WO2005025613A1 - Methods of identifying biomolecular targets for diagnosis and therapy - Google Patents

Abstract

A method of identifying a putative drug target is provided. The method comprising (a) identifying an autoantibody populations which displays higher titer in an abnormal biological sample as compared to a normal biological sample; and (b) identifying a disease-causing biomoelcule targeted by the autoantibody population thereby identifying the putative drug target.

Description

METHODS OF IDENTIFYING BIOMOLECULAR TARGETS FOR DIAGNOSIS AND THERAPY

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to methods of identifying biomolecular targets which can be used for diagnostics and therapy. While the promise of genomics in the biological and pharmaceutical industry is great, its impact on the drug discovery pipeline has not yet been realized. Evidently, extracting novel biologically valid targets from exponentially growing amounts of sequence data requires time and considerable investment in biological research infrastructure. Biomolecules which are associated with disease formation or progression, can serve as diagnostic or therapeutic targets.. Such targets are typically proteins or the DNA or mRNA molecules that encode these proteins, although other biornoecules, such as carbohydrates can also serve as diagnostic or therapeuic targets. The key to determining what genes and proteins do, and which ones represent valid drug targets, lies in bioinformatics and functional genomics technologies. Using these tools, researchers aim to uncover a small list of molecular targets that are, or are likely to be, associated with a disease of interest, or with a condition that predisposes an individual to that disease. Traditionally, drug discovery has been based largely on the modification of older drugs and drug leads to obtain new therapeutic candidates and on a good deal of serendipity. Currently marketed drugs, excluding relatively few recombinant protein and monoclonal-antibody drugs, were nearly all discovered via the traditional drug discovery process and target approximately 450 molecules. Most of the targets of these drugs fall into the following protein families: G-protein-coupled receptors (GPCRs), ion channels, certain types of enzymes (e.g., serine proteases), and nuclear hormone receptors (e.g., steroid receptors). In contrast to traditional drug discovery, genomic-based drug discovery involves sorting through very large numbers of potential targets and very large numbers of target families. Many potential targets are of unknown function, and many of them belong to families of genes and proteins with which the pharmaceutical industry is completely unfamiliar. The lack of sufficient annotation for genomic data is a major obstacle in target selection. This is evident from the sharp decrease in the average number of literature references now availabloe for newly considered targets. As noted in a recent report by Lehman Brothers analysts "Ten years ago, the average number of literature references per target under consideration at a pharmaceutical company was more than 100, now it is eight." It is widely agreed that the cost of failure is the biggest drain on pharmaceutical profits. In this decade, the average drug will take approximately ten years to go from the discovery phase to the clinic, a process which typically costs $400 million to more than $1 billion to complete. Researchers at Millennium Pharmaceuticals determined that failures account for approximately three-fourths of drug development costs. It is well established that the stronger the link between the protein and the disease the lower the chances of failure during the clinical trial process. The genomic target-selection process involves three major tasks: (1) target screening, or identifying molecules that may be associated with a disease process (e.g., upregulation of a particular gene identified through gene expression analysis); (2) target identification, the process of identifying molecules that clearly play a role in a disease process*, and (3) target validation, the process of determining which among these molecules leads to a phenotypic change when modulated, suggesting it may have value as a therapeutic target. While reducing the present invention to practice the present inventor established a novel approach for drug target selection. This functional approach provides highly detailed information on the functions of novel targets in a disease of interest, and highlights the specific pathways and the functional consequences of activating these targets under different conditions. Hence, this approach unifies target identification and validation and thus provides a clever and cost effective approach for developing new drugs.

SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided a method of identifying a putative drug target, the method comprising: (a) identifying an autoantibody populations which displays higher titer in an abnormal biological sample as compared to a normal biological sample; and (b) identifying a disease-causing biomoelcule targeted by the autoantibody population thereby identifying the putative drug target. According to further features in preferred embodiments of the invention described below, the disease-causing biomolecule is a proinflammatory mediator. According to still further features in the described preferred embodiments the disease-causing biomolecule is not a regulatory mediator. According to still further features in the described preferred embodiments the autoantibody population includes IgG antibodies. According to another aspect of the present invention there is provided a method of diagnosing a disease in a subject, the method comprising determining a titer of an autoantibody population directed at an endogenous disease-causing biomolecule in a biological sample obtained from the subject, wherein a titer of the autoantibody population above a predetermined normal titer is indicative of the disease. According to yet another aspect of the present invention there is provided a method of treating a disease in a subject, the method comprising providing to the subject a therapeutically effective amount of at least a portion of a disease-causing biomolecule endogenously produced by the subject, thereby treating the disease in the subject. According to still further features in the described preferred embodiments the at least a portion represents an immuniogenic non-active portion of the disease causing biomolecule. According to still further features in the described preferred embodiments the disease causing biomolecule is a polypeptide. According to still further features in the described preferred embodiments providing the protein is effected by: (i) administering the polypeptide to the subject; and/or (ii) administering an expressible polynucleotide encoding the polypeptide to the subject. According to still further features in the described preferred embodiments the method further comprising administering an antibody directed against the disease- causing biomolecule. The present invention successfully addresses the shortcomings of the presently known configurations by providing methods if identifying targets which can be used in diagnosis and therapy. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings: FIG. la is a bar graph depicting the development of autoantibodies to various proinflammatory and regulatory mediators during AA and the ability of targeted DNA vaccines to amplify each potential response. Lewis rats were immunized with CFA to induce active AA and separated into 14 groups of 3 rats each. On day 17 and 19 these groups were injected with plasmid DNA encoding MlP-la, MIF, IP- 10, MCP-1, TNF- α, IL-15, IL-17, IFN-γ IL-4, IL-10, IL-18BP, or FasL. Prior to ligation into pcDNA3 each cDNA was cloned and sequenced. The development of Ab titer to the relevant gene product of each vaccine was determined 3 days later in sera derived from rats treated with DNA plasmids encoding each of the above gene products (black squares) or from control AA rats (gray squares). These titers were also recorded in sera of rats immunized with CFA to induce a local inflammatory response (diagonal stripes) and in sera from naive rats (open squares). Results are shown as mean of 3 samples ± SE.

A direct ELISA assay was used to determine the anti cytokine/chemokine antibody titer in sera of the different groups. Results of triplicates were calculated as log X antibody titer ± SE. As sera were added in serial dilutions from 2⁶ to 2³⁰ the baseline of the sensitivity of the test is log Ab titer = 6. FIG. lb is a graph depicting the ability of DNA vaccination encoding the targets of Figure la to suppress an on going disease. A groups of Lewis rats was subjected to active induction of AA. On day 17 the group was divided to 7 groups of 9 equally sick rats each and subjected to the administration of naked DNA vaccines (days 17 and 19, 300 μg/rat) using DNA constructs encoding, MIF (open circles), IP- 10 (closed diamonds), TNF-α (closed circles), IL-15 (closed triangles), or IL-17 (open triangles), according to the protocol described in details before (13). As additional controls, a plasmid DNA construct encoding soluble intracellular β-actin was used (closed squares, 13) or non treated rats were used (open squares). Clinical manifestation of disease was monitored by an observer blind to the experimental protocol as previously described (13). Clinical scores were also histologically verified (not shown). Results are shown as mean score of 9 rats ± SE. FIG. lc is a graph depicting transfer of autoantibodies produced in DNA vaccinated donors for suppression of an ongoing disease in an recipient subject. AA rates were subjected to plasmid DNA therapy as described in Figure lb (days 17 and 19, 300 μg/rat). Each group included 30 rats. On day 22 all rats were sacrificed and blood sera were purified in two steps; first on IgG purification column, and then on CNBr columns loaded with the gene product of each vaccine. Thereafter each Ab was adoptively transferred to AA rats (100 μg/rat every other day beginning at the onset of disease) as follows: Anti MIF (open circles), Anti IP- 10 (diamonds), Anti TNF-α (closed circles), Anti IL-15 (closed triangles), or Anti IL-17 (open triangles). Control rats were injected with IgG purified from rats that were treated with β-actin construct (closed squares), or were not treated (open squares). An observer blind to the experimental protocol monitored clinical manifestation of the disease. Results are shown as mean score of 6 rats ± SE. FIG. 2 is a bar graph depicting TNF-α amino acid determinants which are recognized by autoantibodies generated in AA rats. TNF-α was subjected to trypsin proteolysis followed by HPLC purification resulting in twenty-four different fragments that constitutes the whole protein. Sequence of each fragment was verified and then used for mapping of Ab binding (ELISA). Groups of 3 rats were subjected to active AA induction. On day 18, just before the peak of disease, these rats were treated (closed squares), or were not treated (open squares) with a TNF-α encoding DNA plasmid. Three days later (day 21) blood serum form each group was tested for its ability to bind each of different TNF-α fragments described above. Results are shown as mean of triplicates ± SE. FIG. 3 a is a graph depicting the ability of anti TNF-α autoantibodies to abolish the cytotoxic activity of TNF-α on U937 T cells. The IgG fraction (protein-G purification) of blood sera obtained from AA rats (peak of disease) was purified on a CNBr-TNF-α purification column and tested for anti TNF-α neutralizing competence in an in vitro system based on (39) with our minor modifications (12). These Ab (closed circles) or purified IgG from naive Lewis rats (open circles), were determined for their competence to inhibit neutral red uptake of U937 cells in the presence of various concentrations of TNF-α (100 μg/well of each Ab, 4xl0⁴ U937 cells/well in a total volume of 100 μl). Another group included wells that were not supplemented with any Ab (open squares). Results are shown as mean O.D. of triplicates at 570 ± SD. FIG. 3b is a graph depicting the ability of TNF-α specific autoantibodies produced in AA rats to suppress ongoing AA. Anti TNF-α Ab described in Figure 3 a (IgG fraction of blood sera obtained from AA rats, purified on a CNBr-TNF-α purification column) were tested for their competence to suppress ongoing AA in an adoptive transfer experiment. Rats with developing AA were separated, one day after the onset of disease, into 3 equally sick groups of 6 rats each, that were administered, every other day, with 100 μg/rat of Ab (closed circles). Control groups were administrated with purified IgG from naive Lewis rats (open circles), or with equal volume of PBS (open squares). Shown are the results of one out of three experiments done under the same conditions with similar results. Results are shown as mean maximal score ± S.D. FIGs. 4a-d are graphs depicting the inability of TNF-α tolerant rats to combat AA. During the first 24 hours after birth, groups of 6 Lewis rats were subjected to a single administration of 10 ng of plasmid DNA encoding TNF-α (closed squares). Control rats were administered, at that time, with an empty plasmid (open squares), or PBS (closed circles). Another group was administered with the same TNF-α encoding DNA plasmid at 4 weeks of age (triangles). At eight weeks of age all rats were subjected to the induction of moderated form of AA and monitored for the development of disease. Figure 4a shows the results of one out of three experiments done under the same conditions with similar results. Results are shown as mean clinical score ± SD. At the peak of disease in control animals (day 22) sera was obtained from all rats and TNF-α specific antibody titer was determined (Figure 4b) as follows: (a) naϊve rats, no disease; (b) AA rats; (c) naϊve rats previously subjected to neonatal administration of TNF-α encoding DNA plasmid; (d) AA rats previously subjected to neonatal administration of TNF-α encoding DNA plasmid; (e) AA rats subjected to "adult life" administration of TNF-α encoding DNA plasmid. Results are shown as log Ab titer ±SE of triplicates. Figure 4c shows the effect of TNF-α antibody administration to rats that were subjected to TNF-αencoding DNA plasmid at the neonatal period and to active induction of AA at eight weeks of age. Following AA^" induction, rats were separated into different group that were, or were not administered (any other day beginning at the onset of disease) with TNF-α antibody (IgG fraction of blood sera obtained from control AA rats, purified on a CNBr-TNF-α purification column). Results are mean maximal clinical score of 6 rats per group ±SE. Figure 4d shows histological evaluation of the experiment described in Figure 4c. 30 days after disease induction joint samples form rats were subjected for histological analysis (12 sections each group) as follows: (a) a naϊve rat; (b) control AA rats; (c) AA rats previously subjected to neonatal administration of TNF-α encoding DNA plasmid; (d) AA rats previously subjected to neonatal administration of TNF-α encoding DNA plasmid and during disease to replacement therapy with TNF-α antibody, Representative samples from each group are presented ( lO). The arrowheads point to the synovial lining (b = bones, S = synovial membrane). FIGs. 5a-b are graphs depicting the levels of anti-TNF-α autoantibodies in healthy individuals and in subjects suffering from RA and OA. Blood sera were obtained from 22 patients suffering from RA, 10 patients suffering from OA and 12 healthy individuals. Synovial fluid was also taken from all RA patients. Figure 5a shows log₂Ab titer of each individual One-way ANOVA test (with the Bonferoni correction) showed a significant elevation (p<0.05) in antibody titer determined in sera RA patients compared to healthy individuals and those suffering from OA. No significant difference could be observed between the last two groups. Figure 5b shows the correlation between the antibody titer to TNF-α developed in blood sera and synovial fluid of the above-RA patients.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of methods of identifying biomolecular targets which can be used for diagnostics and therapy. The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. The recent increase in amount of genomic information has created new challenges for the pharmaceutical industiy. These include, the rapid and efficient identification of target genes responsible for complex disease phenotypes and the use of this information for the development of new and specific classes of drugs. While reducing the present invention to practice the present inventor designed a novel approach for drug target selection. This functional approach provides highly detailed information on the functions of novel targets in a disease of interest, and highlights the specific pathways and the functional consequences of inactivating these targets under different conditions. Hence, this approach unifies target identification and validation to provide a clever and cost effective mode for the development of new drugs. As is illustrated in the Examples section, which follows, the present inventor showed, for the first time, that during a disease state, a beneficial autoimmune response (i.e., antibody) against selected cytokine targets is established and halts disease progression. Specifically, the immune system allows a selective breakdown of tolerance to proinflammatory, but not regulatory mediators. Thus, the immune system identifies specific targets which play a critical role in disease progression and neutralizes them. As is illustrated in Example 1 of the Examples section which follows, DNA vaccines encoding these targets effectively alleviated disease progression and completed the process already initiated by the endogenous immune system. The present inventor utilized this selection machinery of the immune system to identify targets for therapy and to establish methodology, which can be used to identify diagnostic and therapeutic targets. Thus, according to one aspect of the present invention there is provided a method of identifying a putative drug target. As used herein a "putative drug target" refers to a disease causing biomolecule, typically a protein encoded by a gene, a DNA sequence, an RNA sequence or a small molecule (e.g., IP3), which can interact with a drug or a drug candidate to thereby at least partially arrest disease onset or progression. The disease causing biomolecule of the present invention is endogenously produced by the individual and is critical for disease onset and/or progression such that elimination of an activity or expression thereof is expected to at least partially alleviate disease symptoms. The disease causing biomolecule of the present invention is preferably a proinflammatory mediator. As used herein "proinflammatory mediator" refers to soluble mediators which are secreted in- and support the onset and/or progression autoimmune, inflammatory or cancer diseases. Examples of proinflammatory mediators include, but are not limited to, chemokines and cytokines [see U.S. Pat. Appl. No. 60/534,111 entitled "COMPOSITIONS AND METHODS FOR DIAGNOSING AND TREATING PROSTATE CANCER" (Attorney docket no. 27145) assigned to the same assignee hereof and contain subject matter related, in certain respects, to the subject matter of the instant application, the teachings of all of which are incorporated herein by reference and Example 1 of the Examples section which follows]. Proinflammatory mediators are distinct from regulatory mediators, which down regulate inflammatory responses. Examples of regulatory mediators include, but are not limited to, such as IL-10, IL-4 and TGF-β. As used herein the term "disease" refers to a pathological condition which is dependent for onset and/or progression upon an endogenous biomolecule against which autoantibodies can be formed. Examples of such diseases include but are not limited to autoimmune diseases such as rheumatoid arthritis, type I diabetes and multiple sclerosis; cancer diseases; chronic inflammatory conditions such as intestinal bowels diseases such as Crohn's disease and ulcerative colitis. In all these diseases inflammatory chemokines have a direct role in the pathogenesis, and as such are putative candidates for "beneficial autoimmune" production of autoantibodies. The method according to this aspect of the present invention is effected by identifying an autoantibody population which displays higher titer in an abnormal biological sample as compared to a normal biological sample; and identifying a disease-causing biomolecule targeted by this autoantibody population, thereby identifying the putative drug target. As used herein the phrase "an autoantibody population" refers to a population of antibodies which are produced by an individual and are directed against a self- antigen, i.e., an endogenously produced biomolecule. The autoantibody population of the present invention can be of different specificities (i.e., recognizing different epitopes on a single self-antigen), immunoglobulin classes (e.g., IgA, IgD, IgG, IgM, IgE, IgY) or subclasses (e.g., IgAl, IgA2, IgGl , IgG2, IgG3 and IgG4). As used herein an "abnormal biological sample" refers to an antibody- containing sample of cell, tissue or fluid derived from a subject having a disease of interest, or suspected of having the disease. As used herein a "subject" refers to any subject who may benefit from the present invention such as a mammal (e.g., canine, feline, ovine, porcine, equine, bovine, human), preferably a human subject. The abnormal biological sample can also be derived from cells tissues or fluids of an animal model of the disease. Antibodies present in the sample are typically found within cytoplasmic membrane -bound compartments (e.g., endoplasmic reticulum and Golgi apparatus) and on the surface of B lymphocytes (which synthesize antibody molecules) and immune effector cells such as, mononuclear phagocytes, natural killer (NK) cells and mast cells, which express specific receptors for binding antibody molecules. Antibodies are also present in the plasma (i.e., fluid portion) of the blood and in the interstitial fluid of the tissues. Antibodies can also be found in secretory fluids such as mucus, synovial fluid, sperm and milk into which certain types of antibody molecules are specifically transported. As used herein a "normal biological sample" refers to an antibody-containing sample of cell, tissue or fluid isolated from a healthy individual. Procedures for obtaining biological samples (i.e., biopsying) from individuals are well known in the art. Such procedures include, but are not limited to, blood sampling, joint fluid biopsy, cerebrospinal biopsy and lymph node biopsy. These and other procedures for obtaining tissue or fluid biopsies are described in details in http://www.healthatoz.com/healthatoz/Atoz/search.asp. Regardless of the procedure employed, once the biological samples (i.e., normal and abnormal) are obtained, the titer (number) of antibody molecules for specific antigens (i.e., at least an immunogenic portion of the disease-causing biomolecules) in each biological sample is determined. It will be appreciated that such titer data may also be available in pre-existing publications (i.e., literature based analysis). Antibody titer can determined by techniques which are well known in the art such as ELISA and dot blot using an immobilized antigen (see for Example Abbas, Lichtman and Pober "Cellular and Molecular Immunology". W.B. Saunders International Edition 1994 pages 56-59). Specifically, an antigen of interest is preferably immobilized on a solid support. To avoid non-specific binding of antibodies, the solid support is preferably coated with a nonantigenic protein as well. A peptide is typically immobilized on a solid matrix by adsorption from an aqueous medium, although other modes of immobilization applicable to proteins and peptides well known to those skilled in the art can be used. Useful solid matrices are also well known in the art. Such materials are water insoluble and include cross-linked dextran (e.g., SEPHADEX™, Pharmacia Fine Chemicals, Piscataway, N.J.), agarose, polystyrene beads about 1 μm to about 5 mm in diameter, polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrene or polyvinylchloride. Once the antigens is immobilized antibody-containing samples are added in serial dilutions until binding can no longer be observed. The antibody containing samples can be either a crude sample or immunoglobulin purified samples (e.g., ammonium sulfate precipitated fraction and/or chromatography isolated). Immunocomplexes are allowed to form and the support is washed to remove non- specifically bound antisera. Detection of immunocomplexes can be effected by adding labeled antibody-binding molecules such as staphylococcal protein A. The label can be an enzyme such as horseradish peroxidase (HRP), glucose oxidase, or the like. In cases where the major indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to indicate that an immunocomplex has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2,- azino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS). Radioactive labels may also be used in accordance with the present invention. An exemplary radiolabeling agent is a radioactive element that produces γ ray 1 emissions, such as I. Methods of protein labeling are well-known in the art and described in details by Galfre et al., Meth. Enzyol., 73:3-46 (1981). The techniques of protein conjugation or coupling through activated functional groups are also applicable. See, for example, Aurameas et al., Scand. J. Immunol., 8(7):7-23 (1978); Rodwell et al, Biotech, 3:889-894 (1984); and U.S. Pat. No. 4,493,795. When higher antibody titers towards an endogenous biomolecule is detected in the abnormal biological sample as compared to the normal biological sample, such a biomolecule is considered a disease causing biomolecule and identified as a putative drug target. Further validation of such putative drug targets can be effected as described in Examples 2-5 of the Examples section. Using the above-described methodology, the present inventor identified a number of drug targets which can be used for the diagnosis and treatment of numerous diseases, several of which are described hereinunder in the Examples section which follows. Once autoantibodies to disease causing biomolecules are correlated with a specific disease they can be used as markers for diagnosing the disease in a subject, as described hereinabove. Essentially, diagnosis according to this aspect of the present invention is effected by determining a titer of an autoantibody population directed at the disease- causing biomolecule in a biological sample obtained from the subject, where a titer above a predetermined normal titer (i.e., in a non-pathological sample) is indicative of the disease. When the disease-causing biomolecule is a polypeptide (i.e., protein) it is often preferable to use an antigenic portion thereof for the evaluation of antibody titer. This may simplify the procedure of antigen production, and even allow in-vivo diagnosis of the disease at much lower costs. Parameters for selection of antigenic portions of a protein are further described hereinbelow. The novel concept of disease fighting autoantibodies, suggests that contrary to traditional approach, in some cases, it can be advantageous to introduce disease- causing biomolecules to an individual in order to treat a disease. Thus, according to another aspect of the present invention, there is provided a method of treating a disease in a subject. As used herein the term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of the diseases described herein. The method according to this aspect of the present invention is effected by providing to the subject a therapeutically effective amount of at least a portion of a disease-causing biomolecule endogenously produced by the subject, thereby treating the disease in the subject. Preferably, a portion of a disease causing biomolecule represents an immunogenic non-active portion thereof. Essentially, a segment, which promotes a specific immunogenic response but is not active and thus does not contribute to disease progression or severity. Examples of immunogenic non-active portions of trypsin proteolysed TNF-α are provided in Example 2 (SEQ ID NOs: 15-17) of the Examples section. To promote specific immune response, specificity of the immunizing agent can be determined by comparing the composition of the biomolecule to biomolecules which are known in the art. For example a portion of a polypeptide sequence can be compared to all known protein databases, employing a sequence alignment algorithm such as BLAST (Basic Local Alignment Search Tool, available through www.ncbi.nlm.nih.gov/BLAST) or the Smith- Waterman algorithm. Parameters which can be used to determine an immunogenicity of a biomolecule include: Molecular size - there is a correlation between the size of an amino acid sequence and its immunogenicity. As such, amino acid sequences having a molecular mass of at least 1000 daltons (Da) are favored as immunogens. Chemical composition and heterogeneity - In general, homopolymers (i.e., polymers composed of a single amino acid) tend to lack immunogenicity, regardless of their size. Copolymers of sufficient size, containing two or more different amino acids, are immunogenic. Furthermore, all four levels of protein organization-primary, secondary, tertiary and quaternary-contribute to the structural complexity of a polypeptide and hence affect its immunogenicity. Susceptibility to antigen processing and presentation- Basically, polypeptides that cannot be degraded and presented with MHC molecules are poor immunogens. Moreover, large insoluble molecules are more immunogenic than small soluble ones, because they are more readily phagocytosed and processed. Post-translational modifications - Generally, preferred peptide products are those lacking any post-translational modification sites, since post-translationally modified amino acid sequences are often difficult to purify, and are frequently poor immunogens. Various sequence analysis software are known in the art, which provide an immunogenicity index according to, for example, the Jameson- Wolf algorithm. Examples include, but are not limited to, Sciprot (available from www.asiaonline.net.hk/~twcbio/ DOCS/1 /scPrtein.htm) and Macvector (available from www.accelrys.com products/macvector/) as well as the widely utilized GCG package (Genetics Computer Group, Wisconsin). It will be appreciated that the immunogenic non-active polypeptide can be directly administered to the subject, or it can be expressed from a nucleic acid construct including a polynucleotide encoding the polypeptide (see DNA vaccination in the Examples section which follows). To enhance an immune response the biomolecule is preferably administered with an immunostimulant in an immunogenic composition. An immunostimulant may be any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes into which the compound is incorporated (see e.g., U.S. Pat. No. 4,235,877). Vaccine preparation is generally described in, for example, M. F.

Powell and M. J. Newman, eds, "Vaccine Design (the subunit and adjuvant approach)," Plenum Press (NY, 1995). As mentioned, immunogenic compositions may contain a polynucleotide encoding the immunogenic polypeptide as described above, such that the polypeptide is generated in situ. The DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems (see the Examples section which follows), bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the subject (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Suitable systems are disclosed, for example, in Fisher-Hoch et al, Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al, Ann. N.Y Acad. Sci. 569:86-103, 1989; Flexner et al. Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,1 12, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al. Science 252:431-434, 1991; Kolls et al, Proc. Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al, Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993; Guzman et al. Circulation 88:2838-2848, 1993; and Guzman et al, Cir. Res. 73:1202-1207, 1993. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al. Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. It will be appreciated that an immunogenic composition may comprise both a polynucleotide and a polypeptide component. Such immunogenic compositions may provide for an enhanced immune response. Any of a variety of immunostimulants may be employed in the immunogenic compositions of this invention. For example, an adjuvant may be included. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2,-7, or -12, may also be used as adjuvants. The adjuvant composition may be designed to induce an immune response predominantly of the Thl type. High levels of Thl-type cytokines (e.g., IFN-γ, TNF- α, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. Preferably, high levels of Th2-type cytokines (e.g., IL-4, IL- 5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of an immunogenic composition as provided herein, the subject will support an immune response that includes Thl- and Th2-type responses. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145- 173, 1989. Preferred adjuvants for use in eliciting a predominantly Thl-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O- acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt. MPL adjuvants are available from Corixa Corporation (Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Thl response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al. Science 273:352, 1996. Another preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, Mass.), which may be used alone or in combination with other adjuvants. For example, an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in- water emulsion and tocopherol. A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210. Other preferred adjuvants include Montanide ISA 720 (Seppic, France), SAF (Chiron, Calif, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. patent application Ser. Nos. 08/853,826 and 09/074,720. A delivery vehicle may be employed within the immunogenic composition of the present invention to facilitate production of an antigen-specific immune response that targets tumor cells. Delivery vehicles include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature

392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmernan and Levy, Ann. Rev. Med. 50:507-529, 1999). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes

(dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T cell responses. Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within an immunogenic composition (see Zitvogel et al. Nature Med. 4:594- 600, 1998). Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNF-α to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNF-α, CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells. Dendritic cells are categorized as "immature" and "mature" cells, which allows a simple way to discriminate between two well characterized phenotypes. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fey receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB). APCs may generally be transfected with at least one polynucleotide encoding a polypeptide of the present invention, such that variant II, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a composition comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to the subject, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al.

Immunology and cell Biology 75:456-460, 1997. Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with a polypeptide of the present inventio, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule) such as described above. Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide. The disease-causing biomolecule of the present invention or a construct encoding same can be provided to the subject per se (or as described above), or as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier. As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable earners and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. Herein the term "active ingredient" refers to the polypeptide or antibody preparation, which is accountable for the biological effect. Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979). Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co, Easton, PA, latest edition, which is incorporated herein by reference. Suitable routes of administration may, for example, include oral, rectal, transmucosal, transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections. Alternately, one may administer the preparation in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body. Thus, for example, the preparation may be directly injected into a joint of an RA patient by intra-articular administration. Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For oral administration, the compounds can be formulated readily by ^• combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use. The preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g, conventional suppository bases such as cocoa butter or other glycerides. Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans. Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g, Fingl, et al, (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p.l]. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. It will be appreciated that the above-described methodology can be combined with administration of antibodies directed at the biomolecule causing disease using the above-described administration routes (e.g, intraveoneously). The term "antibody" refers to whole antibody molecules as well as functional fragments thereof, such as Fab, F(ab')₂, and Fv that are capable of binding with antigenic portions of the target polypeptide. These functional antibody fragments constitute preferred embodiments of the present invention, and are defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) (Fab') , the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')₂ is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and

(5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule as described in, for example, U.S. Patent 4,946,778. Purification of serum immunoglobulin antibodies (polyclonal antisera) or reactive portions thereof can be accomplished by a variety of methods known to those of skill including, precipitation by ammonium sulfate or sodium sulfate followed by dialysis against saline, ion exchange chromatography, affinity or immunoaffmity chromatography as well as gel filtration, zone electrophoresis, etc. (see Goding in, Monoclonal Antibodies: Principles and Practice, 2nd ed, pp. 104-126, 1986, Orlando, Fla, Academic Press). Under normal physiological conditions antibodies are found in plasma and other body fluids and in the membrane of certain cells and are produced by lymphocytes of the type denoted B cells or their functional equivalent. Antibodies of the IgG class are made up of four polypeptide chains linked together by disulfide bonds. The four chains of intact IgG molecules are two identical heavy chains referred to as H-chains and two identical light chains referred to as L-chains. Additional classes include IgD, IgE, IgA, IgM and related proteins. Methods of generating and isolating monoclonal antibodies are well known in the art, as summarized for example in reviews such as Tramontano and Schloeder, Methods in Enzymology 178, 551-568, 1989. A recombinant polypeptide may be used to generate antibodies in vitro (see the materials and experimental procedures section of the Examples section which follows). More preferably, the recombinant protein is used to elicit antibodies in vivo. In general, a suitable host animal is immunized with the recombinant polypeptide. Advantageously, the animal host used is a mouse of an inbred strain. Animals are typically immunized with a mixture comprising a solution of the recombinant polypeptide in a physiologically acceptable vehicle, and any suitable adjuvant, which achieves an enhanced immune response to the immunogen. By way of example, the primary immunization conveniently may be accomplished with a mixture of a solution of the recombinant polypeptide and Freund's complete adjuvant, said mixture being prepared in the form of a water in oil emulsion. Typically the immunization will be administered to the animals intramuscularly, intradermally, subcutaneously, intraperitoneally, into the footpads, or by any appropriate route of administration. The immunization schedule of the immunogen may be adapted as required, but customarily involves several subsequent or secondary immunizations using a milder adjuvant such as Freund's incomplete adjuvant. Antibody titers and specificity of binding to the polypeptide can be determined during the immunization schedule by any convenient method known in the art, such as described hereinabove. When suitable antibody titers are achieved, antibody-producing lymphocytes from the immunized animals are obtained, and these are cultured, selected and cloned, as is known in the art. Typically, lymphocytes may be obtained in large numbers from the spleens of immunized animals, but they may also be retrieved from the circulation, the lymph nodes or other lymphoid organs. Lymphocytes are then fused with any suitable myeloma cell line, to yield hybridomas, as is well known in the art. Alternatively, lymphocytes may also be stimulated to grow in culture, and may be immortalized by methods known in the art including the exposure of these lymphocytes to a virus, a chemical or a nucleic acid such as an oncogene, according to established protocols. After fusion, the hybridomas are cultured under suitable culture conditions, for example in multi-well plates, and the culture superaatants are screened to identify cultures containing antibodies that recognize the hapten of choice. Hybridomas that secrete antibodies that recognize the recombinant polypeptide are cloned by limiting dilution and expanded, under appropriate culture conditions. Monoclonal antibodies are purified and characterized in terms of immunoglobulin type and binding affinity. Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') . This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, in U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety (see also Porter, R. R, Biochem. J, 73: 119-126, 1959). Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. Fv fragments comprise an association of V_H and V_L chains. This association may be noncovalent, as described in Inbar et al. (Proc. Nat'l Acad. Sci. USA 69:2659- 62, 1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise V_H and V_L chains connected by a peptide linker. These single- chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the V_H and V_L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow and Filpula, Methods, 2: 97-105, 1991; Bird et al. Science 242:423-426, 1988; Pack et al, Bio/Technology 11:1271-77, 1993; and Ladner et al, U.S. Pat. No. 4,946,778, all of which are hereby incorporated, by reference, in entirety. Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, for example, Larrick and Fry Methods, 2: 106-10, 1991). Humanized forms of non-human (e.g, murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non- human residues. Humanized antibodies may also comprise residues, which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al. Nature, 321:522- 525 (1986); Riechmann et al. Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol, 2:593-596 (1992)]. Methods for humanizing non-human antibodies are well known in the art.

Generally, a humanized antibody has one or more amino acid residues introduced into it from a source, which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al. Nature, 321:522-525 (1986); Riechmann et al. Nature 332:323-327 (1988); Verhoeyen et al. Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No.. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al, J. Mol. Biol, 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Li^'ss, p. 77 (1985) and Boerner et al, J. Immunol, 147(l):86-95 (1991)]. Similarly, human monoclonal antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al, Bio/Technology 10, 779-783 (1992); Lonberg et al. Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al. Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995). As used herein the term "about" refers to + 10 %. Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al, (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M, ed. (1994); Ausubel et al, "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al, "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531 ; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E, ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E, ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co, New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J, ed. (1984); "Nucleic Acid Hybridization" Hames, B. D, and Higgins S. J, eds. (1985); "Transcription and Translation" Hames, B. D, and Higgins S. J, Eds. (1984); "Animal Cell Culture" Freshney, R. I, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B, (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al, "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE 1 Treatment ofAA with autoantibodies to proinflammatory cytokines and complementary treatment using DNA vaccines for similar targets The ability of targeted DNA vaccines of various pro-inflammatory and regulatory mediators involved in AA, to amplify autoantibodies expression, thus suppress an ongoing AA disease was evaluated. MA TERIALS AND METHODS: Animals: Female Lewis rats of about six weeks old were purchased from Harlan

(Jerusalem, Israel) and maintained under clean conditions in the Technion animal facility (Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel). Immunizations and active disease induction: Rats were immunized subcutaneously in the base-tail with 0.1 ml of CFA (incomplete Freund's adjuvant supplemented with 10 mg/ml heat-killed

Mycobacterium tuberculosis H37Ra in oil, Difco laboratories Inc., Detroit, MI) to induce active AA. Rats were then monitored for clinical signs daily by an observer blind to the treatment protocol. Severity of the disease was quantified subjectively by scoring each limb on a scale of 0-4 to indicate the severity of peripheral joint swelling and erythema: 0 = no signs of disease, 1= disease evident in a small number of distal joints of the limb, 2 = disease evident in all of distal joints of the limb, 3 = disease evident in all the limb, 4 = severe disease evident in all the limb. The arthritic clinical score was determined as the sum of the scores of all four limbs from each animal (0- 16). In addition to clinical evaluation in all experiments, severity of disease was also assessed histologically as described bellow. DNA vaccine: Plasmid DNA vaccines encoding MlP-lα (GenBank Accession No. NM_002983), MCP-1 (GenBank Accession No. NM_002982), TNF-α (GenBank Accession No. NP_ 000585), and FasL (GenBank Accession No. NP_000630) were constructed as we described before (7, 13-16, 18). Plasmid DNA vaccines encoding rat MIF (GenBank Accession No. NM_031051), rat IL-15 (GenBank Accession No. AF015719), IL-17 (GenBank Accession No. NM_002190), IFN-γ (GenBank Accession No. NM_000619), IL-10 (GenBank Accession No. NM_000572), rat IL-4 (GenBank Accession No. NM_201270), IL-18BP (GenBank Accession No. NM_010531) were constructed based on the published sequence of each cytokine/chemokine encoding gene using the primers described in Table 1.

Table 1 Encoded Sense primer SEQ ID NO: Anti-sense primer SEQ ID NO: gene MIF CATGCCTATGTTCATCGTGAACACCSEQ ID NO:l rCAAGCGAAGGTGGAACCGTTCCA SEQ ID NO:2 IL-15 CATGAAATTTTGAAACCAT SEQ ID NO:3 ΓCAGGACGTGTTGATGAAC SEQ ID NO:4 IL-17 AGCACCAGCTGATCAGGA SEQ ID NO:5 GGGGTTTCTTAGGGTCA SEQ ID NO:6 IL-10 CATGCTTGGCTCAGCACTGCT SEQ ID NO:7 ΓCAATTTTTCATTTTGAGTG SEQ ID NO:8 IL-4 CATGGGTCTCAGCCCCCACCT SEQ ID NO:9 ΓTAGGACATGGAAGAGCAGG SEQ ID NO: 10 IFN-γ CATGAGTGCTACACGCCGCGT SEQ ID NO: 1 1 ΓCAGCACCGACTCCTTTTCCG SEQ ID NO:12 IL-18BP CATGAGACACTGTGGCTGTGC SEQ ID NO: 13 ΓCATGGGGCCCCTGGGCCTGCT SEQ ID NO:14

Each sequenced PCR product was cloned and sequence has been verified before being transferred into a pcDNA3 vector (Invitrogen, San Diego, CA). Large- scale preparation of plasmid DNA was conducted using Mega prep (Qiagen Inc., Chatsworth, CA). DNA vaccination was performed as described before (7). Before being administered to AA rats, each DNA vaccine was injected to naϊve Lewis rats. Four-five days later RT-PCR was applied on tibialis anterior muscle samples to verify- that the relevant insert of each gene is transcribed in the injected muscle. Rats vaccination: Rats were separated into 14 groups of 3 rats each. On day 17 and 19 these groups were injected with plasmid DNA encoding MlP-lα, MIF, IP-10, MCP-1, TNF-α, IL-15, IL-17, IFN-γ, IL-4, IL-10, IL-18BP, or FasL. Production and purification of recombinant gene products: PCR product of each of the constructs described above was re-cloned into a pQE expression vector, expressed in E. coli (Qaigen, Chatsworth, CA) and then purified by an NI-NTA-supper flow affinity purification of 6xHis proteins (Qaigen, Chatsworth, CA). After purification the purity of the recombinant protein was verified by gel electrophoresis followed by sequencing (N - terminus). Evaluation of antibody titer in sera samples: A direct ELISA assay was used to determine the anti cytokine/chemokine antibody titer in sera of AA rats. ELISA plates (Nunc, Roskilde, Denmark) were coated with 50ng/well commercially available recombinant rat MlP-lα, rat TNF-α, rat IL-4, rat IL-10, rat MCP-1, rat IFN-γ, murine IP- 10, murine IL-15 (PeproTech, Rocky Hill, NJ), murine IL-17, murine IL-18BP and murine FasL and human MIF (R&D systems, Minneapolis, NM). All results obtained using murine and human gene products have been verified using recombinant rat gene products which were generated from each cloned product using a PQE expression vector as described bellow. Detected sera samples were added in serial dilutions from 2⁶ to 2³⁰ to wells that were, or were not, pre-coated with the relevant recombinant gene product. Goat anti-rat IgG alkaline phosphatase conjugated antibody (Sigma) was used as a labeled antibody. p-Nitrophenyl Phosphate (p-NPP) (Sigma) was used as a soluble alkaline phosphatase substrate. Calculation of each titer was done by comparing the O.D. measured in wells coated with the appropriate cytokine/chemokine to those not coated with this recombinant gene product. Results of triplicates were calculated as log₂Ab titer ± SE. As sera were added in serial dilutions from 2⁶ to 2³⁰ the baseline of the sensitivity of the test was log₂Ab titer = 6. Histopathology: Joints were removed, fixed with 10 % buffered formalin, decalcified in 5 % ethylenediaminetetraacetic acid in buffered formalin, embedded in paraffin and sectioned along the midline through the metatarsal region (19). Sections were stained with hematoxylin and eosin and analyzed by a histopathologist who was a blind observer to the experimental procedure. Evaluation was made based upon inflammatory mononuclear cell infiltrate in the synovial membrane, thickness of the synovial lining, joint space narrowing and periosteal new bone formation. Histological score was determined as follows: 0 = no evidence of disease; 1 = mild lymphocytic infiltrate, 2 = widespread mononuclear of inflammation and thickening of the synovial lining and 3= severe bone destruction, new bone formation and destruction of the synovial lining (19). Statistical analysis: Significance of differences was examined using Student's t-test. A value of PO.05 was considered significant. Mann- Whitney sum of ranks test was used to evaluate significance of differences in mean of maximal clinical score. Value of P<0.05 was considered significant. RESULTS: The development of autoimmunity response to various pro-inflammatory and regulatory mediators during AA, and the ability of targeted DNA vaccines encoding each gene product to amplify each potential response was evaluated (Figure la). During the course of induced AA disease, the immune response generates autoimmunity only to pro-inflammatory mediators including: MlP-lα, MIF, IP- 10, MCP-1, TNF-α, IL-15, and IL-17, (Figure la, log₂Ab titer of 14.3±0.26. 16±0.45, 14.3 ±0.26 13±0.45, 16±0.45, 14.3±0.26 and 14.3±0.26 respectively, compared to control sera from naϊve donors: log₂Ab titer of 6.6±0.26, 6.6±0.26, 6±0, 6.6±0.26, 6.6±0.26, 6.6±0.26 and 6±0, p<0.01 for each reciprocal comparison). Each antibody titer developed in AA rats was significantly higher even from the corresponding one in rats immunized with CFA in hint footpads to develop a local inflammatory process (p<0.05). AA rats never mounted a notable response (over background of log₂Ab titer =6) against regulatory cytokines such as IL-10, I 18 binding protein (IL-18BP) and IL-4 (Figure la). Even in response to IFN-γ, that plays a pleautropic role in the regulation of autoimmunity 20, was at background levels. Moreover, the immune response could distinguish two members of the same family: TNF-α that is a key pro- inflammatory mediator from Fas-L that plays a dual role in the regulation of autoimmunity (18, 21), and mounted autoantibody titer only in response to the pro- inflammatory one (Figure la). In accordance with previous observations (13, 14, 16), administration of plasmid DNA vaccines encoding each pro-inflammatory mediator, during an ongoing disease, could very rapidly, accelerate autoantibody production to the relevant mediator (Figure la, p<0.01 for each comparison) and at the same time suppress the ongoing disease (Figure lb, Mean Max score of 13.2±1 and 14±0.8 in control groups Vs 4±0.5, 4.8±0.6, 6.5 ±0.9, 6±0.9 and 4.5±0.8 in rats treated with plasmid DNA encoding TNF-α, MIF, IL-15, IL-17 or IP-10. pO.Ol respectively). The beneficial effect of each vaccine could be adoptively transferred by autoantibodies produced in DNA vaccinated rats against each relevant gene product

(Figure lc, p<0.01 for the comparison of each treated group with each of the two control groups). These results together with previous observations (13, 14, 16) strongly suggest that DNA vaccines amplify a pre-existing regulatory response aimed to restrain the pathological consequences of autoimmunity.

EXAMPLE 2 Autoantibodies are produced against selected TNF- a fragments Autoantibodies against TNF-α were shown to bind to selected and unique fragments of the TNF-α protein. MA TERIALS AND METHODS: Animals: Female Lewis rats, were obtained and maintained as described in Example 1 of the Examples section. TNF- a autoantibodies: Groups of 3 rats were subjected to active AA induction (induced as described in Example 1 of the Examples section). On day 18, just before the peak of disease, these rats were treated, or were not treated (control) with a TNF-α encoding DNA plasmid (produced as described in Example 1 of the Examples section). Three days later (day 21) blood serum from each group was used to test its TNF-α autoantibodies ability to bind each of different TNF-α fragments described below. Results are shown as mean of triplicates ± SE. Production, purification, and proteolysis of recombinant TNF-a: Recombinant TNF-α was produced as described in Example 1 of the

Examples section. After purification, TNF-α was subjected to trypsin fragmentation (The Protein Service Center of the Technion, Haifa, Israel) followed by HPLC purification, resulting in twenty-four different fragments that constitutes the whole protein. Sequence of each fragment was verified and then used for mapping of antibodies binding by ELISA as described in Example 1 of the Examples section. Statistical analysis: See Example 1 of the Examples Section. RESULTS: In order to explore the hypothesis that DNA vaccines amplify a pre-existing regulatory response aimed to restrain the pathological consequences of autoimmunity, one of the pro-inflammatory cytokines, TNF-α, was chosen for further investigations. TNF-α has been selected for its key function not only in experimental models of autoimmunity, but also in human RA and several related autoimmune diseases (22- 24). The determinants that autoantibodies from AA rats, subjected to a TNF-α encoding DNA vaccine, bind on were mapped. Just before the peak of active AA rats were, or were not subjected to TNF-α encoding DNA vaccine. Three days later blood serum form each group was tested for its ability to bind different TNF-α fragments. Out of 24 different fragments of TNF-α autoantibodies produced in AA rats bound only 3 (Figure 2): Fragment 6 (pi 45- 160, GQGCPDYVLLTHTVSR; SEQ ID NO: 15), Fragment 8 (p207-218, GDLLSAEVNLPK; SEQ ID NO: 16) and Fragment 11 (pl61-176, FAISYQEKVSLLSAIR; SEQ ID NO: 17). Targeted DNA vaccine encoding TNF-α amplified only the response to these 3 fragments (Figure 2). The sequence of each peptide fragment was analyzed in the protein data bank (ENTREZ) and no matching sequence to each of these fragments in any known protein (except for TNF-α) could be found. This further suggests that targeted DNA vaccination encoding TNF-α is highly specific and so effective since it amplifies a pre-existing response.

EXAMPLE 3 TNF- a antibodies neutralize cytotoxic activity ofTNF-ccin-vitro and suppress AA in-vivo The contribution of TNF-α antibody produced during the autoimmune condition to the natural regulation of disease was evaluated by complementary experiments. MA TERIALS AND METHODS: Animals: Female Lewis rats, were obtained and maintained as described in Example 1 of the Examples section. TNF- a cytotoxic activity: The IgG fraction (protein-G purification) of blood sera obtained from AA rats

(peak of disease) was purified on a CNBr-TNF-α purification column and tested for anti TNF-α neutralizing competence in an in vitro system based on (39) with minor modifications (12). These antibodies (100 μg/well), purified IgG from na^'ϊve Lewis rats, or no antibodies (controls), were determined for their competence to inhibit neutral red uptake of U937 cells (4xl0⁴ U937 cells/well; Wallach, D. 1984.

Preparations of lymphotoxin induce resistance to their own cytotoxic effect. J

Immunol 132:2464-2469) in the presence of various concentrations of TNF-α (at concentrations of 0-300 pg/ml) in a total volume of 100 μl. Results are shown as mean O.D. of triplicates at 570nm± SD. Immunizations and active disease induction: Rats were immunized and monitored for clinical signs as described in

Example 1 of the Examples section. A doptive transfer of TNF- a autoantibodies: Rats with developing AA were separated, one day after the onset of disease, into 3 equally sick groups of 6 rats each, that were administered, every other day, with

100 μg/rat of antibodies (IgG fraction of blood sera obtained from AA rats, purified on a CNBr-TNF-α purification column). Control groups were administrated with purified IgG from naϊve Lewis rats, or with equal volume of PBS. The results shown are of one out of three experiments done under the same conditions with similar results. Results are shown as mean maximal score ± S.D. Statistical analysis: See Example 1 of the Examples Section. RESULTS: The contribution of TNF-α antibodies produced during the autoimmune condition to the natural regulation of disease was evaluated. At first the IgG fraction of blood sera obtained from AA rats (peak of disease) was purified on a CNBr-TNF-α purification column and tested for TNF-α neutralizing competence in an in vitro system. Figure 3a shows that these TNF-α antibodies could effectively abolished the cytotoxic activity of TNF-α on U937 human T cells (pO.OOl). Then these antibodies were tested for their competence to suppress AA in adoptive transfer experiments. Figure 3b clearly shows that TNF-α specific antibodies produced in AA rats (even without being subjected to plasmid DNA therapy) can effectively suppress ongoing AA (mean maximal score of 4.66±1 Vs 10.3±1.5 and lO±l in control groups, p<0.01). EXAMPLE 4 TNF- tolerant mice are highly sensitive to AA The importance of TNF-α autoantibodies to the natural regulation of AA disease was evaluated by abolishing their in vivo elicitation. Animals: Female Lewis rats, were obtained and maintained as described in Example 1 of the Examples section. Abolishment of TNF-a autoantibodies elicitation: TNF-α autoantibodies elicitation in the natural regulation of AA disease was abolished by neonatal administration of a self-antigen (i.e. neonatal tolerance) (25) using plasmid DNA vaccines (26). During the first 24 hours after birth, groups of 6 Lewis rats were subjected to a single administration of 10 ng of plasmid DNA encoding TNF-α. Control rats were administered, at that time, with an empty plasmid, or PBS. Another group was administered with the same TNF-α encoding DNA plasmid at 4 weeks of age. Immunizations and active disease induction: Rats were immunized and monitored for clinical signs as described in

Example 1 of the Examples section. At eight weeks of age all rats were subjected to the induction of moderated form of AA and monitored for the development of disease.

Presented results are one out of three experiments done under the same conditions with similar results. Results are shown as mean clinical score ± SD. Evaluation of antibody titer in sera samples: At the peak of disease in control animals (day 22) sera was obtained from all rats and TNF-α specific antibody titer was determined by direct ELISA assay in all rat groups as described in Example 1 of the Examples section. Results are shown as log₂Ab titer ±SE of triplicates. Adoptive transfer of TNF-a autoantibodies: Rat that were subjected to TNF-α encoding DNA plasmid at the neonatal period and to active induction of AA at eight weeks of age, were separated into different groups. One group was administered (any other day beginning at the onset of disease) with TNF-α antibodies (IgG fraction of blood sera obtained from AA rats, purified, on a CNBr-TNF-α purification column). Control groups were administrated with purified IgG from naϊve Lewis rats, or with equal volume of PBS. Results are mean maximal clinical score of 6 rats per group ±SE. Histopathology: Histological analysis was performed as described in Example 1 of the Example section. 30 days after disease induction, 12 sections from each group of rats were examined. Statistical analysis: See Example 1 of the Examples Section. RESULTS: In order to explore the role of TNF-α autoantibodies in the natural regulation of AA disease, their in vivo elicitation was abolished by neonatal administration of a self-antigen (i.e. neonatal tolerance) (25). This approach has recently been extended to plasmid DNA vaccines (26). The TNF-α encoding DNA plasmid was used to induce neonatal tolerance to its gene product and the consequences of this abolishment on the development and progression of AA were evaluated (Figure 4). Since immunization of 1 mg of MT in lOOμl oil resulted in a very severe form of disease (Figure lb, Figure lc) the AA system was re-optimized in order to obtain a moderated form of disease, which could potentially been aggravated. Administration of 0.2-0.4 mg of MT in lOOμl oil was found to induce a moderated form of AA

(Figure 4a). Rats that were subjected to a neonatal administration of TNF-α encoding DNA plasmid failed to elicit TNF-α antibody titer during AA (Figure 4b, log₂Ab of

14.2±0.23 in AA rats that were not subjected to neonatal tolerance Vs 6.2±0.6 in those neonatally administered with TNF-α encoding DNA vaccine, b Vs d, p<0.01) and as a result developed an extremely severe form disease (Figure 4a, mean maximal score of

13.3±0.9 Vs 5.3±0.6 in control AA rats). This group mounted similar levels of antibodies titers to MCP-1 or IP- 10 to those measured in AA rats that were not subjected to neonatal administration of TNF-α encoding DNA (data not shown).

Thus, abrogation of the competence to elicit antibody titer to TNF-α was Ag specific. Replacement therapy with TNF-α specific antibodies reversed the effect of neonatal tolerance (Figure 4c). Clinical scores were verified histologically. Rats that were subjected to a neonatal administration of TNF-α encoding DNA vaccines exhibited an extremely enhanced inflammatory process in the joints compared to control AA rats [Figure 4d (c) Vs 4d (b)] that could be reversed by TNF-α specific autoantibodies (Figure 4d). Administration of plasmid DNA encoding TNF-α after birth (at 4 weeks of age, 4 weeks before induction of disease) led to an enhanced production of antibody to TNF-α [Figure 4b (e), p<0.001 compared to control AA rats] and abolishment of disease (Figure 4a). Interestingly, administration of TNF-α DNA vaccine at 4 weeks of age to rats that have been subjected to neonatal administration of the same vaccine led to a partial recovery in antibody production, and as a result to development of a moderated form of disease (data not shown), suggesting that neonatal tolerance alters an immunological balance rather than eliminating autoreactive cells (27-29). Taken together the above results further emphasize the contribution of naturally produced autoantibodies against pro-inflammatory mediators to the regulation of autoimmunity.

EXAMPLE 5 TNF-a autoantibody titer in human patients suffering from RA The relevance of the results obtained using a rat model were evaluated using human patients suffering from RA in comparison with those suffering from osteoarthritis. MA TERIALS AND METHODS: Blood sera and synovial fluid: Blood sera were obtained from 22 patients suffering from RA, 10 patients suffering from OA and 12 healthy individuals. Synovial fluid was taken from all 22 RA patients. Evaluation of TNF-a antibody titer in sera samples and synovial fluid: See Example 1 of the Examples section. Statistical analysis: One-way ANOVA test (with the Bonferoni correction) was preformed on log₂Ab titer of each individual. RESULTS: To determine the relevance of the findings of this study to human autoimmunity the possible appearance of anti-self antibodies to TNF-α in patients suffering from RA in comparison with those suffering from osteoarthritis, or control healthy subjects was monitored. Figure 5a shows that the vast majority of RA patients (72%), none of the control subjects and only 20% of osteoarthritis patients developed a notable (>log₂=8) antibody titer to TNF-α. One-way ANOVA test (with the Bonferoni correction) showed a significant elevation (p<0.05) in antibody titer determined in sera of RA patients compared to healthy individuals and those suffering from Osteoarthritis. No significant difference could be observed between the last two groups. Very high correlation (R=0.098) was observed between the titer measured in sera and synovial fluid of each RA patient. These observations are highly interesting since neutralizing TNF-α suppresses human RA and displays no effect on Osteoarthritis. Thus, likewise experimental models, during an autoimmune condition the human immune system generate a response that neutralizes pro-inflammatory mediators only when it is beneficial for the patient. Two interesting concepts that the present study explore are the ability of the immune system to distinguish pro-inflammatory mediators to which it should mount beneficial anti self-response, from regulatoiy cytokines to which it should not, and at the same time to distinguishes a local inflammatory process (i.e. CFA immunization to hind foot pads) from an autoimmune disease, resulting from the immunization of the same CFA to base tail, while mounting a significantly higher antibody titer to each detected pro-inflammatory mediator during the autoimmune condition. The biological significance of both features is apparent. Turning on an autoimmune antibody response to pro-inflammatory mediators is beneficial during an autoimmune condition but could be disadvantageous to the host during microbial inflammation. Anti regulatory cytokine immunity could be detrimental during an autoimmune conditions since it might block the beneficial function of regulatory T cell that blunt the pathological consequences of these diseases (36, 37). The autoantibodies produced in response to each pro-inflammatory mediator were IgG. Additional characterization reviled that the vast majority of antibodies produced during the early and acute phase of disease against TNF-α were of the IgG2a isotype (Thl dependent in rodents) that switched during the chronic phase to a

Th2 dependent production of IgG 1 (data not shown). This implies that breakdown of tolerance to pro-inflammatory mediators is a T dependent antibody mediated response. In the process of T dependent antibody production professional antigen presenting cells (APC, i.e. dendritic cells and macrophages) present self-pro- inflammatory mediators in association with MHC II to CD4+ T cells that assist antibody production by B cells. Why would dendritic cells and macrophages present self-pro-inflammatory mediators? These cells express high levels of receptors to TNF- α and to each of the pro-inflammatory chemokines discussed above, and to a much lesser extent, if at all to regulatory cytokines. Previous studies showed that following binding their target receptors on macrophages/dendritic cells chemokines undergo rapid internalization (38). It is possible that this mechanism that is primarily important for desensitization and intracellular signaling, also enable an effective presentation of these inflammatory mediators by MHC II and thus play a major role in the initiation of beneficial autoimmunity. It is suggested that this mechanism is turned on so effectively during autoimmunity and its function can be amplified so rapidly by DNA vaccines, due to the persistence of a pre-existing regulatory network selected to restrain the pathological consequences of autoimmunity. From the practically oriented perspective the finding that human patients suffering from RA develop autoantibody response to TNF-α implies for the significance of the findings in the experimental model. It also suggests the possible use of targeted DNA vaccines as a potential therapeutic way for this and other diseases. The major advantageous of such therapy over engineered chimeric antibody/soluble receptor therapy are apparent. An ideal way of therapy would be dependent on teaching the immune system how to use its own tools to restrain its harmful activities.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

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Claims

WHAT IS CLAIMED IS:

1. A method of identifying a putative drug target, the method comprising: (a) identifying an autoantibody populations which displays higher titer in an abnormal biological sample as compared to a normal biological sample; and (b) identifying a disease-causing biomoelcule targeted by said autoantibody population thereby identifying the putative drug target.

2. The method of claim 1, wherein said disease-causing biomolecule is a proinflammatory mediator.

3. The method of claim 1 , wherein said disease-causing biomolecule is not a regulatory mediator.

4. The method of claim 1, wherein said autoantibody population includes IgG antibodies.

5. A method of diagnosing a disease in a subject, the method comprising determining a titer of an autoantibody population directed at an endogenous disease- causing biomolecule in a biological sample obtained from the subject, wherein a titer of said autoantibody population above a predetermined normal titer is indicative of the disease.

6. A method of treating a disease in a subject, the method comprising providing to the subject a therapeutically effective amount of at least a portion of a disease-causing biomolecule endogenously produced by the subject, thereby treating the disease in the subject.

7. The method of claim 6, wherein said at least a portion represents an immuniogenic non-acti e portion of said disease causing biomolecule.

8. The method of claim 6, wherein said disease causing biomolecule is a polypeptide.

9. The method of claim 8, wherein providing said protein is effected by: (i) administering said polypeptide to the subject; and/or (ii) administering an expressible polynucleotide encoding said polypeptide to the subject.

10. The method of claim 6, further comprising administering an antibody directed against said disease-causing biomolecule.