the non-apoptotic cell extract, it can be determined whether phosphoproteins or phosphoprotein complexes present in apoptotic cell extracts were recognized which were not recognized in non-apoptotic cell extracts. Such a difference in binding patterns indicates the presence of autoantibodies in the patient's serum which are specific for phosphoproteins or phosphoprotein complexes (since it may be that the patient's serum recognizes proteins bound to or associated with phosphoproteins instead of the phosphoproteins themselves) found in apoptotic cells but not non-apoptotic cells, and can link the autoimmune disease of the patient, or a risk for an autoimmune disease, to the presence of such autoantibodies. The assaying step may be carried out using any suitable procedure for detecting the binding of the anti-phosphoprotein antibody to the reaction plate. One preferred means involves labeling the anti-phosphoprotein antibody via linkage to an enzyme which is then detected in an enzyme immunoassay (EIA) (Voller, "The Enzyme Linked Immunosorbent Assay (ELISA)", Diagnostic Horizons 2:1-7, 1978, Microbiological Associates Quarterly Publication, Walkersville, MD; Voller, et al., J. Clin. Pathol. 31 :507-520 (1978); Butler, Meth. Enzymol. 73:482-523 (1981); Maggio, (ed.) Enzyme Immunoassay, CRC Press, Boca Raton, FL, 1980; Ishikawa, et al., (eds.) Enzyme

Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection of the anti-phosphoprotein antibody may also be accomplished using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassay s, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a γ counter or a scintillation counter or by autoradiography.

It is also possible to label the anti-phosphoprotein antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. The antibody can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA). The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent- tagged antibody is then determined by detecting luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound may be used to label the antibody. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

Monoclonal antibodies capable of recognizing phosphoproteins of the invention can be prepared using methods well known in the art. Such methods are described, for example, in detail in US 4,942,131 and US 5,583,053, the contents of which are incorporated by reference herein. The term "monoclonal antibody," as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of a phosphoprotein, such as phosphoserine residue of ppl7, pp23, pp34, pp42, pp46, pp54, pp90, pp200, SRp54, SRp40, ASF/SF2, SC35, U2AF35 or SRp20. A monoclonal antibody composition thus typically displays a single binding affinity for a particular phosphoprotein or phosphoprotein complex with which it immunoreacts.

Monoclonal antibodies useful in the methods of the invention are directed to an epitope of a phosphoprotein, such that complex formed between the antibody and the phosphoprotein (also referred to herein as ligation) can be recognized in any of the assays described above. A monoclonal antibody to an epitope of a phosphoprotein can be prepared by using a technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497), and the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, Inc., pp. 77-96), and trioma techniques. Other methods which can effectively yield monoclonal antibodies useful in the present invention include phage display techniques (Marks et al. (1992) J Biol Chem 16007-16010).

In one embodiment, the antibody preparation applied in the subject method is a monoclonal antibody produced by a hybridoma cell line. Hybridoma fusion techniques were first introduced by Kohler and Milstein (Kohler et al. Nature (1975) 256:495-97; Brown et al. (1981) J Immunol 127:539-46; Brown et al. (1980) JBiol Chem 255:4980- 83; Yeh et al. (1976) PNAS 76:2927-31 ; and Yeh et al. (1982) Int. J. Cancer 29:269-75). Thus, the monoclonal antibody compositions of the present invention can be produced by immunizing an animal with a phosphoprotein protein such as ppl7, pp23, pp34, pp42, pp46, pp54, and pp200, or peptide thereof. The immunization is typically accomplished by administering the phosphoprotein immunogen to an immunologically competent mammal in an immunologically effective amount, i.e., an amount sufficient to produce an immune response. Preferably, the mammal is a rodent such as a rabbit, rat or mouse. The mammal is then maintained for a time period sufficient for the mammal to produce cells secreting antibody molecules that immunoreact with the phosphoprotein immunogen. Such immunoreaction is detected by screening the antibody molecules so produced for immunoreactivity with a preparation of the immunogen protein. Optionally, it may be desired to screen the antibody molecules with a preparation of the protein in the form in which it is to be detected by the antibody molecules in an assay, e.g., a membrane-associated form of phosphoprotein. These screening methods are well known to those of skill in the art, e.g., ELISA and/or flow cytometry.

A suspension of antibody-producing cells is then removed from each immunized mammal secreting the desired antibody is then prepared. After a sufficient time, the mouse is sacrificed and somatic antibody-producing lymphocytes are obtained.

Antibody-producing cells may be derived from the lymph nodes, spleens and peripheral blood of primed animals. Spleen cells are preferred, and can be mechanically separated into individual cells in a physiologically tolerable medium using methods well known in the art. Mouse lymphocytes give a higher percentage of stable fusions with the mouse myelomas described below. Rat, rabbit and frog somatic cells can also be used. The spleen cell chromosomes encoding desired immunoglobulins are immortalized by fusing the spleen cells with myeloma cells, generally in the presence of a fusing agent such as polyethylene glycol (PEG). Any of a number of myeloma cell lines may be used as a fusion partner according to standard techniques; for example, the P3-NSl/l-Ag4-l, P3- x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md.

The resulting cells, which include the desired hybridomas, are then grown in a selective medium, such as HAT medium, in which unfused parental myeloma or lymphocyte cells eventually die. Only the hybridoma cells survive and can be grown under limiting dilution conditions to obtain isolated clones. The supernatants of the hybridomas are screened for the presence of antibody of the desired specificity, e.g., by immunoassay techniques using the antigen that has been used for immunization. Positive clones can then be subcloned under limiting dilution conditions and the monoclonal antibody produced can be isolated. Various conventional methods exist for isolation and purification of the monoclonal antibodies so as to free them from other proteins and other contaminants. Commonly used methods for purifying monoclonal antibodies include ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography (see, e.g., Zola et al. in Monoclonal Hybridoma Antibodies: Techniques And Applications. Hurell (ed.) pp. 51-52 (CRC Press 1982)). Hybridomas produced according to these methods can be propagated in vitro or in vivo (in ascites fluid) using techniques known in the art. Generally, the individual cell line may be propagated in vitro, for example in laboratory culture vessels, and the culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by decantation, filtration or centrifugation. Alternatively, the yield of monoclonal antibody can be enhanced by injecting a sample of the hybridoma into a histocompatible animal of the type used to provide the somatic and myeloma cells for the original fusion. Tumors secreting the specific monoclonal antibody produced by the fused cell hybrid develop in the injected animal. The body fluids of the animal, such as ascites fluid or serum, provide monoclonal antibodies in high concentrations. When human hybridomas or EBV- hybridomas are used, it is necessary to avoid rejection of the xenograft injected into animals such as mice. Immunodeficient or nude mice may be used or the hybridoma may be passaged first into irradiated nude mice as a solid subcutaneous tumor, cultured in vitro and then injected intraperitoneally into pristane primed, irradiated nude mice which develop ascites tumors secreting large amounts of specific human monoclonal antibodies. Media and animals useful for the preparation of these compositions are both well known in the art and commercially available and include synthetic culture media, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al. (1959) Virol. 8:396) supplemented with 4.5 gm/1 glucose, 20 mM glutamine, and 20% fetal caf serum. An exemplary inbred mouse strain is the Balb/c.

When antibodies produced in non-human subjects are used therapeutically in humans, they are recognized to varying degrees as foreign and an immune response may be generated in the patient. One approach for minimizing or eliminating this problem, which is preferable to general immunosuppression, is to produce chimeric antibody derivatives, i.e., antibody molecules that combine a non-human animal variable region and a human constant region. Such antibodies are the equivalents of the monoclonal and polyclonal antibodies described above, but may be less immunogenic when administered to humans, and therefore more likely to be tolerated by the patient.

Chimeric mouse-human monoclonal antibodies (i.e., chimeric antibodies) reactive with phosphoproteins of the invention can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the constant region of a murine (or other species) anti-phosphoprotein antibody molecule is substituted with a gene encoding a human constant region, (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al. (1987) Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl Cancer Inst. 80:1553-1559). A chimeric antibody can be further "humanized" by replacing portions of the variable region not involved in antigen binding with equivalent portions from human variable regions. General reviews of "humanized" chimeric antibodies are provided by Morrison, S. L. (1985) Science 229:1202-1207 and by Oi et al. (1986) BioTechniques 4:214. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of an immunoglobulin variable region from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from an anti- phosphoprotein antibody producing hybridoma. The cDNA encoding the chimeric antibody, or fragment thereof, can then be cloned into an appropriate expression vector. Suitable "humanized" antibodies can be alternatively produced by CDR or CEA substitution (see U.S. Patent 5,225,539 to Winter; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141 :4053-4060).

As an afterntive to humanizing a monoclonal antibody or fragment thereof from a mouse or other species, a human a monoclonal antibody (mAb) or fragment thereof directed against a human protein can be generated. Transgenic mice carrying human antibody repertoires have been created which can be immunized with human phosphoproteins of the present invention or peptides thereof. Splenocytes from these immunized transgenic mice can then be used to create hybridomas that secrete human mAbs specifically reactive with human phosphoproteins (see, e.g., Wood et al. PCT publication WO 91/00906, Kucheriapati et al. PCT publication WO 91/10741; Lonberg et al. PCT publication WO 92/03918; Kay et al. PCT publication 92/03917; Lonberg, N. et al. (1994) Nature 368:856-859; Green, L.L. et al. (1994) Nature Genet. 7:13-21; Morrison, S.L. et al. (1994) Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. (1993) Year Immunol 7:33-40; Tuaillon et al. (1993) PNAS 90:3720-3724; Bruggeman et al. (1991) Ewr J Immunol 2k 1323-1326).

Monoclonal antibodies or fragments thereof suitable for use in the present invention (i.e., which recognize and specifically bind to phosphoproteins or phosphoprotein complexes) can also be produced by other methods well known to those skilled in the art of recombinant DNA technology. Such alternative methods include the "combinatorial antibody display" method which identifies and isolates antibodies and antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal anti-phosphoprotein antibodies (for descriptions of combinatorial antibody display see e.g., Sastry et al. (1989) PNAS 86:5728; Huse et al. (1989) Science 246:1275; and Orlandi et al. (1989) PNAS 86:3833). After immunizing an animal with a phosphoprotein immunogen as described above, the antibody repertoire of the resulting B-cell pool is cloned. Methods are generally known for directly obtaining the DNA sequence of the variable regions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and PCR. For instance, mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework 1 (FR1) sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification of the heavy and light chain variable regions from a number of murine antibodies (Larrick et al. (1991) Biotechniques 11:152-156). A similar strategy can also been used to amplify human heavy and light chain variable regions from human antibodies (Larrick et al. (1991) Methods: Companion to Methods in Enzymology 2:106- 110).

In an illustrative embodiment, RNA is isolated from activated B cells of, for example, peripheral blood cells, bone marrow, or spleen preparations, using standard protocols (e.g., U.S. Patent No. 4,683,202; Orlandi, et al. PNAS (1989) 86:3833-3837; Sastry et al., PNAS (1989) 86:5728-5732; and Huse et al. (1989) Science 246:1275- 1281.) First-strand cDNA is synthesized using primers specific for the constant region of the heavy chain(s) and each of the K and λ light chains, as well as primers for the signal sequence. Using variable region PCR primers, the variable regions of both heavy and light chains are amplified, each alone or in combinantion, and ligated into appropriate vectors for further manipulation in generating the display packages. Oligonucleotide primers useful in amplification protocols may be unique or degenerate or incorporate inosine at degenerate positions. Restriction endonuclease recognition sequences may also be incorporated into the primers to allow for the cloning of the amplified fragment into a vector in a predetermined reading frame for expression.

The V-gene library cloned from the immunization-derived antibody repertoire can be expressed by a population of display packages, preferably derived from filamentous phage, to form an antibody display library. Ideally, the display package comprises a system that allows the sampling of very large variegated antibody display libraries, rapid sorting after each affinity separation round, and easy isolation of the antibody gene from purified display packages. In addition to commercially available kits for generating phage display libraries (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene Swr ZΛRTM phage display kit, catalog no. 240612), examples of methods and reagents particularly amenable for use in generating a variegated anti-phosphoprotein antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 1:81-85; Huse et al. (1989) Science 246:1275-1281 ; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.

In certain embodiments, the V region domains of heavy and light chains can be expressed on the same polypeptide, joined by a flexible linker to form a single-chain Fv fragment, and the scFV gene subsequently cloned into the desired expression vector or phage genome. As generally described in McCafferty et al., Nature (1990) 348:552-554, complete Vjj and VL domains of an antibody, joined by a flexible (Gly4-Ser)3 linker can be used to produce a single chain antibody which can render the display package separable based on antigen affinity. Isolated scFV antibodies immunoreactive with phosphoprotein can subsequently be formulated into a pharmaceutical preparation for use in the subject method.

Once displayed on the surface of a display package (e.g., filamentous phage), the antibody library is screened with a phosphoprotein protein, or peptide fragment thereof, to identify and isolate packages that express an antibody having specificity for phosphoprotein (e.g., phosphoserine residues). Nucleic acid encoding the selected antibody can be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques.

An alternative method of obtaining an anti-phosphoprotein monoclonal antibody or fragment thereof for use in the present invention is to screen commercially available cell lines (e.g., ATCC cell lines) which generate monoclonal antibodies directed against phosphate groups. Such antibodies can be screened for specific recognition of autoantigens phosphorylated during apoptosis, such as ppl7, pp23, pp34, pp42, ρp46, pp54, and pp200, using standard assays such as Western blotting. In this assay, proteins in extracts from apoptotic and non-apoptotic cells are separated by PAGE, transferred to a blotting surface and then screened with radiolabeled antibody as will be familiar to one of ordinary skill in the art.

In another embodiment of the invention, an in vitro kinase activity assay is used to detect in the biological sample of an individual suspected of having or being at risk for an autoimmune disease an antibody specific for one or more protein kinases, preferably active protein kinases, or protein complexes containing an active protein kinase, which are present in apoptotic but not non-apoptotic cells. Such protein kinases may be recognized directly by antibodies from the individual's serum, or may be recognized indirectly in the form of a protein complex (e.g., kinase-substrate complex) which can be immunoprecipitated using the individual's serum and then tested for kinase activity. In the latter situation, the antibodies may, for example, recognize a substrate which is bound to the protein kinase.

Screening a patient's biological sample for antibodies (e.g., IgG, IgM and IgA) which recognize protein kinases or protein kinase complexes can be accomplished using any of the screening assays previously discussed for identifying antibodies specific for phosphoproteins (e.g., ELISA, RIA, Western Blot). An in vitro kinase activity can then be used as described below to compare the difference in kinase activity between immunoprecipitates from apoptotic and non-apoptotic cell extracts. By comparing this difference, it can be determined whether the patient has developed antibodies which recognize active protein kinases, or complexes containing active protein kinases, which are present in apoptotic but not non-apoptotic cells. It is believed that such kinases are involved in phosphorylation of autoantigens during apoptosis and, therefore, their identification provides both diagnostic value and therapeutic value in that the activity of such kinases can be inhibited using a variety of kinase inhibitors known in the art.

Accordingly, in one embodiment, the invention provides a method for diagnosing an autoimmune disease in an individual which involves collecting a biological sample from the individual which is contacted separately (e.g., divided, for example, on a microtiter plate and tested side by side) with an extract from human apoptotic cells and an extract from human non-apoptotic cells under conditions which promote binding of proteins in the extracts by antibodies the samples. The identical procedures as were previously described above can be used to (a) bind immunoglobulins from the patient's serum onto a solid substrate, such a microtiter plate, (b) remove unbound proteins, (c) incubate apoptotic and non-apoptotic cell extracts on the plate so that antibody-protein complexes are formed, and (d) remove unbound proteins, leaving only proteins which were recognized and bound by the patient's antibodies (e.g., immunoglobulins). To test the immunoprecipitates for the presence of one or more active protein kinases, a variety of standard in vitro kinase activity assays can be employed. For example, a kinase buffer containing labeled ATP can be incubated on the plate containing the antibody-protein complexes. The reaction can then be terminated (e.g., by addition of buffer and boiling for approximately 5 minutes), proteins separated on an SDS-PAGE gel and transferred to a solid support such as polyvinylidene difluoride (PVDF). The presence of one or more active kinases can then be determined by assaying the separated proteins for labeled substrate (e.g., phosphoprotein) or labeled ADP (released from ATP) using a variety of detection methods well known in the art, such as colorimetric, radioactive, or fluorometric assays.

The kinase activity precipitated with the patient's biological sample from apoptotic cell extracts can then be qualitatively and quantitatively compared to the kinase activity precipitated with the patient's biological sample from non-apoptotic cell extracts. A difference in such activity can be attributed to the recognition by the patient's serum of active protein kinases or complexes containing active protein kinases which are present in apoptotic but not apoptotic cells. As shown in the exemplification below, such active protein kinases which are selectively present in dying, apoptotic cells can be linked to and, therefore, are likely involved in the phosphorylation of autoantigens against which pathogenic autoantibodies are generated in patient's suffering from autoimmune diseases.

An alternative way of assaying for active protein kinases present in apoptotic but not non-apoptotic cells which are recognized by serum from patient's is to use a highly specific labeled antibody, such as a monoclonal antibody or fragment thereof. This can be accomplished in a number of different ways as described above, including ELISA, sandwich ELISA, RIA or Western blots using a monoclonal antibody or antibody fragment directed against a protein kinase. Such antibodies can be generated using the methods previously described (and also described, for example, in US 4,942,131 and US 5,583,053). Protein for use in generating such antibodies can be obtained from immunoprecipitates from, for example, apoptotic cell extracts obtained as described above. Alternatively, cell lines which produce antibodies directed against particular protein kinases can be purchased commercially from sources such as the American Type Culture Collection (ATCC).

In a particular embodiment of the invention, antibodies (e.g., monoclonals) specific for kinases which phosphorylate SR splicing factors associated with Ul-snRNP complex, such as SRp54, SRp40, ASF/SF2, SC35, U2AF35, and SRp20, are used to assay for the presence of an active protein kinase in the screening assays described above. Such SR kinases are known in the art and include, for example, the Ul-70 kD snRNP-associated serine kinase (Woppmann et al. (1993) Nucleic Acids Res. 21:2815), the SR protein kinase- 1 (SRPK-1) which is capable of phosphorylating multiple different SR proteins on serine residues (Gui et al. (1994) Nature 169:678; Gui et al. (1994) PNAS 91:10824; Colwill et al. (1996) J. Biol. Chem. 271:24569), the Clk/Sty protein kinase (Colwill et al. (1996) J. Biol. Chem. 271:24569; Melcher et al. (1996) J. Biol. Chem. 271:29958), the nuclear envelope associated kinase (Nikolakaki et al. (1996) J. Biol. Chem. 271:8365), and mammalian DNA topoisomerase I (Rossi et al. (1996) Nαtwre 381 :80-82).

In yet another aspect, the present invention provides an assay kit for diagnosing an autoimmune disease in an individual. The kit contains all of the necessary materials and biological reagents for performing the diagnostic methods of the invention as described herein. These materials and reagents may include a solid support, such as a suitable microtiter plate (e.g., one or more 96 well ELISA plates) which is suitable for adsorbing immunoglobulins from human serum (e.g., IgG, IgA, IgM). For example, the solid support may be treated with protein A which anchors immunoglobulins onto the plate in the correct orientation. In addition, the kit may contain extracts from both apoptotic and non-apoptotic human cell cultures. Such extracts are preferably frozen for suitable preservation. The kit may further contain an antibody, such as a monoclonal antibody, an antibody fragment, or a combination of antibodies, which specifically recognize phosphoproteins. Preferred antibodies for inclusion in the assay kit are labeled anti-phosphoserine antibodies which can be obtained from sources or generated by methods previously described herein. The assay kit can also optionally include the necessary reagents for performing an in vitro kinase assay, such as a kinase buffer (e.g., 20 mM Tris, pH 7.6, 10 mM MgCl2) and labeled ATP (e.g., gamma-labeled ATP), as well as a solution for washing unbound proteins from the solid support (e.g., a TBS solution containing 150 mM ΝaCl, 20 mM Tris, pH 7.6.

As previously described, the diagnostic methods and materials of the present invention can be used to determine in a patient having or suspected of having an autoimmune disease the presence of autoantibodies against one or more phosphorylated autoantigens which are found in apoptotic cells but not in non-apoptotic cells.

Alternatively, the patient may be diagnosed by determining the presence of one or more protein kinases which are active in apoptotic cells but not in non-apoptotic cells, and which may be involved in phosphorylation of autoantigens during cell death. Identification of such phosphoproteins and kinases can be used in the selection of appropriate therapies for patients suffering from or at risk for autoimmune diseases. In addition, identification of such phosphoproteins and kinases can also be used in assays to screen, for example, chemical or peptide libraries for effective agents which inhibit the phosphorylation of such phosphoproteins and/or the activity of such kinases.

Accordingly, in still another aspect, the present invention provides a method for treating or preventing an autoimmune disease in an individual by first diagnosing the individual using the methods described herein, followed by treating the individual with one or more protein kinase inhibitors. In patients already exhibiting symptoms of autoimmune disease, the method can be used to treat the disease symptoms. In patients who are suspected of having, or who are at high risk of having an autoimmune disease, the method can be used prophylactically to prevent onset of the disease. In one embodiment, a patient who has or is at risk for having an autoimmune disease is diagnosed in accordance with the procedures described herein for the presence of antibodies specific for one or more phosphoproteins (e.g., ppl7, pp23, pp34, pp42, pp46, pp54, pp200, SRp54, SRp40, ASF/SF2, SC35, U2AF35, and SRp20) or active protein kinases (e.g., SR kinases, such as SRPK-1, Clk/Sty, DNA topoisomerase I, nuclear envelope-associated kinase and Ul-70 kD-associated kinase) which are present in extracts from human apoptotic cells but not in extracts from human non-apoptotic cells. Methods for such diagnosis are described in detail above and so shall not be repeated here. Once the presence of such autoantibodies is determined using, for example, a sandwich ELISA assay, then the individual can be administered a protein kinase inhibitor to inhibit or prevent phosphorylation of autoantigens and/or activity of kinases involved in the generation of such autoantibodies and the consequent manifestations of the individual's disease phenotype.

The protein kinase inhibitor can be any of a variety of kinase inhibitors, either synthetically or genetically produced, which are known in the art. Such inhibitors are described, for example, in US 5,215,888 and in US 5,496,720, the disclosures of which are incorporated by reference herein. Suitable protein kinase inhibitors include, but are not limited to, those which inhibit stress-activated protein kinases (SAP kinases), serine/threonine kinases, protein kinase C, FAST kinase, and cAMP-dependent protein kinase. Other suitable protein kinase inhibitors include those which inhibit activity of SR kinases (i.e., kinases which phosphorylate SR splicing factors), such as SRPK-1 , Clk/Sty, DNA topoisomerase I, nuclear envelope-associated kinase and Ul-70 kD- associated kinase.

Alternatively, in accordance with the methods described below, new kinase inhibitors not previously known in the art may be screened for and used therapeutically or prophylactically in patients diagnosed with autoimmune disease, or diagnosed as being at risk for autoimmune disease as defined herein. For example, the kinase inhibitor can be an antibody or antibody fragment which blocks the active site of a kinase identified according to the methods of the invention, or a kinase involved in phosphorylation of a phosphoprotein identified according to the methods of the invention. Such antibodies may include anti-phosphovitronectin, anti-PKA (cAMP- dependent protein kinase) or anti-vitronectin antibodies as described in US 5,215,888. Alternatively, the kinase inhibitor may be a peptide, such as an octapeptide, which competes with substrate for binding to the active site of the kinase, thereby preventing phosphorylation of the substrate. The generation and selection of such peptides is also described in US 5,215,888.

In a preferred embodiment, the kinase inhibitor is a serine/threonine kinase inhibitor. These particular kinase inhibitors are described, for example, in US 5,496,720 and include, but are not limited to, 6-dimethylaminopurine (DMAP), staurosporine, 2- aminopurine, and sphingosine. The use of such kinase inhibitors can be directed in particular at inhibiting or preventing phosphorylation of phosphoproteins ppl7, pp23, pp34, pp42, pp46, pp54, pp200 and SR splicing factors (e.g., SRp54, SRp40, ASF/SF2, SC35, U2AF35, SRp20). As described in the examples below, these phosphoproteins are generally phosphorylated on serine residues. Thus, preferred serine/threonine kinase inhibitors of the invention are characterized as preventing the phosphorylation of one or more of these seven phosphoproteins as can be determined using immunoprecipitation and screening assays (e.g., Western blot, ELISA or RIA) such as those described below. To screen protein kinase inhibitors which may have therapeutic and/or prophylactic value in patients diagnosed as having or being at risk for autoimmune disease, a variety of screening assays may be employed. For example, in one embodiment, a chemical or peptide library is screened which contains approximately 10- 100 different compounds. Such libraries may be purchased commercially and screened. In one embodiment, human cells are cultured and plated into two 96 well ELISA plates at a density of approximately 2 million cells/well. In 95 of the 96 wells, a test inhibitor from the chemical or peptide library is added, leaving one well as a control. All cells on one of the two ELISA plates are then exposed to an apoptotic trigger, such as UV light, gamma irradiation or Fas-ligand. Lysates from each plate are then prepared and immunoprecipitated using human serum and tested for kinase inhibition, for example, by (a) screening for the presence of phosphorylated proteins such as ppl7, pp23, pp34, pp42, pp46, pp54, and pp200 (using e.g., Western blot analysis as described in the examples below) and comparing the patterns for apoptotic and non-apoptotic cell extracts; or (b) performing an in vitro protein kinase activity assay on the immunoprecipitates (e.g., as described in the examples below) and comparing the kinase activity for the apoptotic and non-apoptotic cell extracts. In either scenario (a) or (b), each well is compared with control for kinase inhibition and those wells showing inhibition may be selected for further testing. It will be expected that extracts from wells on the non-apoptotic cell plate which correspond to wells showing kinase inhibition from the apoptotic cell plate will not show similar inhibition, since the active protein kinases being inhibited in the apoptotic cell extracts would not be expected to be present in the non-apoptotic cell extracts.

The kinase inhibitor is preferably administered to the patient as a composition along with a pharmaceutically acceptable carrier or diluent. The term "pharmaceutically acceptable carrier or diluent" is intended to include any biologically compatible vehicle which does not reduce the activity of the kinase inhibitor and which is physiologically tolerable to the patient. Such agents include a variety of solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. 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 therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Administration of a kinase inhibitor of the invention as described herein can be in any pharmacological form including a therapeutically active amount of kinase inhibitor alone or in combination with another therapeutic molecule (e.g., one or more other kinase inhibitors or an immunosuppressant suitable for treating an autoimmune disease). Administration of a therapeutically active amount of the therapeutic compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired (e.g., reduction of phosphorylation of autoantigens in vivo during apoptosis). For example, a therapeutically active amount of an kinase inhibitor may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

The kinase inhibitor may be administered in a convenient manner such as by injection (subcutaneous, intravenous, intraarticular etc.), oral administration, inhalation, or transdermal application. Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. For example, a kinase inhibitor may be administered to an individual in an appropriate carrier or diluent, co- administered with enzyme inhibitors or in an appropriate carrier such as liposomes.

Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et al., (1984) J Neuroimmunol 7:27). The kinase inhibitor may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

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. In all cases, the composition must be sterile and must 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 polyetheylerie 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, asorbic 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 kinase inhibitor 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 which 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, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (e.g., antibody) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

It is especially advantageous to formulate 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 particular individual 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 (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

The protein kinase inhibitor should be administered for a sufficient time period to alleviate the undesired clinical autoimmune symptoms in a patient and/or to inhibit the undesired molecular autoimmune events which have been shown to occur in the patient (e.g., phosphorylation of autoantigens during apoptosis and the clinical signs associated with the condition being treated). The concentration of active compound in the drug composition will depend on absorption, inactivation, and other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time. A typical daily dose of kinase inhibitor for all of the herein-described conditions is between 0.1 milligrams and 120 grams. The active compounds can be applied in any effective concentration, usually varying between 0.001 % and 100 %> (all percentages are by weight). Alternatively, an effective concentration range is between 0.01 % and 50 %. Alternatively, an effective concentration range is between 1 % and 25 %>. In yet another embodiment, the invention provides a method for altering RNA splicing patterns within a cell by modifying phosphorylation of U-snRNPs and splicing factors which associate with U-snRNPs (e.g., SR proteins) in the cell. The method can be used, for example, to render a cell more or less susceptible to an apoptotic stimulus (e.g., UV or gamma irradiation or chemotherapeutic agents). Specifically, by altering a particular splicing pattern within a cell (e.g., changing the cell's ability to select alternative splice sites), the cell can be induced to produce only selected transcripts which render the cell more or less susceptible to an apoptotic stimulus (e.g., UV or gamma irradiation or chemotherapeutic agents). Thus, the method can be used, for example, to render cancer cells more susceptible to chemotherapy, or to render healthy cells less susceptible to UV or gamma irradiation.

In one embodiment, splicing of pro-survival factors, such as bcl-xL and IchlS, or of proapoptotic factors such as bcl-xS and IchlL, are modified by upregulating or by downregulating SR protein phosphorylation in a cell, thus altering the susceptibility of the cell to an apoptotic trigger. It has been shown that cells expressing the larger splice variant of the bcl-x gene (bcl-xL) are protected against cell death, while cells expressing the short form lacking the highly conserved BH1 and BH2 interaction domains (bcl-xS) have an increased susceptibility to cell death (Boise et al., Curr Top Microbiol. Immunol. 200:107-121; Fang et al. (1994) J. Immunol. 151:4388-4398; Boise et al. (1993) Cell 74:597; Yang et al. (1996) Blood 88:386-401). It has been shown that reversible phosphorylation of SR proteins (e.g. ASF/SF2) can alter their ability to select alternative mRNA splice sites (Ge et al. (1991) Cell 66:373-382, Krainer et al. (1990) Genes and Development 4:1158-1171; Kuo et al. (1991) Science 251 :1045-1050). Accordingly, in one embodiment of the invention, a cell (e.g., a cancer cell) which expresses bcl-xL and is resistant to death by apoptotic triggers (e.g., UV or gamma irradiation or chemotherapy) is induced to express alternative transcripts, such as bcl-xS, by administering to the cell an agent (e.g., drug) which upregulates SR protein phosphorylation (e.g., activates or upregulates activity of an SR protein kinase) or an agent which downregulates SR protein phosphorylation (e.g., downregulates activity of an SR protein kinase inhibitor). By doing so, the cell can be rendered susceptible to apoptotic triggers (e.g., UV radiation or chemotherapy), since the bcl-xS gene product, in contrast to bcl-xL, does not provide cellular resistance to apoptotic triggers. Suitable kinases and kinase inhibitors (e.g., SR serine kinases) for which the activity can be modified include those previously described herein. Administration of drugs which modify kinase activity and kinase inhibitors in a subject (e.g., a human patient or other mammal) can be achieved using methods and guidelines also previously described herein. In addition, the methods and compositions of the invention provide tools for studying the underlying mechanisms and interrelation of cellular apoptosis, de novo protein phosphorylation and related autoimmune diseases. For example, specific antibodies of the invention directed against phosphoproteins, active protein kinases, or complexes thereof, associated with autoimmune diseases can be used to determine whether novel peptide epitopes are generated during apoptosis to which T cells have not been rendered tolerant. This may be useful in studying the phenomenon of "epitope spreading" whereby an immune response to one component of a particle, such as the Ul- snRNP, promotes the formation of antibodies reactive with other components of the particle (Craft et al. (1997) Arthritis Rheum 40:1374-1382), such as caspase-cleaved U 1 -70 kD and/or phosphorylated SR derived peptides.

The invention shall be further described in the following working examples: EXAMPLES Materials and Methods

Cell culture. Jurkat cells were grown in 5% CO2 at 37°C using RPMI 1640 (Bio Whittaker, Walkersville, Maryland) supplemented with 10% Heat-inactivated fetal calf serum (HI-FCS) (Tissue Culture biologicals, Tulare, California) and penicillin and streptomycin (Mediatech, Herndon, Virginia). Cells were grown and harvested at mid- log phase. Jurkat T cells overexpressing bcl-2 (or empty vector), a kind gift from John Reed, were grown in RPMI medium as described above, supplemented with G418 (Gibco, Grand Island, New York) at a final concentration of 500 μg/ml. Protein overexpression was confirmed by Western blotting prior to each experiment.

Metabolic Labeling. Jurkat cells were incubated at a density of 2 x 10 > cells/ml in labeling medium containing the following 45%> RPMI 1640, 45% RPMI 1640 lacking either phosphate (Gibco, Grand Island, New York), or methionine and cystein (Gibco, Grand Island, New York), 2 mM glutamine (Mediatech, Herndon, Virginia), 5% HI- FCS, and 5% HI-FCS that had been dialyzed to equilibrium against 10 mM Hepes buffer (Sigma, St. Louis, Missouri). 2p_ι_ab_eι_e(j orthorphosphate or 35s-labeled methionine and cysteine (Dupont, New England Nuclear (NEN), Boston MA) was added at a concentration of 0.1 mCi/ml. Cells were incubated at 37°C for 10-12 hours to allow the cells to reach steady-state before each treatment, unless otherwise indicated. For 2D tryptic phosphopeptide mapping experiments, cells were labeled for 2 hours, followed by a 3 hour stimulation with anti-Fas antibodies (7C11) in labeling media composed of 90%> RPMI 1640 lacking phosphate, 2 mM glutamine, 10% dialyzed HI-FCS, and 0.15 mCi/ml32p orthophosphate.

Cell Lysis. Lysis of cells was performed using Nonidet P40 (NP40) (Sigma, St. Louis, Missouri) lysis buffer (1% NP40, 150mM NaCl, 50mM Tris pH 7.8, 1 mM EDTA). NP40 lysis buffer was supplemented immediately before use with 1 mM sodium vandate (Sigma, St. Louis, Missouri) and a 100X protease inhibitor cocktail prepared by dissolving 10 mg chymostatin, 1.5 mg leupeptin, 7 mg pepstatin A, 850 mg phenylmethylsulfonyl fluoride, 500 mg benzamidine, and 5 mg aprotonin in 50 ml of ethanol by stirring overnight. The solution was sterilized by filtration and stored at room temperature (Karwan et al. (1991) Genes Dev. 5:1264-1276). All chemicals were purchased form Sigma (St. Louis, Missouri). After addition of 1 ml lysis buffer, the lysate was incubated on ice for 30 minutes, centrifuged in a refrigerated Eppendorf 5402 microfuge (Eppendorf, Hamburg, Germany) at 14000 rpm for 15 minutes, and the supernatant used immediately for each experiment.

Ultraviolet (UV) Irradiation. Labeled Jurkat cells were plated on 100 x 15 mm polystyrene petri dishes (Nunc, Thousand Oaks, California) at a concentration of 2 x 10°^" cells/ml and irradiated in a Stratalinker 2400 (stratagene, La Jolla, California) at a distance of 9 cm for 12 seconds. After irradiation, cells were incubated at 37°C for the indicated times prior to harvesting.

Gamma Irradiation. Labeled cells were placed in a 50 ml conical tube and irradiated at a dose of 300 rad from a Cesium 137 source using a Gammacell 1000 irradiator (Atomic Energy of Canada, Limited). After irradiation, cells were placed in culture dishes at 37°C and incubated for the indicated times prior to harvesting.

Cellular Activation. Labeled Jurkat cells were treated with the following antibodies: anti-Fas antibody 7C11 (kindly provided by Michael Robertson) from hybridoma supernatant at a final dilution of 1 :500; anti-CD3 antibody (Coulter

Immunology, Hialeah, Florida) at a concentration of 5 μg/ml followed by goat anti- mouse antibody (Jackson Immunologic Labs, West Grove, Pennsylvania) at the same concentration. Cells were incubated at 37°C for the indicated times prior to harvesting. Immunoprecipitation and Western blot analysis. Lysates were precleared once with 25 μl of a 50%> solution of Protein A Sepharose (Pharmacia Biotech, Uppsala, Sweden) in phosphate buffered saline (PBS) and 5 μg Rabbit anti-mouse (RAM) IgG (Jackson Immunologic Labs, West Grove, Pennsylvania) for 1 hour, followed by two preclears with Protein A Sepharose overnight. Mouse monoclonal antibodies (5 μg) and 5 μg RAM, or 3.5-5 μl patient serum alone were used in precipitation experiments. Immunoprecipitation experiments using anti-Ul A human antibody fragments were performed as described in de Wildt et al. (1996) Eur. J. Immunol. 26:629. Human polyclonal antibodies were obtained from the following sources and stored at -70°C until used: Arthritis Foundation/CDC Reference Sera, Atlanta, Georgia: anti-Ro, anti-La, anti-Sm, anti-Jo-1, anti-nucleolar, anti-centromere, anti-Scl-70, anti-DNA, and anti-Ul RNP; T. Medsger and N. Fertig, University of Pittsburgh School of Medicine,

Pittsburgh, Pennsylvania: anti-Th/To, anti-U3-fibrillarin, anti-SRP, anti-PL-7, and anti- PL-12; J. Craft, Yale University School of Medicine, New Haven, Connecticut: two different anti-Ul/U2 monospecific sera (Sera Ya and Ga) (22), and anti-SRP; E. Tan and C. Casiano, The Scripps Institute, La Jolla, California: anti-NuMA (serum AS), and anti-UBF (serum JO); M. Kuwana, Keio University Medical School, Tokyo, Japan: anti-RNA polymerase I/III and II (serum KA), anti-polymerase I,III (serum IM), anti- Th To, anti-U3-fibrillarin, anti-Ku, and anti-Scl-70; Immunovision Inc., Springdale, Arizona: anti-ribosomal P and anti-histone/U-RNP; A. Rosen, The Johns Hopkins University School of Medicine, Baltimore, Maryland: anti-Ul-70 kD snRNP protein; D. Bloch, Massachusetts General Hospital, Boston, Massachusetts: anti-spl40, and anti- splOO; W. van Venrooij, University of Nijmegen, Nijmegen, The Netherlands: 7 different antibodies specific for the Ul-snRNP complex (B83, B152, B175, H34, H165, K4, and L41), and a control serum specific for both UI and U2-snRNPs (V26) (Sillekens et al. (1989) Nucleic Acids Res. 17:1893-1906). Serum from patients with SLE and MCTD with high titers of antibodies against Sm and RNP components, respectively, were generously provided by P.H. Schur, Brigham and Women's Hospital, Boston, Massachusetts. Mouse monoclonal antisera were obtained from the following sources and stored at -70°C until used: E.G. Nigg, University of Geneva, Geneva, Switzerland: anti-lamin B (E3), and anti-lamin A+B (E6); Calbiochem, San Diego, California, anti- lamin B; Zymed Laboratories, Inc., South San Francisco, California: anti-PCNA; D. Weaver, Dana Farber Cancer Institute, Boston, Massachusetts: anti-DNA-PK; C. Zhang, Dana Farber Cancer Institute, Boston, Massachusetts: two monoclonal anti-Ku antibodies; D. Bloch, Massachusetts General Hospital, Boston, Massachusetts: anti- Ki67; S. Hoch, the Agouron Institute, La Jolla, California: anti-Ul -70 kd (Billings et al. (1982) J. Immunol. 128:1176-1180); J. Craft, Yale University School of Medicine, New Haven, Connecticut: anti-Sm Y16; W. van Venrooij, University of Nijmegen, Nijmegen, The Netherlands: anti-Ul A/U2B" 9A9 (Habets et al. (1989) J. Immunol.

141:2560), anti-U2B" 4G3 (Habets et al. (1989) J Immunol. 141:2560), anti-Ro60 2G10 (Veldhoven et al. (1995) Clin. Exp. Immunol. lp_l:45), anti-Ro52, and anti-La SW5 (Pruijn et al. (1995) Europ. J. Biochem. 232:611); R. Reed, Harvard University School of Medicine, Boston, Massachusetts: mAb 104 monoclonal directed against SR proteins (Roth et al. (1990) J. Cell Biol. 111:2217); Sigma Company, Inc., St. Louis, Missouri: anti-SC35; K.M. Pollard, Scripps Institute, La Jolla, California: anti-fibrillarin monoclonal antibodies (72B9.D31 and 17C12.G9) and 2 monoclonal antibodies directed against other U3-snRNP components (7G3.B7 and 6G10.D3). Serum from all Brigham and Women's Hospital Arthritis Center patients who had a serum sample submitted to the Brigham and Women's Hospital Clinical Immunology Laboratory over an 8 month period was collected and stored at -20°C until used. Serum from healthy control patients was a gift from P. Fraser. Diagnoses and serum characterization were confirmed by chart review. Immunoprecipitations were performed after addition of 5% bovine serium albumin (BSA) (Intergen Company, Purchase, New York) in PBS to a total volume of 500 μl, and rotation in a 4°C cold room for 2-24 hours. Comparison of precipitates showed no difference between incubation times for periods of up to 72 hours. Precipitates were harvested by centrifuging for 15 seconds at 14000 rpm in a refrigerated Eppendorf microfuges, washing 3 times with NP40 lysis buffer supplemented with the protease inhibitor cocktail, resuspending in SDS loading buffer with 9% 2- mercaptoethanol, boiling for 5 minutes, and electrophoresing on SDS polytacrylamide geles as described (Laemmli et al. (1970) Nature 227:680-685). Proteins were transferred to nitrocellulose (Schleicher and Scheull, Keene, New Hampshire) for Western blotting experiments or to Polyvinylidene difluoride (PVDF), (Dupont, New England Nuclear, NEN, Boston, MA) for phosphoaminoacid analysis, and either exposed for autoradiography or subjected to Western blot analysis as indicated (Harlow et al.(1988) Immunoblotting. In Antibodies: A laboratory manual. Cold Spring Harbor Laboratory Publications, Cold Spring Harbor, N.Y. 474-510.). The mouse monoclonal antibody 4D7, anti-bcl-2 (Pharmingen, San Diego, California) was used for blotting studies at a dilution of 1 : 1000. Nitrocellulose blots were blocked with 3% BSA in PBS overnight at 4°C. Bands were visualized using RAM conjugated to horse-radish peroxidase (Amersham, Arlington Heights, Illinois) at a dilution of 1 :7500 in 1% BSA in PBS, and developed using ECL chemiluminescence performed according to the manufacturer's instructions (Amersham, Arlington Heights, Illinois).

Phosphoaminoacid analysis. Immunoprecipitates that had been electrophoresed and transferred to PVDF were rinsed thoroughly with water, exposed for radiography, and appropriate bands excised with a razor blade. The radiolabeled bands were then subjected to acid hydrolysis as described (Coligan et al. (1994) Curr. Prot. Immunol. 1:11.2.1-11.2.8) with the exception that two-dimensional electropheresis was performed at 14°C rather than at 4°C.

DNA Fragmentation. Unlabeled Jurkat cells were induced to undergo apoptosis using the above triggers in parallel experiments to those using radiolabelled cells. Cells were collected at the indicated times and centrifuged for 5 minutes at 1000 rpm. The cell pellet was lysed by adding 500 μL DNA Lysis Buffer (20 mM Tris, pH 7.4, 5 mM EDTA, and 0.4%> Triton X-100) and incubating on ice for 15 minutes, mixing several times. After centrifuging at 4°C, 14000 rpm for 5 minutes, supernatants were extracted with a 25 phenol: 24 chloroform: 1 isoamyl alcohol mixture (Gibco, Grand Island, New York). Next, 100 μl 5 M NaCl and 500 μl isopropanol were added to each tube prior to incubating overnight at -70°C. Samples were thawed and centrifuged at 14000 m for 5 minutes, washed once with 70% enthanol, and dried in a Speed-Vac. Pellets were resuspended in 30 μl of Tris-EDTA buffer containing 0.1 mg/ml RNase A (Sigma, St. Louis, Missouri) and incubated at 37°C for 30 minutes. After the addition of 10 μl loading buffer, 10 μl of each sample, corresponding to 1 million cells per lane, was separated on 0.8% agarose gels and visualized by ethidium bromide staining under UV light.

In vitro kinase assays. Individual immunoprecipitates were washed three times in NP40 lysis buffer, then once with TBS (150 mM NaCl, 20 mM Tris, pH 7.6) before resuspending in 30 μl kinase buffer (20 mM Tris, pH 7.6, 10 mM MgCl2, and

20 μCi[32p] _gamma ATP (Dupont, NEN, Boston, Massachusetts, 150 mCi/ml)) for 30 minutes at 30°C. The reactions were terminated by addition of sample buffer and boiling for 5 minutes. Proteins were separated on an SDS-PAGE gel prior to transfer to PVDF and autoradiography for 2-5 minutes (Tian et al. (1995) J Exp. Med. 182: 865- 874).

RNA Isolation and Identification: Immunoprecipitates from 32p_i_abeled Jurkat cells were prepared as described above. Following the third NP-40 lysis buffer wash, the immunoprecipitate was digested in a volume of 300 μl for one hour at 37°C in a solution containing 50 μg/ml proteinase K (Sigma Chemical Co.), 10 mM Tris pH 7.8, 10 mM EDTA, and 0.5% SDS. The RNA was isolated following two extractions with a 25 phenol: 24 chloroform: 1 isoamyl alcohol mixture (Gibco, Grand Island, New York). The RNA was precipitated overnight at -70°C following the addition of 20 μl 3M sodium acetate, 400 μl ethanol, and 10 μg transfer RNA (Sigma Chemical Co.) as a carrier. The pellet was obtained following a 15 minute centrifugation in an Eppendorf centrifuge maintained at 4°C. The pellet was washed once with 70% ethanol, dried in a fume hood, and subjected to PAGE on 6% sequencing gels. A small amount of whole cell lysate was also processed as above and included as an internal standard on each gel. Two Dimensional Phosphopeptide Analysis: Two dimensional tryptic phosphopeptide mapping was performed as described (Medley et al. (1996) PNAS 93:685) using trypsin (Worthington Biochemical Corporation, Freehold, New Jersey) at a concentration of 0.1 mg/ml in 50 mM ammonium bicarbonate. Plates were exposed to film at -70°C with an intensifying screen for 2 days.

Autoimmune Sera Recognize Proteins Phosphorylated During Stress-Induced Apoptosis

Serum from 12 random patients with positive tests for antinuclear antibodies (ANA, defined as > 1 :20 titer on immunofluorescence staining using Hep2 cells as a substrate), as well as serum from 10 healthy control patients, 5 rheumatoid arthritis patients, and 15 patients with diseases considered to be unassociated with autoantibodies (including fibrositis, tendonitis, bursitis, chronic fatigue syndrome, carpal tunnel syndrome, and osteoarthritis), were chosen from the sera collected as described above in Materials and Methods. All patients with a positive ANA test were further screened by ELISA for antibodies against DNA, the Smith complex, Ro, La and ribonucleoprotein (RNP), and the patients' charges were reviewed to obtain clinical data sufficient to establish a diagnosis (von Muhlen et al. (1995) Sem. in. Arthritis Rheum. 24: 323-258).

As summarized in Table 1 below, most patients (10/12) met published criteria for either SLE or lupus in association with a second inflammatory condition (referred to as SLE overlap syndrome) (Tan et al. (1982) Arthritis Rheum. 25: 1272-1277). Other conditions were also represented, included Sjδgrens disease (SD) (patient 6), and undifferentiated connective tissue disease (UCTD) (patient 9). One patient with RA (patient 13) and a patient with fibrositis (patient 14) are also presented for comparison.

Individual patient sera are identified by the numbers above each column. Abbreviations used are as follows: ANA, antinuclear antibody titer; Pattern, immunofluorescence staining pattern using Hep 2 cells as substrate (P, peripheral; D, diffuse or homogeneous; C, cytoplasmic; N, homogenous nuclear; S, speckled; Nu, nucleolar); RF, rheumatoid factor; Ro, RNA binding protein Ro; La, RNA binding protein La; Sm, Smith antigen; dsDNA, double-stranded DNA; ssDNA, single- stranded DNA; RNP, ribonuclear protein; APLA, antiphospholipid antibody, determined by anticardiohpin ELISA assay; Comp, complement determined by CH 50 assay. Test results are labeled as positive (+); negative (-); normal (NI); not done (ND); increased (t); or decreased (I). Diseases are abbreviated as follows: SLE, systemic lupus erythematosus; Over, SLE overlap syndrome; SD, Sjogren's syndrome; UCTD, undifferentiated connective tissue disease; RA, rheumatoid arthritis; Fib, fibrositis. The relative migration of phosphoproteins precipitated using sera derived from individual patients (derived from Figure 1 A) are as indicated.

Jurkat cells metabolically labeled with 32p_₀rthorphosphate were cultured for 2.5 hours in the absence or presence of a monoclonal antibody reactive with Fas (anti- 7C11), solubilized in NP40 lysis buffer, and immunoprecipitated using the polyclonal autoimmune or control sera indicated in Figure 1A. Immunoprecipitates were separated on 12% SDS polyacrylamide gel, transferred to nitrocellulose, and subjected to autoradiography. Figure 1 A shows that 9/12 ANA+ autoimmune sera, representing 9/10 SLE or SLE overlap patients, precipitated at least one new phosphoprotein from cells undergoing Fas-mediated apoptosis compared to untreated cells. The phosphorylation of these proteins did not result from a nonspecific, general increase in kinase activity following Fas engagement, as 32p_iabeled, whole cell extracts prepared from untreated and apoptotic cells were identical when compared on SDS-PAGE gels. The relative migration of individual phosphoproteins precipitated by several of the patient sera are strikingly similar in profile but variable in intensity of phosphorylation. For example, serum from patients 1, 2, 3, 4, 8, 11, and 12 precipitates a protein of ~54 kDa (pp54) that is weakly phosphorylated in untreated cell lysates and strongly phosphorylated in lysates from apoptotic cells. Similarly, a 34 kDa protein (pp34) was precipitated using serium derived from patients 3, 8, 11 and 12; and a doublet of approximately 42 kDa (pp42) was precipitated using serum derived from patients 3, 8, 11 and 12. None of these phosphoproteins were precipitated using ANA (-) sera derived from patients 13 or 14, nor using sera derived from 12 healthy control patients or 4 additional patients with RA. The level of phosphorylation of pp42, pp34, and ppl7 differed significantly between patients (patients 3, 8, 11 and 12) and was independent of the ANA titer as detected by immunofluorescence (Table 1), suggesting that these phosphoproteins may be novel and independent of the major proteins responsible for the immunofluoresence detectable as an ANA. In addition to the phosphoproteins described above, 3 other new phosphoproteins can be seen as bands migrating at the following positions: 17 kDa doublet (ppl7, patients, 1, 4 5, 8 and 11); 23 kDa (pp23, patients 3, 8 and 11); and 46 kDa (pp46, patient 7). A seventh protein migrating between 96 and 200 kDa (pp200) was observed for patient 1 (Figure 2A-C).

The preferential inclusion of phosphoproteins in precipitates prepared from apoptotic vs. non-apoptotic lysates could result from de novo phosphorylation of autoantigens, increased extractability of the phosphoproteins during the detergent lyss, or recruitment of pre-existing or new phosphoproteins to the autoantigen complex during apoptosis. To differentiate between these three possibilities, the experiment shown in Figure IB was performed using cells that were metabolically labeled with 5s methionine and cysteine in a manner identical to the experiment depicted in Figure 1A, which utilized cells labeled with 32.p orthophosphate. In most cases, immunoprecipitates prepared from apoptotic and non-apoptotic lysates contained similar 35s-labeled proteins. Two exceptions were observed. A 60 kDa protein and a > 200 kDa protein were included in immunoprecipitates prepared from apoptotic, but not non- apoptotic lysates using sera derived from patients 10 (Figure IB, lane 20), and patient 11 (Figure IB, lane 22), respectively (indicated with arrows on the right side of the panel). Although neither of these proteins clearly corresponded to the phosphoproteins identified in Figure 1 A, 35s-labeled proteins (Figure IB) migrating similarly to the phosphoproteins identified in Figure 1 A were observed in all cases. Taken together, these results are most consistent with de novo phosphorylation of autoantigens during apoptosis.

Table 1: Characterization of Autoimmune Sera

To investigate further the nature of the phosphoproteins coprecipitated using polyclonal autoimmune sera (e.g., pp54, pp42, pp34 and pp23), monospecific sera reactive with known autoantigens were tested for their ability also to precipitate these proteins (or complexes) from extracts prepared from 5s-labeled Jurkat cells, as detected by SDS PAGE followed by autoradiographic exposure. Sera that precipitated ambiguous patterns of proteins were subjected to further analysis by Western blotting of both whole cell Jurkat extracts and immunoprecipitates prepared as above, to confirm that the well-characterized monospecific sera precipitated the expected target antigen. Most of the monospecific sera were derived from patients with autoimmune disease. In addition, six murine monoclonal antibodies reactive with known autoantigens

(Ku, DNA-dependent protein kinase (DNA-PK), lamins A and B, Ki67, and proliferating cell nuclear antigen (PCNA)) were also included in this screen.

Jurkat cells metabolically labeled with 32p._or hophosphate were cultured for 3 hours in the absence or presence of a monoclonal antibody reactive with Fas (anti- 7C11), solubilized in NP40 lysis buffer, and immunoprecipitated as described above using the aforementioned monospecific sera or monoclonal antibodies. Immunoprecipitates were separated on a 12% SDS polyacrylamide gel, transferred to nitrocellulose, and subjected to autoradiography. Most sera did not precipitate unique phosphoproteins from apoptotic lysates. For example, sera reactive with nuclear proteins implicated in DNA replication, binding, or repair (i.e., Ku, DNA-PK, PCNA, Scl-70, and histone) failed to reproducibly precipitate new phosphoproteins from apoptotic Jurkat cell extracts (data not shown). Similarly, proteins present in the nuclear matrix or involved in mitosis (i.e., lamins A and B, centromere A and B, Ki-67, NuMA, spl40, and splOO), the nucleolus (i.e., Th/To, UBF/NOR-90, RNA polymerase I, II, and III, and U3-snRNPs), or cytoplasmic components of the translational apparatus (i.e., Jo- 1, Pl-7, PI- 12, signal recognition particle (SRP), and ribosomal P) were unmodified in these experiments (data not shown).

In contrast, several of the monospecific sera specific for U-snRNP complexes precipitated phosphoproteins of 54, 42, 34, and 23 kD from apoptotic Jurkat cell extracts (Figure 7A). It was confirmed that the phosphorylation of these proteins did not result from a nonspecific, general increase in kinase activity following Fas engagement, as 32p-labeled, whole cell extracts prepared from untreated and apoptotic cells were identical when analyzed by SDS PAGE. Moreover, this pattern was similar to that observed using sera from patients 1, 8, 11, and 12 described above capable of precipitating phosphoproteins pp54, pp42, pp34, and pp23. These results strongly indicate that these 4 proteins (pp54, pp42, pp34, and pp23) are previously unrecognized components of U-snRNP complexes. As shown herein, the constitutive phosphorylation of La (Figure 7 A, Ro and La panels) is unaltered in cells undergoing apoptosis.

Autoimmune Sera Co-Precipitate U-snRNP Proteins Immunoprecipitates were prepared from 35s-labeled lysates prepared before

(-) or 3 hours after (+) Fas ligation using the five monospecific sera shown in Figure 7B. Although immunoprecipitates prepared from apoptotic and non-apoptotic lysates contained 35s-labeled proteins migrating similarly to the phosphoproteins identified in Figure 7A, the relationship between the 32p. and 35s-labeled proteins was not able to be established. Nevertheless, the appearance of new 35s-labeled proteins corresponding to components of the phosphoprotein complex was observed. Moreover, all five sera precipitated U-snRNP components (e.g. the A, B, B', and C proteins, indicated by arrows) from labeled Jurkat cell extracts (Figure 7B and Figure 8). Six other sera from patients with SLE and mixed connective tissue disease (MCTD) possessing high titers of antibodies against Sm or RNP components (as determined by ELISA assay), also precipitated U-snRNP components from 3 s-labeled Jurkat cell extracts, as well as all four phosphoproteins from apoptotic cell extracts prepared from 32p_ι_abeled Jurkat cells. These results demonstrate that autoimmune sera capable of precipitating U-snRNP proteins co-precipitate a phosphoprotein complex containing pp54, pp42, pp34, and pp23 from apoptotic Jurkat cell lysates.

Autoimmune Sera Co-Precipitate the Ul-snRNA Molecule and pp54, pp42, pp34, and pp23

To further establish that pp54, pp42, pp34, and pp23 are components of U- snRNP complexes, the five monospecific sera shown in Figure 7B, and the four polyclonal sera shown to precipitate these four phosphoproteins (patients 1, 8, 11, and 12) shown in Figure 1 A, were used along with a control sera to identify the RNA molecules present in individual immunoprecipitates. Jurkat cells metabolically labeled with 32P-orthophosphate were solubilized in NP40 lysis buffer, RNA was extracted from washed immunoprecipitates, separated on a 6% polyacrylamide gel, and subjected to autoradiography. As shown in Figure 8, all sera capable of precipitating the phosphoprotein complex also precipitate the Ul-snRNA. A similar result was obtained for another well-characterized serum previously shown to recognize the Ul-70 kD protein (lane 6, U-serum 6, a gift of A. Rosen) (Casciola-Rosen et al. (1994) J. Biol. Chem. 269(49)30757-30760. One sample (U-serum 2, lane 9) specifically precipitated the UI- but not the U2- snRNA molecule, suggesting that pp54, ρp42, pp34, and pp23 may be components of the Ul-snRNP complex. To confirm this observation, 7 additional monospecific human sera previously shown to specifically precipitate the Ul- snRNP complex and serum V26 (which precipitates both the UI- and U2- snRNPs) (Sillekens et al. (1989) Nucleic Acids Res. 17:1893-1906) were screened for their ability to coprecipitate pp54, pp42, pp34, and pp23 from 32p_l_abeled, apoptotic Jurkat cell extracts.

All seven sera coprecipitated the Ul-snRNA (Figure 9 A) and all four phosphoproteins (Figure 9B). Identical results were obtained using serum V26. None of the control antibodies directed against Ro, La, ribosomal P, or signal recognition particle (SRP) precipitated either the U-snRNAs or any of the four phosphoproteins. Several sera also precipitated phosphoproteins between 96 and 200 kD that were no longer detected following Fas stimulation (e.g., B152, H34, and K4, Figure 9B, lanes 3, 7, and 11). It is unknown if this represents dephosphorylation, caspase cleavage, or dissociation of these phosphoproteins from the immunoprecipitate following the apoptotic stimulus. These results suggest that the Ul-snRNP is a dynamic particle that is altered by caspases (Ul-70 kD protein (Casciola-Rosen et al. (1994) J. Biol. Chem. 269(49)30757-30760)) and potentially by kinases (pp54, pp42, pp34, and pp23) and phosphatases (the high molecular weight protein complex shown in Figure 9B during apoptosis).

Monoclonal Antibodies Directed Against Ul-snRNP Components Precipitate pp54, pp42, pp34, and pp23 From Apoptotic Jurkat Cell Lysates

To confirm the results described above using autoimmune serum, and to determine whether pp54, pp42, pp34, and pp23 exist in both the UI- and U2- snRNP splicing complexes, a series of monoclonal antibodies directed against proteins common or unique to each U-snRNP complex were used. Specifically, antibodies directed against the Ul-70 kD component of the Ul-snRNP, the U2B" protein of the U2-snRNP (4G3), the anti-Sm monoclonal antibody Y16, and a monoclonal antibody (9A9) that recognizes an epitope common to both UI A and U2B", were tested for their ability to precipitate pp54, pp42, pp34, and pp23 from apoptotic extracts. Jurkat cells metabolically labeled with 32p._or hophosphate were cultured for 3 hours in the absence or presence of a monoclonal antibody reactive with Fas (anti- 7C11), solubilized in NP40 lysis buffer, and individually immunoprecipitated using each of these antibodies; control antibodies directed against other RNA binding proteins included monoclonal antibodies against Ro60, Ro52, La, and the anti-TIAR antibody 6E3 (Taupin et al. (1995) PNAS 92:1629-1633) As shown in Figure 10(a), the Smith antibody Y16, and the 9A9 monoclonal antibody specific for the UI A and U2B" proteins (both of which recognize components common to both UI- and U2-snRNPs) precipitate all four phosphoproteins, pp54, pp42, pp34, and pp23, from apoptotic Jurkat cell lysates, as well as pp90 (lanes 2 and 8). These bands are absent in the lanes corresponding to the immunoprecipitation using anti-U2B" (lane 6) and anti-Ul -70 kD monoclonal antibodies (lane 4, see Discussion). Interestingly, increased phosphorylation of a 90 kD protein is observed following Fas stimulation when immunoprecipitates are prepared using anti-U2B" (9A9) antibody (lane 6), and on a short exposure of the lanes corresponding to the U1A/U2B" immunoprecipitate (lanes 7 and 8) suggesting that a specific phosphoprotein (referred to herein as pp90) is associated with the U2-snRNP during apoptosis. Bands corresponding to pp90, pp54, pp42, pp34, and pp23 are absent using monoclonal antibodies directed against TIAR (6E3, lane 10), Ro60 (lane 12), Ro52 (lane 14), La (lane 16), or the putative apoptosis effector TIA-1, another autoantigen that is known to be reversibly phosphorylated during Fas-mediated apoptosis, but at an earlier time point (Tian et al. (1991) Cell 67:629-639; Tian et al. (1995) J Exp. Med. 182:865- 874). The same experiment performed in cells labeled with 5s methionine and cysteine (Figure 10B) demonstrates no difference between immunoprecipitates prepared from apoptotic and nonapoptotic cell extracts, consistent with the results shown in Figure 7B. Phosphoamino acid analysis of all four proteins precipitated using anti-Ul A/U2B" (9A9) demonstrates exclusive phosphorylation of pp54, pp42, pp34, and pp23 on serine residues (Figure 10C).

The failure of the Ul-70 kD monoclonal antibody to precipitate pp54, pp42, pp34, and pp23 from apoptotic lysates appeared to be inconsistent with the hypothesis that these phosphoproteins are specifically associated with the Ul-snRNP during apoptosis. To address this apparent paradox, two previously-described human variable domain antibody fragments directed against a different, unique component of the Ul- snRNP (the UI A protein) were used in immunoprecipitation experiments, as previously described (de Wildt et al. (1996) Eur. J. Immunol. 26:629-639). Both antibodies (Figure 4D, lanes 1-4) coprecipitate a phosphoprotein complex containing pp54, pp42, pp34, and pp23, but not pp90. A control antibody fragment directed against bovine serum albumin precipitates only a faint, nonspecific 60 kD protein (Figure 4D, lanes 5 and 6). Taken together, these results demonstrate an association between a phosphoprotein complex (containing pp54, pp42, pp34, and pp23) and the Ul-snRNP during apoptosis, and suggests that pp90 may be associated specifically with the U2-snRNP during apoptosis.

Phosphorlvation of Autoantigens and Association of pp54, pp42, pp34 and pp23 With U-snRNPs Accompanies Apoptosis, But Not T Cell Receptor Stimulation The results shown in Figure 1 indicate that autoimmune sera (e.g., from patients shown on Table I) preferentially precipitate proteins phosphorylated in response to Fas ligation. To determine whether these proteins are also phosphorylated during apoptosis triggered by stimuli other than Fas ligation, selected patient sera were used to precipitate 32p_iabeled Jurkat lysated prepared from cells subjected to apoptotic stimuli or an activation stimulus for various times.

This kinetic analysis reveals that phosphorylation of autoantigens is induced between 1 and 2.5 hours following Fas ligation (Figure 2A), between 2.5 and 4.5 hours following gamma irradiation (Figure 2B), and between 1 and 2.5 hours following UV irradiation (Figure 2C). Individual autoantisera precipitate a similar cadre of phosphoproteins regardless of the apoptotic trigger. In contrast, ligation of the T cell receptor complex using a monoclonal antibody reactive with CD3, a stimulus that induces IL-2 production and enhances proliferation in these cells, induced neither new protein phosphorylation nor DNA fragmentation over the course of this experiment (Figure 2D and 3D). Control sera derived from an individual without autoimmune disease did not precipitate phosphoproteins from apoptotic lysates nor from lysates prepared from CD3 -stimulated cells (Figure 2A-D, right panels). The kinetics of DNA fragmentation induced by apoptotic or activation stimuli was also determined. As shown in Figure 3A-D, the onset of DNA fragmentation is approximately coincident with the phosphorylation of autoantigens regardless of the apoptotic stimulus.

In addition to the phosphorylation of autoantigens during apoptosis, selected phosphoproteins appear to be rapidly dephosphorylated, and then rephosphorylated in a reproducible manner over the course of the kinetic assay (ppl7 and pp 23; Figure 2B, lanes 1-4). The level of basal phosphorylation of several autoantigens, particularly pp34, pp23 and ppl7 was somewhat variable in each experiment (e.g., patient 1, Figure 2A-D), and appeared to be related to the initial density of the cells at the time of labeling, with less dense, presumably more active cells labeling more uniformly. Overall, these results indicate that, in addition to death induced by Fas ligation, phosphorylated autoantigens are also immunoprecipitated during apoptosis triggered by other stimuli including gamma and ultraviolet (UV) irradiation, but not by T cell receptor stimulation.

To confirm this, the aforementioned experiments were repeated using the anti- Ul A/U2B" (9A9) monoclonal antibody in immunoprecipitation experiments using 32p_ labeled Jurkat lysates prepared from cells subjected to apoptotic stimuli or an activation stimulus over a 5 hour time course (Figure 11). This analysis revealed that phosphorylated autoantigens are precipitated beginning at the 3 hour time point following Fas cross-linking (Figure 5, lanes 1-4) or UV irradiation (Figure 5, lanes 11- 14), and much less intense bands were observed 5 hours following gamma irradiation (Figure 11, lanes 5-7), consistent with the initial results. In contrast, ligation of the T cell receptor complex using a monoclonal antibody reactive with CD3, a stimulus that induced IL-2 production and enhanced proliferation in these cells, induced neither precipitation of phosphoproteins (Figures 11, lanes 8-10) nor DNA fragmentation over the course of this experiment.

Phosphoaminoacid Analysis of Autoantigens

As both tyrosine kinases and serine-threonine kinases have been implicated in signaling Fas-mediated apoptosis (Kyriakis et al. (1994) Nature 169: 156-160; Kyriakis et al. (1996) Bioassays 18: 567-577; Verheij et al. (1996) Nature 180:75-79; Tian et al. (1995) J. Exp. Med. 182: 865-874; Eischen et al. (1994) J. Immun. 153: 947-1954; Lahti et al. (1995) Mol. Cell. Biol. 15:1-11; Migita et al. (1995) Immunology 85:550-555; Xia et al. (1995) Science 270:1326-1331), all 7 phosphoprotein autoantigens described in the sections above and shown on Table I were subjected to phosphoamino acid analysis. In each case, phosphorylation was restricted to serine residues (Figure 4A-G), implicating one or more serine/threonine protein kinases in the phosphorylation of these autoantigens.

Bcl-2 Overexpression Blocks Gamma Irradiation-Induced Apoptosis and Phosphorylation of pp 54, pp46, pp42, pp34 and pp23

The following experiment was performed to determine whether the phosphorylation of pp54, pp46, pp42, pp34 and pp23 could be blocked by overexpression of the bcl-2 protein, which has been shown to efficiently block apoptosis induced by multiple apoptotic stimuli, including gamma irradiation and UV irradiation (Boise et al. (1995) Curr. Top. Micro. Immunol. 200: 107-121 ; Itoh et al. (1993) J

Immunol. 151:621-627; Reed (1994) J. Cell. Biol. 124:1-6; Sentman et al. (1991) Cell 67:879-888). Jurkat T cells stably transformed with either bcl-2 (Figure 6, left panels) or empty vector (Figure 6, right panels) were labeled with p orthophosphate and subjected to Fas ligation, gamma irradiation, or UV irradiation. Cells were solublized at the indicated times. In a first study, the lysates were precipitated using serum derived from patient 7 (Table I). In a second study, lysates were precipitated using the anti- U1A/U2B" (9A9) monoclonal antibody.

As shown in Figure 6B, phosphorylation of pp46 is rapidly induced in Jurkat (neo) control cells in response to gamma-irradiation (Figure 6B, right panel), however pp46 is absent from Jurkat (bcl-2) transformants treated with this same stimulus (Figure 6B, left panel). Qualitatively similar results are seen with UV irradiation (Figure 6C) although a small amount of pp46 is observed in Jurkat (bcl-2) transformants beginning at 4.5 hours. In separate experiments, overexpression of bcl-2 effectively inhibited apoptosis in response to these triggers, as judged by the induction of DNA fragmentation. In contrast, phosphorylation of pp46 following Fas ligation was relatively unaffected by overexpression of bcl-2 (Figure 6A). The induction of DNA fragmentation following Fas ligation was similarly unaffected by overexpression of bcl- 2 in these cells, supporting the correlation between phosphorylation of pp46 and the induction of apoptosis. A similar inhibitory effect of bcl-2 following gamma and UV irradiation but not anti-Fas treatment, on the phosphorylation of pp54, pp34, and ppl7 (Figure 1 A and Table 1) recognized by serum from patient 11 (Table I), was also observed. Taken together, these results demonstrate that the in vivo phosphorylation of all 4 autoantigens that were tested correlated with the induction of apoptosis, and is downstream of the inhibitory effects of bcl-2.

The pattern of immunoprecipitation using 9A9 monoclonal antibody (anti- Ul A/U2B") indicated that, whereas precipitation of phosphorylated pp54, pp42, pp34, and pp23 is rapidly induced in Jurkat (neo) control cells in response to gamma- irradiation (Figure 12, lanes 5-8), the intensity of the bands corresponding to the 4 phosphoproteins is unchanged in Jurkat (bcl-2) transformants treated with this same stimulus (Figure 12, lanes 1-4). Short exposure of the gel in Figure 12 showed that bcl-2 overexpression also inhibited the appearance of pp90. Ectopic expression of bcl-2 effectively inhibited apoptosis in response to gamma irradiation, as judged by the induction of DNA fragmentation. Taken together, these results demonstrate that the coprecipitation of all 5 phosphorylated snRNP factors correlates with the induction of apoptosis, and is downstream of the inhibitory effects of bcl-2.

Monoclonal Antibodies Specific for SR Slicing Factors Precipitate a

Phosphoprotein Complex Containing pp54, pp42, pp34, and pp23 From Apoptotic Jurkat Cell Lysates

The identification of pp54, pp42, pp34, and pp23 as components of the Ul- snRNP complex enabled the search for known phosphoprotein components of these splicing particles. The serine/arginine (SR) family of splicing factors were selected as candidates. Six of the eight most abundant SR proteins are virtually identical in molecular weight to ρp54, pp42, pp34, and pp23 (i.e., SPp55, SRp40, SC35, ASF/SF-2, SRp30, and SRp20), and are phosphorylated on serine residues in vivo and in vitro (Gui et al. (1994) Nature 369:678-682). To test the possibility that pp54, pp42, pp34, and pp23 might be SR proteins, the experiment shown in Figure 13 was performed. Jurkat cells metabolically labeled 32p. orthophosphate were cultured for 3 hours in the absence (-) or presence (+) of a monoclonal antibody reactive with Fas (anti-7Cl 1), solubilized in NP40 lysis buffer, and immunoprecipitated using 9A9 (anti-Ul A/U2B") mAb 104 (a monoclonal antibody specific for the phospho-SR domain of these factors (Roth et al. (1990) J. Cell Biol. 111:2217-2223), or anti-SC35. Labeled proteins were separated by SDS PAGE and transferred to nitrocellulose. Figure 13 A demonstrates that both mAb 104 and anti-SC35 coprecipitate the same phosphoprotein complex from apoptotic lysates. To confirm that the respective proteins precipitated with each antibody are identical, bands corresponding to pp54, pp42, pp34, and pp23 (from immunoprecipitates prepared independently using anti-SC35 and 9A9, lanes 2 and 4, Figure 13 A) were localized by autoradiography, excised, and subjected to two dimensional phosphotryptic mapping. As shown for one of the bands (pp34), the phosphotryptic maps are identical (Figure 13B). Similar results were obtained when comparing phosphopeptide maps corresponding to pp54, pp42, and pp23. Taken together, these results demonstrate that a phosphoprotein complex is selectively associated with the Ul-snRNP complex during apoptosis, and identify members of the SR family of splicing factors as likely components of this phosphoprotein complex.

A Protein Kinase Activity is Precipitated From Apoptotic Lysates Using Selected Patient Sera A cascade of stress-activated serine/threonine kinases has been implicated in signaling apoptotic cell death (16, 17, 19, 25, 31 Kyriakis et al. (1994) Nature 369:156- 160; Kyriakis et al. (1996) Bioassays 18:567-577; Verheij et al. (1996) Nature 380:75- 79; Tian et al. (1995) J. Exp. Med. 182: 865-874; Gjertsen et al. (1995) Biophys. Ada. 1269: 187-199). Individual kinases within this cascade are regulated, in part, by phosphorylation. It is therefore possible that stress activated kinases may be recognized directly by sera derived from patients with autoimmune disease, or may be recruited during apoptosis to preexisting complexes.

To test this possibility, lysates from untreated or anti-Fas treated Jurkat treated cells were precipitated with individual patient sera, and subjected to an in vitro kinase assay as described (Tian et al. (1995), supra.). Five sera were chosen to encompass all seven phosphoproteins that had been identified in the initial screen using in v vo-labeled apoptotic Jurkat cells (Figure 1 A and Table 1). In addition, sera from a healthy control patient and patient 6, whose serum is monospecific for the Ro protein, were included for comparison. Figure 5 A shows that 4/5 ANA+ patient sera (i.e., patients 3, 7, 8 and 11) precipitate a kinase whose activity is increased in apoptotic cell extracts compared to untreated cell extracts. The healthy control patient and patient 6 were devoid of kinase activity in this assay. Phosphoproteins migrating at 34 kDa (lanes 4, 6, 8 and 10), 23 kDa (lanes 4, 6, 8 and 10), and 46 kDa (lane 6) were identified in this assay. The relative migration of these phosphoproteins is similar to that of prominent phosphoproteins identified in the in vivo phosphorylation assay shown in Figure 1 A. The kinetics with which the kinase (precipitated using serum from patient 7) was activated following Fas ligation was correlated with the induction of DNA fragmentation in the experiment shown in Figures 5B. In this experiment, Jurkat cells were cultured in the presence of anti-Fas monoclonal antibodies for the indicated times prior to processing for DNA fragmentation and in vitro kinase activity. The first appearance of pp46 in the in vitro kinase assay was observed at 90 minutes (Figure 5B), while DNA fragmentation was first observed at 120 minutes following Fas ligation. Phosphoaminoacid analysis of pp46 shows that the in vitro phosphorylation of pp46 is restricted to serine residues (Figure 5C) consistent with the in vivo results shown in Figure 4C. A similar kinetic analysis targeting pp34 and pp23 using serum from patient 11 (Figure 5 A, lanes 9 and 10) gave similar results (data not shown). These results are consistent with the less rigorous time courses presented in Figures 2 and 3, and suggest that a serine/threonine kinase activated by Fas stimulation is present in the immunoprecipitates from patients 7 and 11 at a time that precedes the onset of DNA fragmentation.

Conclusions

The results of the foregoing studies suggest that pp54, pp42, pp34, and pp23 are components of the Ul-snRNP. Autoimmune sera from patients 1, 8, 11 and 12 simultaneously precipitate all four phosphoproteins (Figures 7 and 10B) together with the UI RNA (Figures 8 and 10B), from lysates prepared from Fas-treated Jurkat cells. In addition, two different monoclonal antibodies (Y16 and 9A9) that recognize core (Sm) components of the Ul-snRNP complex also precipitate these same four phosphoproteins from extracts prepared from apoptotic Jurkat cells, whereas monoclonal antibodies directed against six other RNA binding proteins do not (Figure 11). Further, two human variable domain antibody fragments directed against overlapping epitopes of the UI A protein coprecipitate pp54, pp42, pp34, and pp23 from apoptotic Jurkat cell extracts (Figure 1 ID). Finally, the anti-UlA/U2B" (9A9) monoclonal antibody precipitates all four phosphoproteins from extracts prepared from cells subjected to multiple different apoptotic stimuli but not following engagement of the T cell receptor, and the association of these phosphoproteins with the U-snRNPs is blocked in cells engineered to overexpress bcl-2 (Figures 12 and 13). Thus, all of the experiments using SLE sera were replicated using the anti-UlA/U2B" (9A9) monoclonal antibody. The foregoing results further suggest that pp90 is a component of the U2-snRNP complex (e.g., U2B"), as it was precipitated from apoptotic cell extracts using both the anti-UlA/U2B" monoclonal antibody and the anti-U2B" monoclonal antibody.

The identification of pp54, pp42, pp34, and pp23 as SR proteins is also indicated by the observation that the respective SR proteins SRp54, SRp42, SC35, SRp30, ASF/SF2, and SRp20 have similar migration patterns on SDS PAGE and are phosphorylated exclusively on serine residues (Gui et al. (1994) Nature 369:678-682). SR proteins also interact with components of the spliceosome and copurify with the Ul-snRNA during gel filtration analysis (Staknis et al. (11994) Molecular and Cellular Biology 14:7670-7682; Staknis et al. (1995) Nucleic Acids Research 21:4081-4086), and sucrose gradient centrifugation. All four proteins (pp54, pp42, pp34, and pp23) comigrate with their respective SR counterparts during 2D gel electrophoresis, and anti-SC35 is capable of coprecipitating the Ul-snRNA. Finally, an identical phosphoprotein complex is precipitated by two monoclonal antibodies specific for the phosphorylated forms of SR proteins (Figure 13).

EQUIVALENTS

Although the invention has been described with reference to its preferred embodiments, other embodiments can achieve the same results. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments described herein. Such equivalents are considered to be within the scope of this invention and are encompassed by the following claims.

INCORPORATION BY REFERENCE

All references and patents cited herein are hereby incorporated by reference in their entirety.

Claims

What is claimed is:

1. A method of diagnosing an autoimmune disease in an individual comprising: collecting a biological sample from the individual; contacting the sample separately with an extract from human apoptotic cells, and an extract from human non-apoptotic cells, under conditions which promote binding of proteins in the extracts by antibodies in the sample; removing unbound proteins from the sample; determining the presence of a bound phosphoprotein in the sample by contacting the sample with a labeled anti-phosphoprotein antibody or fragment thereof, removing unbound antibody, and detecting the presence of the label; and comparing the label patterns for said extract from human apoptotic cells and said extract from human non-apoptotic cells to determine the presence of an antibody in the sample specific for a phosphoprotein which is present in said human apoptotic cell extract, but which is not present in said human non-apoptotic cell extract.

2. The method of claim 1 wherein the labeled anti-phosphoprotein antibody is a monoclonal antibody.

3. The method of claim 1 wherein the labeled anti-phosphoprotein antibody is directed against a phosphoprotein selected from the group consisting of ppl7, pp23, pp34, pp42, pp46, pp54, pp90 and pp200.

4. The method of claim 1 wherein the labeled anti-phosphoprotein antibody is directed against a phosphoprotein selected from the group consisting of SRp54, SRp40, ASF/SF2, SC35, U2AF35, SRp20, UI A and U2B".

5. The method of claim 1 wherein the labeled anti-phosphoprotein antibody is directed against a component of the UI- or U2-snRNP complex which is associated with a phosphoprotein or a protein kinase.

6. The method of claim 5 wherein the phosphoprotein is selected from the group consisting of ppl7, pp23, pp34, pp42, pp46, pp54, pp90 and pp200.

7. The method of claim 5 wherein the phosphoprotein selected from the group consisting of SRp54, SRp40, ASF/SF2, SC35, U2AF35, SRp20, U1A and U2B".

8. The method of claim 7 wherein the protein kinase is selected from the group consisting of SRPK-1, Clk/Sty, DNA topoisomerase I, nuclear envelope- associated kinase and Ul-70 kD-associated kinase.

9. The method of claim 1 wherein the biological sample is serum from blood.

10. A method of diagnosing an autoimmune disease in an individual comprising determining the presence of an anti-phosphoprotein antibody or fragment thereof specific for a phosphoprotein in a biological sample of the individual, wherein said phosphoprotein is present in a human apoptotic cell extract but is not present in a human non-apoptotic cell extract.

11. The method of claim 10 wherein the anti-phosphoprotein antibody is specific for a phosphoserine residue.

12. The method of claim 10 wherein the presence of said antibody specific for a phosphoprotein is detected using a sandwich ELISA assay.

13. The method of claim 12 wherein the sandwich ELISA assay employs a monoclonal antibody or fragment thereof directed against a phosphoprotein selected from the group consisting of ppl7, pp23, pp34, pp42, pp46, pp54, and pp200.

14. The method of claim 7 wherein the sandwich ELISA assay employs a monoclonal antibody or fragment thereof directed against a phosphoprotein selected from the group consisting of SRp54, SRp40, ASF/SF2, SC35, U2AF35, and SRp20.

15. The method of claim 10 wherein the biological sample is serum from blood.

16. A method of diagnosing an autoimmune disease in an individual comprising: collecting a biological sample from the individual; contacting the sample separately with an extract from human apoptotic cells, and an extract from human non-apoptotic cells, under conditions which promote binding of proteins in the extracts by antibodies in the sample; removing unbound proteins from the sample; assaying for protein kinase activity in the sample; and comparing the kinase activity for the sample contacted with said extract from human apoptotic cells and the sample contacted from said extract from human non- apoptotic cells to determine the presence of an antibody in the sample which recognizes an active protein kinase which is present in said human apoptotic cell extract, but which is not present in said human non-apoptotic cell extract.

17. The method of claim 16 wherein the kinase activity is measured using an assay selected from the group consisting of a colorimetric assay, a radioactive assay and a fluorometric assay.

18. The method of claim 16 wherein the protein kinase is selected from the group consisting of SRPK-1, Clk/Sty, DNA topoisomerase I, nuclear envelope- associated kinase and U 1 -70 kD-associated kinase.

19. The method of claim 16 wherein the biological sample is serum from blood.

20. A method of diagnosing an autoimmune disease in an individual comprising determining the presence in a biological sample of the individual of an antibody specific for an active protein kinase which is present in a human apoptotic cell extract but which active kinase is not present in a human non-apoptotic cell extract.

21. The method of claim 20 wherein the protein kinase is a serine/threonine kinase.

22. The method of claim 20 wherein the protein kinase is selected from the group consisting of SRPK-1, Clk/Sty, DNA topoisomerase I, nuclear envelope- associated kinase and Ul-70 kD-associated kinase.

23. The method of claim 20 wherein the biological sample is serum from blood.

24. An assay kit for diagnosing an autoimmune disease in an individual comprising: a solid support capable of adsorbing human immunoglobulin; a first extract from an apoptotic cell culture; a second extract from a non-apoptotic cell culture; and an antibody or fragment thereof specific for a phosphoprotein or a protein kinase.

25. The assay kit of claim 24 wherein the antibody is a monoclonal antibody directed against a phosphoserine residue.

26. The assay kit of claim 24 wherein the monoclonal antibody is directed against a protein kinase.

27. The assay kit of claim 24 wherein the monoclonal antibody is directed against a phosphoprotein selected from the group consisting of ppl7, pp23, pp34, pp42, pp46, pp54, pp90 and pp200.

28. The assay kit claim 24 wherein the monoclonal antibody is directed against an SR protein selected from the group consisting of SRp54, SRp40, ASF/SF2, SC35, U2AF35, and SRp20.

29. The assay kit of claim 24 further comprising a kinase buffer and labeled

ATP.

30. The assay kit of claim 24 further comprising a solution for washing unbound protein from said solid support.

31. A method of treating or preventing an autoimmune disease in an individual comprising: detecting in a biological sample from the individual the presence of an antibody or fragment thereof specific for one or more phosphoproteins which are present in extracts from human apoptotic cells but not in extracts from human non-apoptotic cells; administering to the individual a composition comprising a protein kinase inhibitor and a pharmaceutically acceptable carrier.

32. The method of claim 31 wherein the phosphoprotein is selected from the group consisting of ppl7, pp23, pp34, ρp42, pp46, pp54, pp90 and pp200.

33. The method of claim 31 wherein the phosphoprotein is an SR protein selected from the group consisting of SRp54, SRp40, ASF/SF2, SC35, U2AF35, and SRp20.

34. The method of claim 31 wherein the kinase inhibitor inhibits the activity of an SR protein kinase.

35. The method of claim 31 wherein the kinase inhibitor inhibits the activity of a kinase selected from the group consisting of SRPK-1, Clk/Sty, DNA topoisomerase I, nuclear envelope-associated kinase, and U 1 -70 kD-associated kinase.

36. The method of claim 31 wherein the presence of said antibody specific for one or more phosphoproteins is detected using a sandwich ELISA assay.

37. The method of claim 36 wherein the sandwich ELISA assay employs a monoclonal antibody or fragment thereof directed against a phosphoserine residue.

38. The method of claim 36 wherein the sandwich ELISA assay employs a monoclonal antibody or fragment thereof directed against a phosphoprotein selected from the group consisting of ppl7, pp23, pp34, pp42, pp46, pp54, pp90 and pp200.

39. The method of claim 36 wherein the sandwich ELISA assay employs a monoclonal antibody or fragment thereof directed against a protein kinase.

40. A method of treating or preventing an autoimmune disease in an individual comprising: detecting in a biological sample from the individual the presence of an antibody specific for one or more protein kinases which are present in extracts from human apoptotic cells but not in extracts from human non-apoptotic cells; administering to the individual a composition comprising a protein kinase inhibitor and a pharmaceutically acceptable carrier.

41. The method of claims 40 wherein the protein kinase is a serine/threonine kinase.

42. The method of claim 40 wherein the biological sample is serum from blood.

43. A method of screening a chemical library for a compound which inhibits a protein kinase involved in phosphorylation of autoantigens during apoptosis comprising: contacting an extract from human apoptotic cells with a compound to be tested for protein kinase inhibition; immunoprecipitating the extract with a biological sample from a human containing an antibody or fragment thereof directed against a phosphoprotein or an active protein kinase which is present in apoptotic cells but not in apoptotic cells; determining protein kinase activity for the immunoprecipitated proteins to determine any reduction in the protein kinase activity compared to a control.