Pi Kappa Alpha Risk Awareness Handbook Of Pharmaceutical Excipients

Provided are novel binding molecules of human origin, particularly human antibodies as well as fragments, derivatives and variants thereof that recognize antigens such as native endogenous proteins associated with, e.g., immune response, autoimmune disorders, inflammatory diseases, metabolic disorders, vascular function, neurodegenerative diseases or tumors. More particularly, a human Auto-Immunosome and corresponding monoclonal antibody reservoir are provided. In addition, pharmaceutical compositions, kits and methods for use in diagnosis and therapy of are described. The antibody or antigen-binding fragment of any one of claim 34 to 39, 43 or 44, the anti-idiotypic antibody of claim 45 or the peptide or peptide-based compound of claim 46 for use in treating or preventing the progression of an inflammation or an autoimmune disorder; for the amelioration of symptoms associated with an inflammation or an autoimmune disorder; for diagnosing or screening a subject for the presence or for determining a subject's risk for developing an inflammation or an autoimmune disorder. The method of claim 56, wherein said disorder is selected from the group consisting of Acne vulgaris; Arthritis such as Gouty arthritis, Systemic lupus erythematosus (SLE), Osteoarthritis, Psoriatic arthritis, Rheumatoid arthritis; Asthma; Celiac disease; Chronic prostatitis; Dermatitis; Diabetes mellitus Type 1; Glomerulonephritis; Hypersensitivities; Myocarditis; Multiple sclerosis; Inflammatory bowel diseases; Pelvic inflammatory disease; Polymyositis; Psoriasis; Sarcoidosis; Vasculitis; Interstitial cystitis or the inflammation occurs due to reperfusion injury or transplant rejection. The present invention generally relates to novel binding molecules of mammal, preferably human origin, particularly human monoclonal antibodies as well as fragments, derivatives and variants thereof that recognize antigens such as native endogenous proteins associated with, e.g., immune response, autoimmune disorders, inflammatory diseases, metabolic disorders, vascular function, neurodegenerative diseases or tumors.

In addition, compositions comprising such binding molecules and mimics thereof, methods of screening for novel binding molecules, which may or may not be antibodies, targets and drugs useful in the treatment and diagnosis of disorders are described. Furthermore, the present invention relates to autoantibodies as agents for use in immunotherapy as well as targets in the therapeutic intervention of autoimmune and autoinflammatory disorders as well as malignancies. More specifically, the present invention relates to monoclonal autoantibodies isolated from B cells derived from subjects affected with an impaired central and/or peripheral tolerance or loss of self-tolerance typically due to a mutation in a gene involved in immune regulation.

BACKGROUND OF THE INVENTION•. Inappropriate responses of the immune system may cause stressful symptoms to the involved organism. Exaggerated immune answers to foreign substances or physical states which usually do not have a significant effect on the health of an animal or human may lead to allergies with symptoms ranging from mild reactions, such as skin irritations to life-threatening situations such as an anaphylactic shock or various types of vasculitis. Immune answers to endogenous antigens may cause autoimmune disorders such as systemic lupus erythematosus, idiopathic autoimmune hemolytic anemia, pernicious anemia, type 1 diabetes mellitus, blistering skin diseases and different kinds of arthritis.

Aug 18, 2017. - Pi Kappa Alpha Risk Awareness Handbook Of Pharmaceutical Excipients; - Multicharts 8 7 Crack Full; - A Volte Ritornano Pdf To Excel. A hedge is an investment position intended to offset potential losses or gains that may be incurred by a companion investment. Jul 27, 2017. A pharmaceutical composition comprising the anti-Sortilin antibody of claim 94, and a pharmaceutically acceptable carrier. A method of preventing, reducing risk, or treating an individual having arthritis, comprising administering to the individual a therapeutically effective amount of the anti-Sortilin.

Cytokines are secreted soluble proteins, peptides and glycoproteins acting as humoral regulators at nano- to picomolar concentrations behaving like classical hormones in that they act at a systemic level and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. Cytokines differ from hormones in that they are not produced by specialized cells organized in specialized glands, i.e. There is not a single organ or cellular source for these mediators as they are expressed by virtually all cells involved in innate and adaptive immunity such as epithelial cells, macrophages, dendritic cells (DC), natural killer (NK) cells and especially by T cells, prominent among which are T helper (Th) lymphocytes.

At this time, Th1, Th2, Th17 and iTreg T-cell subclasses have been defined depending on the specific spectrum of cytokines expressed, and respective immunological responses modulated by them. In this respect, principal cytokine products of Th1-cells are IFN-gamma, lymphotoxin-alpha and Interleukin 2 (IL-2). Th1-cells mediate immune responses against intracellular pathogens and are responsible for some autoimmune diseases (Mosmann et al., Annu. 7 (1989), 145-173; Paul and Seder, Cell 76 (1994), 241-251).

Th2-cells produce primarily IL-4, IL-5, IL-9, IL-10, IL-13, IL-25 and amphiregulin. Th2-cells mediate immune responses against extracellular parasites, and are involved in autoimmunity and allergies such as asthma, Type 1 diabetes mellitus, Multiple sclerosis (MS) and Rheumatoid arthritis (RA); see, e.g., Mosmann et al. (1989) and Paul and Seder, (1994), supra. Depending on their respective functions, cytokines may be classified into three functional categories: regulating innate immune responses, regulating adaptive immune responses and stimulating hematopoiesis. Due to their pleiotropic activities within said three categories, e.g., concerning cell activation, proliferation, differentiation, recruitment, or other physiological responses, e.g., secretion of proteins characteristic for inflammation by target cells, disturbances of the cell signaling mediated by aberrantly regulated cytokine production have been found as a cause of many disorders associated with defective immune response, for example, inflammation and cancer.

Central in inflammation and autoimmune diseases are cytokines IL-17A and IL-17F, IL-22, IL-12, IL-23 and subtypes of the IFN-alpha and IFN-omega. Except IL-12, IL-23 and IFNs, said cytokines are expressed by the effector T-cell subset Th17 which have well-described roles in several autoimmune and allergic disorders and in tumour immunology.

Unregulated Th17 responses are associated with chronic inflammation and severe immunopathologic conditions. IL-22, a third Th-17 related cytokine (Zenewicz and Flavell, Eur. 38 (2008), 3265-3268) is a cytokine that acts mainly on epithelial cells. In the skin, it mediates keratinocyte proliferation and epidermal hyperplasia and plays a central role in inflammatory diseases, such as psoriasis.

IL-22 is a signature product of Th17 cells, also secreted at functionally significant levels by other immune cells, especially NKp44/NKp46-expressing natural killer (NK) cells and lymphoid tissue inducer cells after IL-23 stimulation. Interleukin-22 (IL-22) is a member of the IL-10 family of anti-inflammatory cytokines that mediates epithelial immunity. IL-22 expression is enhanced in inflamed colon mucosa in individuals with inflammatory bowel disease. Importantly, IL-22 does not serve the communication between immune cells. It mainly acts on epithelial cells and e.g. Hepatocytes, where it favors the antimicrobial defense, regeneration, and protection against damage and induces acute phase reactants and some chemokines (Wolk et al., Semin.

32 (2010), 17-31). IL-23 is a key cytokine in promotion of chronic inflammation, secreted by activated inflammatory cells like macrophages and dendritic cells (see for review Langrish et al., Immunological Reviews 202 (2004), 96-105; Ferraccioli and Zizzo, Discov. 60 (2011), 413-24; further Langrish et al., J. 201 (2005), 233-40; Vaknin-Dembinsky et al., J. 195 (2008), 140-145; Melis et al., Ann. 69 (2010), 618-23). Initially, IL-23 was found to have a role in supporting the expansion and maintenance of Th17 cells (Aggarwal et al., J.

278 (2003), 1910-1914; Weaver et al., Annu. 25 (2007), 821-852). However, it was shown as having multiple effects on the immune response, including restraining the activity (intestinal accumulation) of Foxp3-positive regulatory T-cells (Barnes and Powrie, Immunity 31 (2009), 401-411; Izcue et al., Immunity 28 (2008), 559-570; Ahern. Immunity 33 (2010), 279-88) and inducing the expression of Th17-type cytokines from non-T-cell sources (Buonocore et al., Nature 464 (2010), 1371-1375; Morrison et al., Immunology 133 (2011), 397-408). It contributes to inflammation, e.g., in psoriasis and inflammatory bowel diseases (see publications supra and Duvallet et al., Ann. 43 (2011), 503-511). IL-23 is composed of two subunits, p19 and p40; the latter subunit is shared with IL-12.

Recent therapeutic approaches for chronic inflammatory diseases such as severe RA and PS involve blockage of tumor necrosis factor-α (TNF-α). While this method is highly effective in some cases, many patients are ‘non-responders.’ Furthermore, a sustained neutralization of TNF-α can enhance susceptibility to microbial infections, highlighting the need for alternative and more specific therapeutic approaches. So far, modulation of Th-17 cell function and/or differentiation has been most successful with monoclonal antibodies to IL-12 p40, which target both IL-12 (a heterodimer of p35 and p40) and IL-23 (a heterodimer of p19 and p40, a growth factor for Th17 cells). Such antibodies, ustekinumab and ABT-874 that target IL-12 p40, have been successful in moderate to severe chronic plaque psoriasis and CD (Miossec et al., N.

361 (2009), 888-898, Steinman, Nat. 11 (2010), 41-44). The most feasible way to control the biologic effects of Th17 cells and cells with related properties, e.g., IL-17-producing gamma delta T cells, would be to target them and a selection of the effector cytokines they produce.

This novel approach would enable a blockade of IL-22 in PS, and of IL-17A (and IL-17F) in RA and CD, leaving the other non-pathogenic arms intact for fighting pathogens and, in the case of IL-22, for exerting its tissue reparative and antimicrobial functions. Also, in some initial responders, the efficacy of the anti-TNF therapy drug wanes over time. Thus, severe cases not responding to monotherapy might require combination therapy with two or more biological drugs. Monoclonal antibodies against IL-17A have been developed for clinical application. Phase 2 trials of a monoclonal antibody against IL-17A (AIN457) for PS, CD and psoriatic arthritis are under way. So far, inhibitors of IL-22 have been tested only in pre-clinical models of autoimmune diseases (Miossec et al., N. 361 (2009), 888-898, Steinman, Nat.

11 (2010), 41-44). Due to immunological responses to foreign antibodies, as mouse antibodies in humans (HAMA-response; Schroff et al., Cancer Res.

45 (1985), 879-885; Shawler et al., J. 135 (1985), 1530-1535), mostly humanized versions of antibodies are used in present therapeutic approaches (Chan et Carter, Nature Reviews Immunology 10 (2010), 301-316; Nelson et al., Nature Reviews Drug Discovery 9 (2010), 767-774). One approach to gain such antibodies was to transplant the complementarity determining regions (CDR) into a completely human framework, a process known as antibody humanization (Jones et al., Nature 321 (1986), 522-525).

This approach is often complicated by the fact that mouse CDR do not easily transfer to a human variable domain framework, resulting in lower affinity of the humanized antibody over their parental murine antibody. Therefore, additional and elaborate mutagenesis experiments are often required, to increase the affinity of the so engineered antibodies. Another approach for achieving humanized antibodies is to immunize mice which have had their innate antibody genes replaced with human antibody genes and to isolate the antibodies produced by these animals. However, this method still requires immunization with an antigen, which is not possible with all antigens because of the toxicity of some of them. Furthermore, this method is limited to the production of transgenic mice of a specific strain. Another method is to use libraries of human antibodies, such as phage display, as described, for example, for the generation of IL-13 specific antibodies in international application WO 2005/007699.

Here, bacteriophages are engineered to display human scFv/Fab fragments on their surface by inserting a human antibody gene into the phage population. Unfortunately, there is a number of disadvantages of this method as well, including size limitation of the protein sequence for polyvalent display, the requirement of secretion of the proteins, i.e.

Antibody scFv/Fab fragments, from bacteria, the size limits of the library, limited number of possible antibodies produced and tested, a reduced proportion of antibodies with somatic hypermutations produced by natural immunisation and that all phage-encoded proteins are fusion proteins, which may limit the activity or accessibility for the binding of some proteins. Similarly, European patent application EP 0 616 640 A1 describes the production of auto-antibodies from antibody segment repertoires displayed on phage. Phage libraries are generated from unimmunized humans in this respect (see, e.g., Example 1; page 16, lines 43-51; Example 2, at page 17, paragraph [0158], lines 57-58). However, also the methods described in this patent application suffer from above mentioned general disadvantages of antibodies generated from phage libraries, in comparison to antibodies produced and matured in a mammalian, i.e. The present invention generally relates to means and methods for isolating autoantigen binding molecules, i.e. Antibodies from mammals, in particular humans, which are affected with an impaired central and/or peripheral tolerance or loss of self-tolerance which may be due to or associated with a disrupted or deregulated genesis of self-tolerance, preferably caused by a monogenic autoimmune disorder. Examples of mammals which provide a particularly suitable source for autoantibodies in accordance with the present invention are mammals, e.g., humans having a disorder associated with a mutation in the AIRE (Autoimmune Regulator) gene such as Autoimmune polyendocrinopathy syndrome type 1 (APS1) (Peterson et al., Nat.

8 (2008), 948-957). In particular, anti-IL-17F and IL-22 antibodies as well as antigen-binding fragments thereof are provided, which demonstrate the immunological binding characteristics and/or biological properties as outlined for the antibodies illustrated in the Examples.

Where present, the term “immunological binding characteristics,” or other binding characteristics of an antibody with an antigen, in all of its grammatical forms, refers to the specificity, affinity, cross-reactivity, and other binding characteristics of an antibody. 1: A: APS1 patient seroreactivity (as a percentage of total shown number of reactivities that have been measured in at least one patient) against extracellular proteins that have been implicated in physiological functioning of tissues or pathophysiology of human disease. • • B: APS1 patient seroreactivity (total number of distinct proteins recognized by sera of at least one patient) against extracellular proteins that have been implicated in physiological functioning of tissues or pathophysiology of human disease. 2: A: APS1 patient seroreactivity (as a percentage of total shown number of reactivities that have been measured in at least one patient) against extracellular proteins that have been implicated in physiological functioning of tissues.

Seroreactivities are independent of those shown in FIGS. • • B: APS1 patient seroreactivity (total number of distinct proteins recognized by sera of at least one patient) against extracellular proteins that have been implicated in physiological functioning of tissues. Seroreactivities are independent of those shown in FIGS. 5: In-vitro neutralizing capacities of recombinant antibodies of the present invention. A: Neutralizing capacities against human IL-22. IC50 for recombinant antibody 30G1 is 2.5 ng/ml and for 35G11 4 ng/ml using 0.5 ng/ml of IL-22. Recombinant antibodies 41D11 and 51G4 are not neutralizing at tested concentrations.

B: Neutralizing capacities against mouse IL-22. IC50 for recombinant antibody 30G1 is 1.5 ng/ml using 0.3 ng/ml mouse IL-22. Recombinant antibodies 35G11, 41D11 and 51G4 are not neutralizing at tested concentrations. C: Neutralizing capacities against IL17A/IL-17F heterodimer. IC50 for recombinant antibody 24D3 is 6 ng/ml, for 17E3 12 ng/ml using 10 ng/ml IL17A/IL-17F heterodimer.

Recombinant antibody 9A2 is not neutralizing at tested concentrations. D: Neutralizing capacities against IL17F. IC50 for recombinant antibody 24D3 is 12 ng/ml, for 17E3 15 ng/ml and for 9A2 45 ng/ml using 10 ng/ml IL17F.

6: Antibody response against IFN-alpha1 in APS1 patients (A: APS-patients 1-01 to 1-07, B: patients 1-08 to 1-14, C: patients 1-15 to 1-17). Test sera obtained from patients suffering from the genetic condition APS1/APECED were tested for presence of IFN-alpha 1 antibodies in an ELISA-assay at different dilutions ranging from 1:50 to 1:156250, as indicated at the X-axis. Control serum was obtained from healthy laboratory personnel, age matched with the patients. Y-axis indicates OD values.

8: ELISA titers of sera isolated from APS1 patients (APS1-1 to APS1-22; filled diamonds) and controls (healthy subjects; C1 to C8; filled squares) against IL-22. X-axis shows the age of the subject and Y-axis the titer seen on a LOG 10 scale. The large open diamond and open square shows the mean titer in the two subject groups. Note that the median age of the controls and patienst are similar, but titers in patients are approximately 100 times higher that in controls. The age of each individual is indicated on the X-axis and the titer (LOG 10 scale) on the Y-axis. 14: Full-length IL-22 (34-179 aa), N-terminally truncated (74-179 aa) or C-terminally truncated (34-116 aa) IL-22 sequences were cloned into fusion with firefly luciferase at its C-terminus using pPK-CMV-F4 (PromoCell GmbH, Heidelberg; Germany) mammalian expression vector. After transfection into HEK 293 cells, crude protein extracts were used as antigen in immunoprecipitation assay.

The figure shows that the recombinant antibodies specific for IL-22 are binding the full-length IL-22 most efficiently indicating to the predominance of conformational epitopes. LU-light units. 15: Amino acid sequences of the variable region, i.e. Heavy chain and kappa/lambda light chain (VH, VL) of IL-17A, IL17F or IL-22 specific human antibodies of the present invention. A: IL-17F specific antibody 9A2: IgG1, kappa (VH3; Glm17; VK1, Km3).

B: IL-17F and IL-17A specific antibody 17E3: IgG1, kappa (VH1, Glm17; VK1, Km3). C: IL-17F specific antibody 24D3: IgG1, lambda (VH3, Glm17; VL3). D: IL-22 specific antibody 30G1: IgG1, kappa (VH3, Glm17; VK3, Km3 or Km1, 2). E: IL-22 specific antibody 35G11: IgG4, lambda (VH7, IGHG4*01; VL1), F: IL-22 specific antibody 41D11: IgG1, lambda (VH4, Glm3 L410F; VL4) and G: IL-22 specific antibody 51G4: IgG2, lambda (VH1, IGHG2*01; VL3). Framework (FR) and complementarity determining regions (CDRs) are indicated with the CDRs being underlined. Due to the cloning strategy the amino acid sequence at the N-terminus of the heavy chain and light chain may potentially contain primer-induced alterations in FR1, which however do not substantially affect the biological activity of the antibody. In order to provide a consensus human antibody, the nucleotide and amino acid sequences of the original clone are aligned with and tuned in accordance with the pertinent human germ line variable region sequences in the database; see, e.g., Vbase (hosted by the MRC Centre for Protein Engineering (Cambridge, UK).

31: Autoimmune reactivity of sera from APECED patients with: Column (A)—previously reported APECED autoantigens GAD2 (glutamate decarboxylase 2), DDC (dopa decarboxylase (aromatic L-amino acid decarboxylase)) and GAD1 (glutamate decarboxylase 1); Column (B)—novel (unreported) autoantigens SULT4A1 (sulfotransferase family 4A, member 1), CCDC40 (coiled-coil domain containing 40), NAP1L (nucleosome assembly protein 1-like 1); and Column (C)—with cancer antigens ABL1 (c-abl oncogene 1, non-receptor tyrosine kinase), MAGEA3 (melanoma antigen family A3) and SSX4 (synovial sarcoma, X breakpoint 4). The Protoarray results are shown as fluorescence values of Invitrogen Protoarray provided herein probed with sera of 23 Finnish APECED patients (APECED) and 7 healthy control (Ctrl) sera. The mean values with SEM for each patient/control group are shown. 33: Anti-IL22 (30G1)+IMQ psoriasiform lesion in vivo study. Experimental protocol and timeline detailing anti-IL22 antibody administration both pre and post (24 hours after) IMQ induction in C57BL/6J mice. Age at shaving 9 weeks, Non-treated control DOB (Date of birth): at 10 weeks A: Tables indicating the treatment composition of the animal groups A to G.

B: Amounts of the control and anti-IL22 antibody applied to the animals. C: Experimental timeline. LN stim=Lymph node stimulation. Clinical data of animal #19 was excluded from analysis due to severe weight loss. Indications shown here are the same as for experiments shown in FIG.

36: Administration of exemplary aIL-22 antibody 30G1 significantly reduces skin thickening in IMQ-induced mice, even if administered “therapeutically”—24 hours post IMQ induction rather than prior to IMQ induction. Significant difference could be observed in skin thickness between IgG+IMQ and aIL-22+IMQ treated groups at Day 4.

A: duplicate skin thickness measurements on the back of the treated animals in mm. B: fold change in the skin thickness of said animals.

The lines in the graphs are numbered in the figure for a better readability. 37: Administration of exemplary aIL-22 antibody 30G1 significantly reduces skin erythema clinical psoriasis scoring in IMQ-induced mice, even when administered “therapeutically”. (A-C) Significant difference in skin erythema could be observed between IgG+IMQ and aIL+IMQ treated groups at days 4 and 5. Redness (A), Scales (B) and Hardness (C) of the plaques were evaluated according to the Psoriasis Area and Severity Index (PASI) score.

The three features of a psoriatic were each assigned a number from 0 to 4 with 4 being worst. 38: Dose-dependent effect of exemplary anti-IL22 antibody 30G1 of the present invention in IMQ-induced psoriasis. Three different doses—200 μg, 20 μg and 2 μg were administered post IMQ-induction. (A) skin erythema and (B) hardness of the plaques were evaluated as described in previous Figs.

(C) and (D) show the values for respective aIL-22 or control treatment. (E) shows the cumulative scores for the different treatments. The mouse cross-reactive, exemplary anti-IL22 antibody of the present invention 30G1 shows indications of dose dependence, proving less effective when titrated down to 1/100 of the initial dose. 39: Combined results of two independent CytoEar experiments—measurements of ear thickness over time show a reduction of the IL17F induced ear inflammation phenotype by injections of exemplary aIL-17 24D3 antibody of the present invention.

(A) Overview of the results. (B) Control experiments—injection of PBS. (C) Induction of inflammation by IL17A-injections.

(D) Induction of inflammation by IL17F-injections—reduced induction because of 24D3 injections. (E) shows combined graphs of Figs. (F) shows combined graphs of Figs. Significant difference (Day 8) could be observed in ear thickness between the Groups C and F in mice given IL-17F and treated with IgG or 24D3 (D, F).

(D and F) 2-Way ANOVA ns at Day 0 to Day 7 **p. 41: Epitope mapping. Differential binding of exemplary IL-17 and aIL-22 MABs of the present invention to distinct binding sites.

Cross-competition experiments show that anti-IL-17 antibodies 17E3 and 24D3 compete against each other but not against antibody 9A2 indicating a different binding site for the latter (A). Similarly, anti-IL-22 antibodies 30G1, 35G11 and 41D11 compete against each other but not against antibody 51 G4, indicating as well two binding sites, one for the first group of antibodies and a second for antibody 51G4 (B).

The results for the exemplary anti-IL-22 antibodies of the present invention have been verified by sandwich ELISA experiments (C). Antibody 9A5 is a MAGEA3 specific antibody which is non-reactive with IL-17 and IL-22 and used herein as negative control (A, B). 42: Results of the PepStar™ analysis of the binding regions of MABs to their respective antigens—IL17F. Binding to overlapping 20mer peptides (15 amino acid overlap) spotted on micro array is shown covering IL-17F including all known variants with full-length IL-17F as last column The low signal strength observed in respect of the binding to the full length antigen does not necessarily correlate to a lack of binding, since the low signal strength or lack of signal may also occur due to the orientation and use of surface lysines to immobilize the antigen. However, observed binding to full-length IL-17F suggests a preferential binding of the exemplary antibodies 24D3 (A), 9A2 (B) and 17E3 (C) to conformational epitopes. X-axis from left to right: Peptides numbered 1 to 23 and full-length IL-17F •.

43: Results of the PepStar™ analysis of the binding regions of MABs to their respective antigens—IL-22. All antibodies show very weak binding patterns to microarray bound peptides indicating failure to recognize linear peptides derived from their specific antigen.

Observed low signal strength may have the same causes as indicated in FIG. All tested anti-Il-22 antibodies appear to bind preferentially to conformational epitopes. Preliminary binding results are shown for anti-IL-22 antibodies: (A) 30G1, (B) 35G11, (C) 41D11 and (D) 51G4. X-axis from left to right: Peptides numbered 1 to 27 and full-length IL-22. Y-axis—Intensity distribution from 10000 to 60000 in 10000 steps. 44: Results of ProteOn XPR36 based analysis of antibody affinities.

Injection of IL-22 in concentrations of A1: 100 nM, A2: 33.33 nM, A3: 11.11 nM A4: 3.70 nM A5: 1.23 nM and A6: 0 nM (running buffer only). Identifiers A1-A6 are preceded by prefixes L1 to L6 (L1A1 etc.). Same concentrations were used in the following FIGS. Data was referenced using the interspot regions of the sensor chip.

Surfaces coated with exemplary aIL-22 antibodies of the present invention 30G1 and 35G11 show specific, concentration dependent binding reactions of the ligand. 45: Detailed analysis of the sensograms concerning the binding of IL-22 to the aIL-22 30G1 antibody of the present invention. (A) Overlayed graphs for the Langmuir fit and experimental data of the binding reaction indicate a good fit to 1.1 Langmuir model. Residuals plot (B) shows a random scatter with the magnitude of the noise level indicating a good residual fit. Table below the figures shows the kinetic parameters derived from the fitted curves for the association (k a), dissociation (k d), R max and the calculated dissociations constant KD.

50: Schematic representation of a general preferred process for isolating and producing recombinant human monoclonal antibodies derived from human memory B cells obtained from patients which are affected with an impaired central and/or peripheral tolerance or loss of self-tolerance, in particular APECED/APS1 patients secreting IgG antibodies that bind and/or neutralize human autoantigens comprising the methods of the present invention for providing short term oligoclonal cultures of antibody-secreting cells. DETAILED DESCRIPTION OF THE INVENTION•. The principle attempt of cloning human antibodies is known in the art; see, e.g., Traggiai et al. Describing an efficient method to make human monoclonal antibodies from memory B cells, which are capable of neutralization of SARS coronavirus in Nature Medicine (Traggiai et al., Nat. 10 (2004), 871-875), and international applications WO2008/081008, WO2010/069603 and WO2008/110372 describing the cloning of human antibodies, in particular human antibodies recognizing beta-amyloid peptide, alpha synuclein and NY-ESO protein, a tumor specific antigen.

In all these approaches as well as in other previous attempts healthy volunteers or patients have been used as the source of the human antibodies suffering from the disease caused by the infectious agent or associated with one particular antigen but having an unusual stable and mild disease state as indication of the presence of protective autoantibodies. This approach suffers from the drawback that in principle for each antigen of interest a corresponding patient pool has to be identified producing an appropriate antibody such patients however may not always be available. Furthermore, though the antibodies so identified may be protective in those patients from whom they have been isolated they may be not effective or may even do not recognize the antigen in different patient populations, for example due to the presence of different neoepitopes.

Furthermore, for some disorders or clinical pictures such as indicated hereinabove the respective antigen or group of antigens may not be known yet. In this context, experiments performed in accordance with the present invention surprisingly revealed that APECED/APS1 patients display an auto-immunosome, i.e an autoantibody profile which in its variety is outstanding and represents a broad spectrum of binding molecules specific for proteins prone to invoke an autoimmune response and/or potentially associated to disorders related to an undesired autoimmune response. Thus, the provision of molecule members of the auto-immunosome identified in accordance with the present invention may pave the way for novel targets and means for therapeutic intervention and diagnosis of such disorders. APS1 is a rare autoimmune disease caused by mutations in the Autoimmune Regulator (AIRE) gene.

The AIRE protein governs the expression in medullary thymic epithelium of many peripheral self-antigens (e.g., insulin) that are presented by MHC to tolerise developing thymocytes. In APS1, AIRE mutations cause aberrant negative selection, which enables autoreactive T cells to escape to the periphery. Another thymus-associated disorder which surprisingly has been found with similar high titre autoantibodies is thymoma, a tumor of thymic epithelial cells, often associated with the autoimmune disease myasthenia gravis (MG), where apparently due to the disturbed function of the thymus cells, negative selection of autoantibodies can be impaired in a similar manner as in APS1. Of all thymoma cases, 30-45% of patients have MG. Additional associated autoimmune conditions include pure red cell aplasia and Good's syndrome (thymoma with combined immunodeficiency and hypogammaglobulinemia).

Other reported disease associations are with acute pericarditis, Addison's disease, agranulocytosis, alopecia areata, ulcerative colitis, Cushing's disease, hemolytic anemia, limbic encephalopathy, myocarditis, nephrotic syndrome, panhypopituitarism, pernicious anemia, polymyositis, rheumatoid arthritis, sarcoidosis, scleroderma, sensorimotor radiculopathy, stiff person syndrome, systemic lupus erythematosus and thyroiditis. In view of the findings of the present invention, which will be also described further below but without intending to be bound by theory, the present inventors hypothesized that the high titer and diverse profile of autoantibodies observed in associated disorders, in particular APECED/APS1 patients is due to an impaired central and/or peripheral tolerance and loss of self-tolerance, respectively, leading to an humoral immune response including both, i.e.

“autoagressive” autoantibodies which may be responsible for the susceptibility of such patients to infections such as candidasis but at the same time produce “protective” antibodies which provide a sort of autovaccine against common disorders of autoimmune or inflammatory origin as well as neoplastic diseases such as cancer. In addition, experiments performed within the scope of the present invention surprisingly revealed that autoantibodies observed in APECED patients which seem to be responsible for suppressing an appropriate immune response against candida infection at the same time have therapeutic utility in the treatment of chronic inflammatory and immunological diseases; see Examples 5, 6 and 12. In accordance with the present invention the hypothesis could be substantiated by screening the sera of APECED/APS1 patients not only for autoantibodies against mediators of immune response such as interferons and interleukins for which the presence of autoantibodies had already been described but also to antigens which are associated with metabolic disorders, vascular function, neurodegenerative diseases and tumors. Those experiments surprisingly revealed that APECED/APS1 patients also produce autoantibodies which otherwise would be rather expected in for example tumor patients with a stable clinical course of the disease because of the presence of autoantibodies against tumor cells or proteins mediating neoplastic growth of the cells. In particular, in accordance with the present invention protein microarray (ProtoArray®; Invitrogen, Carlsbad USA) have been screened against sera of APECED/APS1 patients and identified a complete set of auto-antigens as many as 3000, which may also be referred to as “AutoImmunosome”; see Example 7 and Tables 1 to 13.

In particular, sera from 23 APECED patients and 7 healthy control subjects were tested against the protein array with in total 9000 recombinant proteins. Antibody activity of the APECED sera compared to controls was positive against 3000 target antigens.

As demonstrated in the Examples, experiments performed in accordance with the present invention also confirmed that known auto-antigen candidates showed sero-reactivity. Further experiments performed in accordance with the present invention were successful in cloning autoantibodies to selected autoantigens, i.e. IL-17F and IL-22; see Example 2. In particular, a method for cloning autoantibodies of desired specificity, i.e. Against any of the identified autoantigen of the mentioned AutoImmunosome is disclosed, thereby providing the first time a common “Mab-AutoImmunosome”, i.e.

A complete set of auto-antibodies and thus reservoir for antibodies and binding molecules useful for therapy and diagnosis of autoimmune disorders as well as diseases caused by or associated with the abberant expression or presence of autoantigens. Hitherto, APECED/APS1 patients were known to show a broad seroreactivity which however could also be assumed to result from a rather unspecific immune response and thus polyclonal antibodies without any particular pronounced biological activity, specificity and affinity. This is particular true for the patient pool preferably selected in accordance with the present invention and illustrated in the Examples, wherein the patients do not only suffer from symptoms known from the clinical picture of common autoantibody disorders but from a general poor physical and/or psychological state of health indicated by enamel defects in teeth, nail and hair abnormalities, loss of skin pigment (vitiligo), loss of hair (alopecia), mental depression. In this context, it is noted that despite similarities in clinical condition, there is a clear distinction between those caused by mutation in gene involved in autoimmune regulation and/or development such as the aire gene, and the solitary components of the diseases, such as Addison's disease and diabetes mellitus that are caused by joint activity of environmental factors affecting individuals with so called risk-HLA haplotypes. For example, APS1, with its mucocutaneous candidiasis, hypoparathyroidism, and Addison's disease, is a monogenic disorder resulting from mutations of the autoimmune regulator gene (AIRE) while in contrast, APS2, similar to most common autoimmune disorders, is a polygenic disease with the dominant susceptibility locus on chromosome 6 within the major histocompatibility complex (MHC).

APS2 was reported to be associated with the class 1 human leukocyte antigen (HLA) allele B8. With the discovery of class II HLA alleles, and their strong association with type 1 diabetes, and linkage disequilibrium of HLA-DR3 with HLA-B8, it was assumed that the association of HLA-B8 with Addison's disease was predominantly due to its association with HLA-DR3 and DQB1*0201, as is the case for type 1 diabetes and celiac disease; for review see, e.g., Baker et al., J. 95 (2010), E263-E270.

As explained above and demonstrated in the Examples, the concept underlying the present invention has been demonstrated with B cell samples from patients suffering from APECED/APS1, i.e. A rare monogenic autoimmune disease caused by mutations in the AIRE gene. In this context, the use of B cell samples from patients suffering from a monogenic disorder responsible for the impaired central and/or peripheral tolerance or loss of self-tolerance is advantageous and preferred over samples from for example patients suffering from polygenic disorders such as APS2 or from thymoma since because being monogenic the clinical picture of the disorder is quite reliable among those patients and does allow easy reproducibility of the method of the present invention. Furthermore, monogenic disorders more easily allow the generation of corresponding animal models such as mice, for example by inducing substantially the same mutation in the corresponding gene such as AIRE. Accordingly, in one preferred embodiment of the method of the present invention the disorder of the patient providing the biological sample is monogenic and, preferably a disorder associated with at least one mutation of the Autoimmune Regulator (AIRE) gene; see also supra. Alternatively, or in addition the monogenic disorder may be associated with at least one mutation in the forkhead box protein 3 (FOXP3) gene; see also infra. Nevertheless, while the method of the present invention is preferably performed with B cell samples obtained from patients affected with a monogenic disorder, under specific circumstances, thymoma patients may be used as an equivalent source for the B cells expressing the antibody of interest, in particular if displaying an aire-deficient phenotype.

However, since varying in humoral immune response due to the nature of tumors in accordance with the present invention it is preferred to select mammals which are not suffering from any disorder associated with tumors, such as thymoma. In some instances, it may be desirable to provide a monoclonal autoantibody from a mammal other than human, for example as research tool in animal experiments or for the purposes of preclinical studies in animal models of given disease. Therefore, in one embodiment of the method of the present invention the mammal is not human but an animal, preferably an animal commonly used in laboratory investigations and animal trials such as dog, cat, horse, preferably rodents such rat, gerbil and mice. Most preferably, the non-human animal is a mouse, preferably an AIRE or FOXP3 deficient mouse (AIRE: Ramsey et al., Hum. 11 (2002), 397-409; Kuroda et al., J. 174 (2005), 1862-1870; FOXP3: Brunkow et al., Nat.

27 (2001), 68-73; Wildin et al., Nature Genetics 27, (2001) 18-20). As postulated in the present application and meanwhile confirmed by Kamer et al. (2012); doi: 10.1111/cei.12024, the disclosure content of which is incorporated herein by reference, aire-deficient mice develop Abs (Antibodies) binding and neutralizing Th17-related cytokines as APECED patients do, substantiating a pathogenetic link with AIRE-deficiency in humans and mice. In particular, neutralizing autoantibodies to IL-17A in aged aire-deficient BALB/c mice were found characteristic to both, human and mouse AIRE-deficiency states. Nevertheless, as illustrated in the Examples, preferably the mammal in the method of the present invention is a human patient, preferably a patient suffering from an autoimmune disorder which is autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) or immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX).

IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked syndrome) is a rare X-linked recessive disease resulting in aggressive autoimmunity and early death which is described in more detail in the “Definitions and Embodiments” section below. In case of APECED the mammal typically suffers from one or more symptoms selected from the group consisting of chronic mucocutaneous candidiasis (CMC), hypoparathyroidism and autoimmune adrenal insufficiency (Addison's disease); see also supra.

As illustrated in the Examples, according to the method of the present invention the sample for isolating the antibody of interest comprises or consists of peripheral blood mononuclear cells (PBMC) and serum for the detection of possible antibody reactivities. The sample derived from the subject may either be directly used for, e.g., testing seroreactivity against one or more of the desired antigen(s) or may be further processed, for example enriched for B lymphocytes. In particular, it is preferred that the sample comprises or is derived from B cells that produce the antibody of interest, most preferably memory B-cells. In principle, B-cells producing antibodies can be immortalized and antibodies can be cloned thereof as described in detail in section “B-cell immortalization” below. However, in the preferred embodiment, the method of the present invention does not comprise the step of immortalization of B-cells. Rather, as described in the Examples, memory B cells are isolated from peripheral blood monocytic cells derived from the peripheral blood of voluntary subjects and are cultured under conditions allowing only a definite life span of the B cells, typically no more than 1 to 2 weeks until singling out the cells from B cell cultures which are reactive against the desired antigen subsequently followed by RT-PCR of single sorted cells for obtaining the immunoglobulin gene repertoire for said antibody; see also the Examples, in particular Example 2. In fact, during experiments performed within the scope of the present invention it turned out that previous methods aiming at B cell immortalization for providing a B cell clone producing the antibody of interest such as those described in international application WO 2004/076677 do not work quite well if at all for B cells of patients suffering from APECED or other autoimmune diseases.

Without intending to be bound by theory, it is believed that this is because that due to the impaired tolerance or loss of self-tolerance of the immune system, B cells, in particular those which are of interest in accordance with the present invention, have been pre-activated or triggered through signaling pathway otherwise which induces or is associated with the induction of apoptosis for which reason those cells have only a limited life span and are no longer effectively amenable to immortalization, at least not under the conditions hitherto reported for EBV-mediated immortalization. In view of the findings made in experiments performed in accordance with the present invention but without intending to be bound by theory it is believed that the simultaneous occurrence of cytokine and anti-cytokine antibodies such as observed in APECED/APS1 patients will lead to immune complex formation which could bind to B cells and activate them, thus explaining an activated state of B cells from APS1 patients and their vulnerability to senescence. However, as illustrated in the Examples and mentioned above, in accordance with the present invention a method is provided to isolate the human antibodies by treating and culturing the memory B cells under short term oligoclonal culture conditions allowing only a definite life span of the B cells during activation with subsequent oligo or single cell harvesting of oligoclonal cultures producing the antibody of interest and cloning the cDNA of the variable region of the antibody. A schematic representation of the preferred method for isolating the human antibodies of the present invention is illustrated in FIG. The term “oligoclonal culture” refers to a culture of cells producing the antibody of interest derived from one or a few cells that have been activated.

Preferably, the oligoclonal culture is derived from one single B cell, which may also be referred to as “B cell clone”. As mentioned above, and unless stated otherwise, the terms “oligoclonal” and “clone” do not imply or refer to immortalized cells.

As illustrated in the Examples, the biological sample is preferably derived from peripheral blood mononuclear cells of a patient whose serum has been screened for the presence of auto-antibodies against the protein of interest. The antibody-producing cells are isolated, stimulated, and proliferated according to the methods of the present invention in bulk cultures for a variable number of hours (e.g. From 1 up to 6 hours, or less preferred for longer periods of time such as 6 to 12 hours) before being subdivided into several pools of about 10 cells per culture for stimulation by the second polyclonal activator, each representing a population of cells, that are cultured separately (e.g.

96-, 384- or 1536 well plates). The bulk, polyclonal population of cells maintained in cell culture conditions may be tested using the assays performed already on sera to select the donor, or any other assay relevant for future use of the cells, in order to confirm the presence of cells. Moreover, some aliquots of the polyclonal population of cells may be put in vials and stored as frozen cells (as normally done for established mammalian cell lines), to be thawed and cultured again later. In this context, it is intended that the same culture supernatant can be tested on several different antigens, possibly all the antigens against which serum reactivity of the biological samples have been determined, e.g., in a protoarray. Aliquots of the cell culture supernatant can be screened for their binding and/or functional activity in a high throughput manner, in order to identify the positive well(s) presenting the desired activity, possibly using a dose-response analysis with serially diluted culture supernatants or partially purified antibody preparations (e.g. Obtained by affinity chromatography on protein A columns) in parallel experiments.

Optionally, the positive pools of cells (i.e. Those showing the desired antigen specificity and/or biological activity) can be then used to generate a new series of pools of cells to further restrict the screening to the level of a single cell culture(s) and consequently isolate the cDNA of the antibody variable regions form the selected cell secreting a monoclonal antibody having the desired specificity and activity, at least at the level of the initial screening assay.

The selected monoclonal antibodies should be then re-evaluated using other more demanding functional assays and characterized at the level of isotype and of V H/V L sequence, after isolating them using the recombinant DNA technologies applicable on B cells. Though EBV and CpG, in particular CpG2006 (ODN 2006 according to Hartmann et al., J. In this context, it is noted that though EBV has been used in the prior art for immortalizing B cells, EBV and like viral immortalizing agents have dual activities, i.e. Besides the capability of immortalizing B cells under appropriate cell culture conditions to also independently activate the B cells inducing both proliferation and Ig secretion. This early function of EBV is distinct from its late function of immortalizing B cell lines as shown by Tsuchiyama et al., Hum. Antibodies 8 (1997), 43-47.

Accordingly, the present invention only makes use of the early function as a polyclonal B cell activator of EBV and of similar viral immortalizing agents but not of the late function as a transforming virus capable of generating immortalized B cell lines that can be maintained in cell culture for several months, thereby only providing and using short term oligoclonal cultures of activated B cells with limited life span as further described below. Making use of only the early function of EBV and like agents can be accomplished by adjusting the time of culturing the cells in the presence of EBV only to the extent necessary to achieve a stimulation of the cells, i.e. Proliferation of the cells and antibody secretion, with subsequent separation of the cells from EBV and like agents or vice versa. As further turned out in the experiments performed within the scope of the present invention and illustrated in the Examples, the presence of a cytokine such as interleukin-2 (IL-2) as taught in European patent EP 1 974 020 B1 and EP 1 597 280 B1 or other costimulatory molecules such transferrin is not necessary. Rather, it turned out that in the method of the present invention the presence of cytokines in the B cell culture have substantially no beneficial effects, probably because of the mentioned preactivation or signaling in the B cells. Accordingly, in a preferred embodiment of the method of the present invention the culture conditions in step (b) and/or step (d) do not comprise a cytokine •.

After activation, the B cells are separated from the first polyclonal B cell activator in step (c), wherein the activator is removed for example by diluting off or washing out. In this context, experiments performed in accordance with the present invention confirmed that the presence of the first polyclonal B cell activator is indeed no longer necessary by ensuring the total removal of any thereof using multiple washing steps before subjecting the stimulated cells to the second polyclonal B cell activator.

Nevertheless, for the purposes of the method of the present invention it is usually efficient to remove the first polyclonal B cell activator by diluting off, i.e. Seeding the B cells in fresh culture medium. Thus, the cell culture subjected to the first polyclonal B cell activator may be placed in fresh culture media containing the second polyclonal activator thereby diluting the medium with the first polyclonal activator to a maximum of about 10%, preferably 5%, more preferably to 1%, and most preferably substantially below 1%, for example 0.5%, 0.1% or less. As mentioned hereinbefore and illustrated in the Examples, in step (d) the stimulated B cells are cultured in the presence of the second polyclonal B cell activator such as CpG no more than one to two weeks until singling out the cells from the B cell cultures which are reactive against the desired antigen. In a preferred embodiment of the method of the present invention, the transferred selected cells are exposed in step (d) to the second polyclonal activator for about eight to fourteen days. Preferably, in step (d) and/or (e) the cells are cultured under oligoclonal conditions with about ten cells per well in eight to fourteen days short term cultures. Though the above described method of the present invention of isolating and culturing B cells in the method of the present invention for isolating and producing a human monoclonal antibody of interest is advantageous and thus preferred, the person skilled in the art nevertheless may use alternative means and methods such as those described in the prior art even though they may be less efficient and may provide not as many B cell clones producing the antibody of interest as the preferred method disclosed herein.

Further methods of isolating human monoclonal antibodies using methods based on hybridoma technology, clonal expansion of peripheral B cells, single-cell PCR, phage display, yeast display and mammalian cell display are known to the person skilled in the art and are reviewed for example in Beerli and Rader, Landes Bioscience 2 (2010), 365-378. A method of efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning is described by Tiller et al., in J. Methods 329 (2008), 112-124. According to the present invention, samples taken from individuals are tested individually or combined in a batch for seroreactivity against antigens of interest with techniques known in the art, such as ELISA; see also the Examples.

The samples can be tested for seroreactivity against a particular antigen or against several antigens, at once or sequentially, wherein the antigens can be chosen as to represent particular groups of molecules. Said groups of molecules may be chosen, depending of, e.g., the involvement of the molecules in a particular biological process such as development, differentiation, adult tissue maintenance or disease. In one embodiment of the present invention the samples display substantially the same seroreactivity against a panel of antigens comprising at least one member of secreted or transmembrane proteins involved in inflammation, an autoimmune disorder, cell growth and differentiation, muscular function and neurodegeneration as indicated in tables 1 to 14 and FIGS. Preferably, the sample or pool of samples for obtaining the antibody in accordance with the method of the present invention displays a seroreactivity substantially the same as shown in FIG. Preferably, the antibody to be isolated in accordance with the method of the present invention is selected based on the frequency of antibody response, strength of binding and/or antibody titer in the protoarray assay, and preferably for its potential physiological effect. For example, if seroreactivity is observed for >10% of the samples of the subjects to a given target protein the putative monoclonal antibody may be decided to be further pursued.

Preferably, >20%, more preferably >30%, still more preferably 40%, still more preferably >50%, particularly preferred >75% and most preferably >90% of the samples of the different subjects show seroreactivity against the desired target antigen. Typically, the sample pool comprises at least samples from 10 different subjects, more preferably at least 15 subjects and most preferably at least 20 subjects. In view of the findings of the present invention seroreactivity of the samples corresponds with the rare clinical conditions found for the subjects such as for APECED patients; see also supra. On the other hand, the use of samples obtained from patients suffering from a monogenic autoimmune disorder described above, in particular APECED as the preferred embodiment of the present invention provides the additional advantage that because of the defined phenotype and symptoms the corresponding patients are clearly defined but nevertheless produce a diversity of antibodies due to individual difference in genetic background, development, environment and challenge to infectious agents. For this reason, a pool of samples from the patients are tested in order to determine and isolate an antibody which is specifically produced only by one patient or patients with a particular condition, which otherwise may not be obtained when testing individuals only. Accordingly, in one preferred embodiment of the method of the present invention, a pool of samples from patients is tested for seroreactivity to the antigen of interest. After the pool of samples has been tested seroreactive to the desired antigen, a corresponding pool may be further processed for isolating the desired antibody or the sample pool may be reduced in order to identify the patient(s) whose sample actually contributed to the seroreactivity of the sample pool.

The subgroup of patients from whom a corresponding pool is generated may be selected depending on different clinical status criteria of the patients, such as gender, age, quantity and/or severity of the disease's symptoms. As exemplary shown in Example 16, a gender difference in antibody response could be observed in the proteoarray data obtained from APECED patients. As indicated in Table 28, several antibodies could be obtained specifically from female or from male patients only. Furthermore, from these differentially expressed antibodies, substantially no antibodies against soluble proteins could be observed in male patients. Therefore, in one embodiment the method of the present invention for isolating a monoclonal antibody with desired antigen specificity is provided, wherein the antibody is specifically isolated from a subject with an impaired central and/or peripheral tolerance or loss of self-tolerance which has been selected depending on different clinical status criteria, such as gender, age, quantity and/or severity of the disease's symptoms.

In one embodiment the subject is selected depending on its gender, i.e. The antibodies are isolated from male or female subjects only.

As described in the appended Examples the antibodies isolated in accordance with the method of the present invention preferably recognize a conformational epitope. Hence, this is a further advantage of the method of the present invention in that due to the fact that the humoral immune response has been elicited against the native antigen in its physiologic and cellular environment typically autoantibodies are produced and can be isolated which recognize a conformational epitope of the antigen due to its presentation in context for example with other cellular components, presentation on a cell surface membrane and/or binding to a receptor. In contrast, conventional methods of generating monoclonal antibodies such as mouse monoclonals, humanized versions thereof or antibodies obtained from phage display typically employ an antigenic fragment of the target protein for immunizing an non-human mammal and detection, respectively, upon which usually antibodies are obtained which recognize linear epitopes or conformational epitopes limited to a two-dimensional structure of the immunogen rather than the presence of the native protein in its physiological and cellular context.

Accordingly, it is prudent to expect that the autoantibodies isolated in accordance with the method of the present invention are unique in terms of their epitope specificity. Therefore, the present invention also relates to antibodies and like-binding molecules which display substantially the same binding specificity as the autoantibodies isolated in accordance with the method of the present invention.

Such antibodies can be easily tested by for example competitive ELISA or more appropriately in a cell based neutralization assay using an autoantibody and a monoclonal derivative, respectively, thereof of the present invention as a reference antibody and the immunological tests described in the Examples or otherwise known to the person skilled in the art. As further illustrated in the Examples, the autoantibodies isolated in accordance with the method of the present invention preferably from human donors, preferably recognize only the human antigen or at least preferentially over the corresponding antigen from other species such as mice. Therefore, in one embodiment of the present invention, the desired antibody is specific for the species it is derived from, in particular human antigen and preferably does not substantially recognize the corresponding antigen of another species such as of murine origin, for example mice in case of a human antibody. Binding characteristics such as specificity and affinity of the antibodies of the present invention have been tested in several experimental assays as described in detail in the Examples 7, 8, 11, 13 and 14. Further indications could be obtained from in vivo experimental data within the tests of therapeutic potential of exemplary antibodies of the present invention, e.g., in induced psoriasis-like skin inflammation (Example 6) and in induced ear inflammation (Example 12). In this connection, specificity towards human IL-17F of the exemplary anti-IL-17 antibody 24D3 of the present invention could be confirmed by its observed therapeutic effect in the ear inflammation experiments induced by human IL-17F injections and described in Example 12, and the lack of a significant effect in the IMQ induced psoriasis-like skin inflammation, as shown in Example 7 (no significant effect on the inflammation mediated by murine IL-17F).

In comparison, the mouse cross-reactive exemplary anti-IL-22 antibody 30G1 has shown a therapeutic effect therein, by reducing clinical symptoms of IMQ-induced psoriasiform lesions. In addition, as illustrated in Example 9 in APECED related autoantigens coiled-coil structures are more frequently present than in the antigen repertoire of a healthy volunteer. The coiled-coil structure is a structural motif where 2-7 a-helices are coiled together, most commonly dimers and trimers. It contains a repeated pattern of heptad repeat HxxHCxC (H-hydrophobic; C-charged aa). Coiled-coil domain proteins are mostly intracellular enzymes.

Exemplary proteins comprising coild-coil domains which have been found as APECED related autoantigens in the proteoarray assay of the present invention are identified in Table 21 in Example 9 and the Protoarray results in concern of two of them in FIG. 31 (CCDC40 and NAP1L).

In one embodiment, the antibody of the present invention recognizes an antigen which displays a coiled-coil structure. As has been further demonstrated for the antibodies isolated in accordance with the method of the present invention, they are capable of neutralizing the biological activity of their target protein. Methods of producing clones of an immortalized human B cell and B memory lymphocyte, comprising the step of transforming human B memory lymphocytes using Epstein Barr Virus (EBV) in the presence of a polyclonal B cell activator are summarized in international application WO2004/076677 and are described in more detail in “B-cell immortalization” section further below. However, initial attempts at the cellular cloning of identified antigen-specific EBV-transformed human memory B cells had not been successful suggesting that the majority of cells are not transformed and not immortalized. Therefore, RT-PCR of single sorted cells is preferably employed for obtaining the immunoglobulin gene repertoire for said antibody; see also the Examples. Another method of obtaining human antibodies using inter alia single cell RT-PCR is described for example in the international application WO2008/110372, the disclosure content of which is incorporated herein by reference, in particular the Supplementary Methods section and Example 2.

In addition, an improved method for producing a clone of an immortalized human B memory lymphocyte, comprising the step of inducing or enhancing telomerase activity in the B lymphocyte in the presence of a polyclonal B cell activator is described in international application WO2010/003529, the disclosure content of which is incorporated herein by reference. The antibodies or antigen-binding fragments, e.g., peptides, polypeptides or fusion proteins of the present invention may be provided, as indicated above, by expression in a host cell or in an in vitro cell-free translation system, for example. To express the peptide, polypeptide or fusion protein in a host cell, the nucleic acid molecule encoding said peptide, polypeptide or fusion protein may be inserted into appropriate expression vector, i.e.

A vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al., Current Protocols in Molecular Biology (1989); see also the sections “Polynucleotides” and “Expressions” further below and literature cited in the Examples section for further details in this respect. Seroreactivity against a variety of antigens has been found in the APS1 patients, which were analyzed according to known or putative pathophysiological functions and narrowed down to therapeutically interesting secreted or membrane bound/associated compounds as indicated in Tables 1 to 13 as molecules of interest within the present invention.

In a further set of experiments, additional data concerning a variety of antigens has been found in respect of APS1-patients and in respect of APS2- and IPEX-patients; see Example 17 and Tables 29 to 31. In one embodiment of the method of the present invention, the antigen is selected from the group consisting of extracellular proteins and proteins, polysaccharides, lipopolyproteins and lipopolysaccharides, which are secreted, associated or attached to a membrane or transmembranous. However, in principle the method of the present invention is capable of providing autoantibodies against any desired antigen. This is because, as already explained before the subjects preferably used in accordance with the invention, i.e. Those whose impaired central and/or peripheral tolerance or loss of self-tolerance is caused by a particular genotype, i.e. A monogenic autoimmune disorder due to the general responsiveness of their humoral immune response on the one hand and their exposure to different internal and external stimuli and conditions, respectively, comprising predisposition for an inherited disorder, toxins, infections, age-related disorders and the like on the other hand provide a pool of autoantibodies ranging from autoantibodies common to most if not all subjects to autoantibodies which are specific for an individual disease or condition. For autoantibodies commonly found in the pool of samples in one embodiment of the method of the present invention the antigen is selected from the group consisting of leukotrienes, lymphokines, cytokines, interleukins, interferons and chemokines.

Another interesting class of antigen target for autoantibodies are tripartite motif-containing proteins (TRIMs) which have emerged as having key roles in antiviral immunity either as viral restriction factors or as regulators of pathways downstream of viral RNA and DNA sensors, and the inflammasome. In particular, in view of the role of inflammasome hyperactivation in the pathogenesis of a range of autoinflammatory disorders, it is prudent to expect that antibodies identified in accordance with the method of the present invention against these targets provide useful tools for therapeutic intervention and the treatment of diseases associated with inflammasome activity. Furthermore, regarding non-human animal models for use in the method of the present invention it is also conceived to expose a subject animal such as AIRE deficient mice to a external stimuli such as a test compound, radiation, abiotic or biotic stress and the like and to isolate an autoantibody specific to said external stimuli. To the extent that such expose is in conformity with the law of ethics and morality, the method of the present invention may be similar performed with human subjects, for example in order to identify and isolate stress-specific autoantibodies or autoantibodies evoked in response to food or medicaments. The isolated antibodies of the present invention may of course not be applied as such to a patient, but usually have to be pharmaceutically formulated to ensure, e.g., their stability, acceptability and bioavailability in the patient. Therefore, in one embodiment, the method of of the present invention is provided, further comprising the step of admixing the isolated monoclonal antibody with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers will be described in detail further below.

As described above, the present invention is based on the finding that mammals suffering from a disorder associated with a disrupted or deregulated genesis of autoimmunity, due to, e.g., mutations in the AIRE gene, constantly produce high titers of specific antibodies against several molecules of interest. In this respect, according to the present invention, B-cells producing said antibodies are isolated from such mammals.

Therefore, in one embodiment a B-cell as defined hereinabove is provided, preferably characterized by at least one mutation in the AIRE gene. Host cells as described above may be used as well in the preceding two methods and as described in detail in the “Host” section of this specification. In this respect, in one embodiment the above two methods are provided, where the expression host is a yeast cell, a plant cell or an animal cell. Besides the recombinant cells, in one embodiment it is also within the scope of the present invention to provide a method for preparing a nucleic acid molecule that encodes an antibody of interest, comprising the steps of: • (i) preparing a B-cell clone obtained by the method of the present invention, as described above; • (ii) sequencing nucleic acid and/or obtaining from the B-cell clone nucleic acid that encodes the antibody of interest.

As explained above, it was within the scope of the present invention to provide binding molecules binding specifically particular antigens of interest. In respect of such a binding molecule, the present invention further provides an antibody or antigen-binding fragment thereof obtainable by the method of the present invention, as described above and in Examples in detail. In a particularly preferred embodiment, this antibody is a human antibody. In one embodiment of the present invention, the antibody is directed against (i) interleukin-17A (IL-17A) and/or interleukin-17F (IL-17F); (ii) interleukin-22 (IL-22); or (iii) an IL-17A/IL-17F heterodimer. As demonstrated in appended Examples 4 and 6, binding molecules, i.e.

Antibodies have been identified and cloned, which display particularly high in vitro neutralizing activity with low inhibitory concentrations (IC50) for tested cytokines IL-17F and IL-22. In this respect, in one embodiment of the present invention antibodies are provided with a high affinity for their respective target molecules, e.g. IL-17 or IL-22, showing an ED50 at concentrations below 100 ng/ml, preferably below 10 ng/ml. For more details in respect of the binding affinity of the antibodies of the present invention see, e.g., section “Binding characteristics” further below.

The present invention exemplifies such binding molecules, i.e. Antibodies and binding fragments thereof, which may be characterized by comprising in their variable region, i.e.

Binding domain at least one complementarity determining region (CDR) of the V H and/or V L of the variable region comprising the amino acid sequence depicted in FIG. 15 of (V H) (SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55) and (V L) (SEQ ID NOs: 9, 17, 25, 33, 41, 49, 57)—see the exemplary CDR sequences underlined in FIG. However, as discussed in the following the person skilled in the art is well aware of the fact that in addition or alternatively CDRs may be used, which differ in their amino acid sequence from those indicated in FIG. 15 by one, two, three or even more amino acids, in particular in case of CDR2 and CDR3.

Thus, in one embodiment the antibody or antigen-binding fragment of the present invention is provided comprising in its variable region at least one complementarity determining region (CDR) of the V H and/or V L of the variable region comprising any one of the amino acid sequences depicted in (a) FIG. 15 (V H) (SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55); and (b) FIG. 15 (V L) (SEQ ID NOs: 9, 17, 25, 33, 41, 49, 57).•. Furthermore, in one embodiment, the antibody or antigen-binding fragment of the present invention comprises an amino acid sequence of the V H and/or V L region as depicted in FIG. Alternatively, the antibody of the present invention is an antibody or antigen-binding fragment thereof, which competes for binding to the IL-17 or the IL-22 antibody with at least one of the antibodies having the V H and/or V L region as depicted in FIG. 15 or as encoded by the corresponding nucleic acids as indicated in Table 17.

Hence, the present invention generally relates to an antibody, preferably human antibody and antigen-binding fragment thereof haing one or more of the above-described functional properties comprising in its variable region • (a) at least one complementarity determining region (CDR) of the V H and/or V L variable region amino acid sequences depicted in • (i) FIG. 15 (V H) (SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55); and • (ii) FIG. 15 (V L) (SEQ ID NOs: 9, 17, 25, 33, 41, 49, 57); • (b) an amino acid sequence of the V H and/or V L region as depicted in FIG. 15; • (c) at least one CDR consisting of an amino acid sequence derived from (a) resulted from a partial alteration of any one of the amino acid sequences of (a); • (d) a heavy chain and/or light variable region comprising an amino acid sequence derived from (b) resulted from a partial alteration of the amino acid sequence of (b); • (e) at least one CDR comprising an amino acid sequence derived from (a) with at least 80% identity to any one of the amino acid sequences of (a); • (f) an amino acid sequence derived from (b) with at least 60% identity to the amino acid sequence of (b). In case of a derived sequence, said sequence shows at least 60% identity, more preferably (in the following order) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and most preferably 95%, at least 96-99%, or even 100% identity to a sequence of the group consisting of those sequences referred to above and identified in the Sequence Listing. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, which is well known to those skilled in the art. The identities referred to herein are to be determined by using the BLAST programs as further referred to herein infra •. As mentioned in a preferred embodiment the present invention relates to substantially fully human antibodies, preferably IgG including at least the constant heavy chain I (C H1) and the corresponding light chain of the constant region, i.e.

Γ-1, γ-2, γ-3 or γ-4 in combination with lamda or kappa. In a particular preferred embodiment the nucleotide and amino acid sequences of those constant regions isolated for the subject antibodies illustrated in the Examples are used as depicted in Table 17 below and in SEQ ID NOs: 10-13, 18-21, 26-29, 34-37, 42-45, 50-53 and 58-61. In accordance with the above, in one embodiment the present invention also provides a polynucleotide encoding at least the variable region of one immunoglobulin chain of the antibody or antigen-binding fragment of the present invention. Typically, said variable region encoded by the polynucleotide comprises at least one complementarity determining region (CDR) of the V H and/or V L of the variable region of the said antibody.

Variable and constant regions of antibodies are described in more detail in the section “IgG structure” below. In a preferred embodiment of the present invention, the polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a polynucleotide sequence of the V H or V L region of an antibody of the present invention as depicted in Table 17 below. In this respect, the person skilled in the art will readily appreciate that the polynucleotides encoding at least the variable domain of the light and/or heavy chain may encode the variable domain of either immunoglobulin chains or only one of them. TABLE 17 Nucleotide and amino acid sequences of the variable and constant regions (V H, V L, C H, C L) of IL-17A, IL17F or IL-22 specific antibodies of the present invention.

IL-17F and IL-17A specific antibody 17E3: IgG1, kappa (VH1, G1m17; VK1, Km3). IL-17F specific antibody 24D3: IgG1, lambda (VH3, G1m17; VL3). IL-22 specific antibody 30G1: IgG1, kappa (VH3, G1m17; VK3, Km3 or Kml, 2). IL-22 specific antibody 35G11: IgG4, lambda (VH7, IGHG4*01; VL1); 35G11-C L: at positions 211-213 TAC may be as well TAT, both coding for Tyr. IL-22 specific antibody 41D11: IgG1, lambda (VH4, G1m3 L410F; VL4). IL-22 specific antibody 51G4: IgG2, lambda (VH1, IGHG2*01; VL3); 41D11-C H: positions 877-879 of the nucleotide sequence may be ctc instead of ttc and position 293 of the corresponding amino acid sequence may be Leu instead of Phe (underlined). Underlined, bold nucleotides or amino acids indicate the CDR coding regions in the variable chain sequence. Underlined, italic nucleotides indicate sequences in the constant chain sequences which have not been sequenced but obtained from database. Nucleotide and amino acid sequences of variable heavy (VH) and variable light (VL), constant Antibody heavy (CH) and constant light (CL) chains.

The person skilled in the art will readily appreciate that the variable domain of the antibody having the above-described variable domain can be used for the construction of other polypeptides or antibodies of desired specificity and biological function. Thus, the present invention also encompasses polypeptides and antibodies comprising at least one CDR of the above-described variable domain and which advantageously have substantially the same or similar binding properties as the antibody described in the appended examples. The person skilled in the art will readily appreciate that using the variable domains or CDRs described herein antibodies can be constructed according to methods known in the art, e.g., as described in European patent applications EP 0 451 216 A1 and EP 0 549 581 A1. Furthermore, the person skilled in the art knows that binding affinity may be enhanced by making amino acid substitutions within the CDRs or within the hypervariable loops (Chothia and Lesk, J. 196 (1987), 901-917) which partially overlap with the CDRs as defined by Kabat. Thus, the present invention also relates to antibodies wherein one or more of the mentioned CDRs comprise one or more, preferably not more than two amino acid substitutions.

Preferably, the antibody of the invention comprises in one or both of its immunoglobulin chains two or all three CDRs of the variable regions as set forth in SEQ ID NOs: 7, 9, 15, 17, 23, 25, 31, 33, 39, 41, 47, 49, 55 or 57 or indicated in FIG. The polynucleotide of the invention encoding the above described antibody may be, e.g., DNA, cDNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination. In a preferred embodiment a vector comprising the above polynucleotide is provided, optionally in combination with said polynucleotide which encodes the variable region of the other immunoglobulin chain of said antibody. Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions. Preferably, the polynucleotide of the invention is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells. Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art.

They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally associated or heterologous promoter regions. In this respect, the person skilled in the art will readily appreciate that the polynucleotides encoding at least the variable domain of the light and/or heavy chain may encode the variable domains of both immunoglobulin chains or one chain only.

Likewise, said polynucleotides may be under the control of the same promoter or may be separately controlled for expression. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the P L, lac, trp or tac promoter in E. Coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GALL promoter in yeast or the CMV-, SV40-, RSV-promoter, CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. Furthermore, depending on the expression system used leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide of the invention and are well known in the art. The leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including a C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), or pSPORT1 (GIBCO BRL). Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the immunoglobulin light chains, heavy chains, light/heavy chain dimers or intact antibodies, binding fragments or other immunoglobulin forms may follow; see, Beychok, Cells of Immunoglobulin Synthesis, Academic Press, N.Y., (1979). Furthermore, the present invention relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a polynucleotide encoding the antigen or preferably a variable domain of an immunoglobulin chain of an antibody of the invention; optionally in combination with a polynucleotide of the invention that encodes the variable domain of the other immunoglobulin chain of the antibody of the invention. Preferably, said vector is an expression vector and/or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector of the invention into targeted cell population.

Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. And Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. Alternatively, the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells.

The vectors containing the polynucleotides of the invention (e.g., the heavy and/or light variable domain(s) of the immunoglobulin chains encoding sequences and expression control sequences) can be transferred into the host cell by well known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for the transformation of other cellular hosts; see Sambrook, supra. In respect to the above, the present invention furthermore relates to a host cell comprising said polynucleotide or vector. Said host cell may be a prokaryotic or eukaryotic cell. The polynucleotide or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally.

The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell; suitable host cells and methods for production of the antibodies of the present invention are described in more detail in the section “Host cells” below. The binding molecules, antibodies or fragments thereof may be directly used as a therapeuticum.

However, in one embodiment the antibody or antigen-binding fragment which is provided by the present invention, is detectably labeled or attached to a drug, wherein the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorophore, a peptide and a heavy metal. Labeled antibodies or antigen-binding fragments of the present invention may be used used to detect specific targets in vivo or in vitro including “immunochemistry/immunolabelling” like assays in vitro. In vivo they may be used in a manner similar to nuclear medicine imaging techniques to detect tissues, cells, or other material expressing the antigen of interest. Labels, their use in diagnostics and their coupling to the binding molecules of the present invention are described in more detail in section “labels and diagnostics” further below.

The antibodies of the present invention are isolated from animals or humans affected by an autoimmune disorder. By binding to endogenous molecules, e.g., proteins as enlisted in tables 1 to 14 or in tables 29-31, some of them can be used in treatment of tumor or neurodegenerative diseases. On the other hand antibodies identified in the present invention may be involved in eliciting inflammation or severely impair the immune system of the affected individual, which is associated with, e.g., symptoms observed in APECED patients as described in more detail hereinbefore and below. Therefore, it is a further aspect of the present invention, to extinguish or at least relieve the pathological reactions of subjecs suffering from autoimmune disorders by providing means and measures to minimize the number of auto-antibodies and/or their effects in a diseased human patient or animal. Therefore, in one embodiment the present invention also provides an anti-idiotypic antibody of an autoantibody of the present invention. A similar effect may be obtained by application of competitive antigens, sequestering and preventing thereby the binding of the autoantibodies to their respective targets.

Thus, in one embodiment the present invention also relates to a synthetic peptide or peptide-based compound comprising an epitope specifically recognized by an autoantibody of the present invention, i.e. Isolated according to the methods of the present invention. As already indicated above, the present invention also relates to the anti-idiotypic antibody or the peptide or peptide-based compound of the present invention for use in the treatment of a disorder as defined above, i.e. A disorder associated with a disrupted or deregulated genesis of self-tolerance. These isolated antibodies or fragments thereof of the present invention can be used as immunogens to generate a panel of monoclonal anti-idiotypes.

For suitable methods for the generation of anti-idiotypic antibodies see Raychadhuri et al., J. 137 (1986), 1743 and for T-cells see Ertl et al., J. 159 (1985), 1776. The anti-idiotypic antibodies will be characterized with respect to the expression of internal image and non-internal image idiotopes using standard•assays routinely practiced in the art as described in detail by Raychaudhuri et al., J. 137 (1986), 1743.

If an anti-idiotipic antibody structurally mimics the antigen of the antibody it is binding to or bound by, it is called the “internal image” of the antigen. Methods of providing molecules which mimic an idiotype of an autoimmune disease-associated auto-antibody (autoantibodies) are described in the art; see, e.g., international application WO03/099868, the disclosure content of which incorporated herein by reference. Moreover, the present invention also relates to compositions comprising the aforementioned antibody or antigen-binding fragment, the polynucleotide, the vector, the cell, the anti-idiotypic antibody or the peptide or peptide-based compound. In one embodiment the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers, administration routes and dosage regimen can be taken from corresponding literature known to the person skilled in the art and are described as well in more detail in sections “Pharmaceutical carriers” and “Dosage regimen” further below. Besides biochemical and cell based in vitro assays therapeutic utility of the antibody of the present invention can be validated in appropriate animal models such as for psoraris, IBD, candidasis; see Examples 5 and 6 and for Inflammatory bowel disease IBD (DSS colitis) model in Wirtz et al, Nature Protocols 2 (2007), 541-546; for a Psoriasis (Imiquimod) model in van der Fits et al, J Immunol 182 (2009), 5836-5845; for a multiple sclerosis (Experimental Autoimmune Encephalomyelitis (EAE)) model in Miller, S. And Karpus, W.

J., Current Protocols in Immunology. 78 (2007.), 15.1.1-15.1.18 and at for a rheumatoid arthritis (Collagen-induced arthritis) model in Julia J Inglis et al, Nature Protocols 3 (2008), 612-618. In one embodiment the pharmaceutical composition further comprises an additional agent useful for treating an inflammation or an autoimmune disorder, selected from the group consisting of Non-Steroidal Antiinflammatory Drugs (NSAIDs), Corticosteroids, Anti-Histamines and combinations thereof.

In addition or alternatively, in a further embodiment the pharmaceutical composition further comprises an additional agent useful for treating an inflammation related disease, selected from the group consisting of immunosuppressive and anti-inflammatory or “anti-rheumatic” drugs. Furthermore, the present invention provides the aforementioned antibody or antigen-binding fragment, the anti-idiotypic antibody or the peptide or peptide-based compound for use in treating or preventing the progression of an inflammatory or an autoimmune disorder; for the amelioration of symptoms associated with inflammation or an autoimmune disorder; for diagnosing or screening a subject for the presence or for determining a subject's risk for developing inflammation or an autoimmune disorder. Due to the multitude of molecules suitable in treatment of, e.g., disorders associated with inflammation, the present invention also provides several methods of treatment of such disorders.

In one embodiment a method of treating a disorder associated with an inflammation or an autoimmune disorder is provided, which method comprises administering to a subject in need thereof a therapeutically effective amount of the aforementioned antibody or antigen-binding fragment, the anti-idiotypic antibody or the peptide or peptide-based compound. Treatment methods based on the use of only one monoclonal antibody specific for an epitope of a particular antigen, which is related or causing a disease may suffer from several shortcomings. For example, difficulties and probably inefficiency of treatment may stem from the multiplicity of the pathogenic mechanisms causing a specific disorder requiring targeting of several antigens simultaneously. Furthermore, the inherent diversity of the patient population has to be taken into account concerning, e.g., polymorphism, heterogeneity of glycosylation or slight denaturation of a given antigen, either in different or in one patient which may lead to a decreased binding efficiency of the monoclonal antibody used at least.

Some of these shortcomings may be circumvented by, e.g., pretreatment screenings to determine whether the antigen is immunologically relevant to the patients intended to be treated and whether there are any epitope changes in the particular patients. However, such screenings are often omitted either due to treatment urgency or to cost restraints. Therefore, the present invention further relates to methods based on the application of more than one type of a binding molecule at once to a patient. These binding molecules may specifically bind to one antigen at different epitopes or each of the binding molecules applied binds specifically another disease related antigen.

In case the binding molecules of the present invention are directed (bind specifically) towards one antigen, their binding specificity is directed towards distinct epitopes of said antigen. The use of such cocktails is in particular envisaged for the treatment of patients suffering from autoimmune disorders such as APS1, who in view of the presence of autoantibodies against about 3000 endogenous antigens are often not amenable to monotherapy with one particular antibody. In such cases, combination therapy with two or more monoclonal antibodies and/or peptides and peptide-based compounds of the present invention with the same or different antigen specificity are expected to achieve at least some relief of the symptoms. In another embodiment the present invention relates to a kit for the diagnosis of a disorder which is accompanied with the presence of a disorder-associated protein as defined in any one of the peptides claimed, said kit comprising the aforementioned antibody or antigen-binding fragment, the anti-idiotypic antibody or the peptide or peptide-based compound, the polynucleotide, the vector or the cell, optionally with reagents and/or instructions for use. Associated with the kits of the present invention, e.g., within a container comprising the kit can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition or alternatively the kit comprises reagents and/or instructions for use in appropriate diagnostic assays.

The compositions, i.e. Kits of the present invention are of course particularly suitable for the diagnosis, prevention and treatment of a disorder which is accompanied with the presence of a inflammation-associated antigen defined above, in particular applicable for the treatment of diseases as mentioned above.

In another embodiment the present invention relates to a diagnostic composition comprising any one of the above described binding molecules, antibodies, antigen-binding fragments, peptides or peptide-based compounds, polynucleotides, vectors or cells of the invention and optionally suitable means for detection such as reagents conventionally used in immuno or nucleic acid based diagnostic methods. The antibodies of the invention are, for example, suited for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.

Examples of immunoassays which can utilize the antibody of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay), flow cytometry and the Western blot assay. The antigens and antibodies of the invention can be bound to many different carriers and used to isolate cells specifically bound thereto. Examples of well known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.

The nature of the carrier can be either soluble or insoluble for the purposes of the invention. There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds; see also the embodiments discussed hereinabove. In this context, the present invention also relates to means specifically designed for this purpose. For example, a protein- or antibody-based array may be used, which is for example loaded with either antigens derived from the mentioned disorder-associated protein and containing the inflammation-associated antigen in order to detect autoantibodies which may be present in patients suffering from a autoimmune diseases, in particular arthritis, type I diabetes or APECED/APS1, or with antibodies or equivalent antigen-binding molecules of the present invention which specifically recognize any one of those inflammation-associated antigens. Design of microarray immunoassays is summarized in Kusnezow et al., Mol.

Cell Proteomics 5 (2006), 1681-1696. Accordingly, the present invention also relates to microarrays loaded with binding molecules or antigens identified in accordance with the present invention. DEFINITIONS AND EMBODIMENTS•. The term “neutralizing” and “neutralizing antibody”, respectively, is used as common in the art in that an antibody is meant that reduces or abolishes at least some biological activity of an antigen or of a living microorganism. For example, an anti-IL-17F antibody of the present invention is a neutralizing antibody, if, in adequate amounts, it abolishes the cytokine activity for example in an assay as described in the Examples.

Neutralization is commonly defined by 50% inhibitory concentrations (IC 50) and can be statistically assessed based on the area under the neutralization titration curves (AUC). For tumor diseases neutralization can be assessed for example by determining inhibition of tumor growth. For example, it is known that oncogenic grafts-induced secretion of interleukin-6 is required for tumorgenesis and that anti-IL-6 antibodies may be used for corresponding cancer therapy; see, e.g.

Ancrile et al., “Genes and development” 21 (2007), 1714-1719. Neutralization of an infectious agent such as a virus is defined as the loss of infectivity through reaction of the virus or its target cell, for example by blocking the receptor of the virus resulting in the reduction of infected cells which can be easily determined in appropriate cell-based assays known by the person skilled in the art. Other biological activities which may be “neutralized” by the antibody of the present invention can be easily ascertained by the person skilled in the art. Central and Peripheral Tolerance•.

Self-tolerance is the process whereby the immune system does not respond to an antigen that is a constituent of that organism. Self-tolerance is achieved by death or inactivation of self-reactive T and B-cells, which may occur as part of central tolerance in a central (generative) immune organ (thymus or bone marrow) or as peripheral tolerance in what are most commonly regarded as secondary immune tissues (e.g. Spleen, lymph node, intestine). Self-tolerance is a central feature of the normal immune system. Failure to establish and/or maintain self-tolerance leads to autoimmunity, which may result in autoimmune diseases that have severe health implications for the host organism. Central tolerance is a form of self-tolerance that is induced in central immune organs such as the thymus and bone marrow. In the process of central tolerance, newly developed T and B-cells recognizing self-antigens are rendered non-reactive to self antigens by cell death, by other forms of inactivation, or by developmental conversion to immunosuppressive regulatory lymphocytes.

Central tolerance for T-cells is induced in the thymus, where developing thymocytes (T-cells in thymus) that recognize self-peptide-MHC complexes with high affinity are mostly deleted. Central tolerance for B-cells is induced in the bone marrow. During their differentiation in thymus and bone marrow, T- and B-cells rearrange their genomic regions respectively containing T-cell receptor and B-cell receptor genes. The somatic gene rearrangement, also called V(D)J recombination, generates a diversity of T-cell receptors and antibodies.

This diversity is the basis for T and B-cell clonal repertoires. After V(D)J recombination, maturing T- and B-cells are capable of antigen recognition. Recognition of antigen at this stage can lead to negative selection that eliminates or alters developing T- and B-cells whose antigen receptors bind strongly to self-antigens present in generative immune organs. Both T and B-cells are susceptible to negative selection within a short period after T-cell receptors and B-cell receptors, respectively, are first expressed on cell surface. T- and B-cells can develop central tolerance towards those antigens that are present in generative immune organs. In the bone marrow, B cells develop tolerance to ubiquitously expressed, bone-marrow specific antigens and to antigens imported by the blood circulation.

In the thymus, thymic medullary epithelial cells can express many hundreds of self-antigens that are presented to developing T-cells. The gene responsible for the broad expression of self-antigens in thymic medullary epithelial cells is AIRE (autoimmune regulator). AIRE activates multiple tissue specific genes that normally are expressed only in particular peripheral organs such as insulin in pancreatic Langerhans islands. In the absence of the functional AIRE gene, antigens are not presented, T cells are not inactivated, and autoimmunity to self-antigens develops, leading to pathology in APECED patients and in Aire deficient mice. Peripheral tolerance is a form of self-tolerance where T and B-cells do not respond to antigens in peripheral tissues. Peripheral tolerance is induced by recognition of antigens without necessary co-stimulatory signals for lymphocyte activation or in the presence of co-inhibitory signals or by repeated and persistent stimulation by these antigens. Peripheral tolerance includes immunological ignorance where self-reactive lymphocytes are not activated because the antigens are expressed in immunoprivileged tissues, which are not under direct immune surveillance such as brain, eye, testis and foetus.

Peripheral tolerance also includes the suppression of autoreactive cells by regulatory T cells. AIRE expression has been reported outside the thymus, in a small population of AIRE-expressing cells in the lymph nodes. Thus, AIRE may play a significant role in self-tolerance via the deletion of autoreactive T cells in periphery. Rare AIRE expressing cells in the lymph nodes share some characteristics with thymic medullary epithelial cells as they act as professional antigen-presenting cells and express tissue-specific antigens. Despite this, the set of AIRE-regulated tissue specific antigens in lymph nodes has little overlap with thymic AIRE-regulated antigens. The lack of overlap suggests that there may be a different order of AIRE-dependent transcriptional regulation of tissue specific antigen expression between the thymus and the lymph nodes.

The term “peptide” is understood to include the terms “polypeptide” and “protein” (which, at times, may be used interchangeably herein) and any amino acid sequence such as those of the heavy and light chain variable region as well as constant region of the present invention within its meaning. Similarly, fragments of proteins and polypeptides are also contemplated and may be referred to herein as “peptides”.

Nevertheless, the term “peptide” preferably denotes an amino acid polymer including at least 5 contiguous amino acids, preferably at least 10 contiguous amino acids, more preferably at least 15 contiguous amino acids, still more preferably at least 20 contiguous amino acids, and particularly preferred at least 25 contiguous amino acids. In addition, the peptide in accordance with present invention typically has no more than 100 contiguous amino acids, preferably less than 80 contiguous amino acids and more preferably less than 50 contiguous amino acids. As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides” such as antibodies of the present invention, and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, “peptides,” “dipeptides,” “tripeptides, “oligopeptides,” “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms.

The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Nevertheless, the term “polypeptide” preferably denotes an amino acid polymer including at least 100 amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure.

Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded. As used herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein via an oxygen-containing or a nitrogen-containing side chain of an amino acid residue, e.g., a serine residue or an asparagine residue. By an “isolated” polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu.

No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. “Recombinant peptides, polypeptides or proteins” refer to peptides, polypeptides or proteins produced by recombinant DNA techniques, i.e. Produced from cells, microbial or mammalian, transformed by an exogenous recombinant DNA expression construct encoding the fusion protein including the desired peptide. Proteins or peptides expressed in most bacterial cultures will typically be free of glycan.

Proteins or polypeptides expressed in yeast may have a glycosylation pattern different from that expressed in mammalian cells. Also included as polypeptides of the present invention are fragments, derivatives, analogs and variants of the foregoing polypeptides and any combination thereof. The terms “fragment,” “variant,” “derivative” and “analog” include peptides and polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of the natural peptide.

The term “sufficiently similar” means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity. For example, amino acid sequences that comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, identical are defined herein as sufficiently similar. Preferably, variants will be sufficiently similar to the amino acid sequence of the preferred peptides of the present invention, in particular to antibodies or antibody fragments, or to synthetic peptide or peptide-based compound comprising epitopes recognized by the antibodies of the present invention or fragments, variants, derivatives or analogs of either of them.

Such variants generally retain the functional activity of the peptides of the present invention, i.e. Are bound by the antibodies of the present invention. Variants include peptides that differ in amino acid sequence from the native and wt peptide, respectively, by way of one or more amino acid deletion(s), addition(s), and/or substitution(s). These may be naturally occurring variants as well as artificially designed ones. The terms “fragment,” “variant,” “derivative” and “analog” when referring to antibodies or antibody polypeptides of the present invention include any polypeptides which retain at least some of the antigen-binding properties of the corresponding native binding molecule, antibody, or polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein. Variants of antibodies and antibody polypeptides of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions.

Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of binding molecules of the present invention, e.g., antibodies and antibody polypeptides of the present invention, are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as “polypeptide analogs”. As used herein a “derivative” of a binding molecule or fragment thereof, an antibody, or an antibody polypeptide refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group.

Also included as “derivatives” are peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

Anti-Idiotypic Antibodies:•. The term “anti-idiotypic antibodies” when referring to antibodies or other binding molecules includes molecules which bind to the unique antigenic peptide sequence located on an antibody's variable region near or at the antigen binding site, inhibiting by this a specific immune response by otherwise caused by the given auto-antibody. In an analogous manner synthetic peptide or peptide-based compound comprising an epitope specifically recognized by an antibody of the present invention may be used. Anti-idiotypic antibodies may be obtained in a similar fassion as other antibodies. The particular anti-idiotypic antibody is detected by any sort of cross-linking, either by agglutination (in turbidimetric or nephelometric assays), precipitation (radial immunodiffusion), or sandwich immunoassays such as ELISAs. Patent application No. 6 provides a method for obtaining anti-idiotypic monoclonal antibody populations directed to an antibody that is specific for a high-concentration, high-molecular-weight target antigen wherein said anti-idiotypic antibody populations have a wide range of binding affinities for the selected antibody specific to said target antigen and wherein a subset of said anti-idiotypic antibody populations can be selected having the required affinity for a particular application.

Patent application No. 6 describes a competitive immunoassay of an antigen using an antibody as coat and an anti-idiotypic antibody as detection or vice-versa. Other references disclosing use of an anti-idiotypic antibody as a surrogate antigen include Losman et al., Cancer Research, 55 (1995) (23 suppl. S):2; Becker et al., J.

Methods 192 (1996), 73-85; Baral et al., International J. Of Cancer, 92 (2001), 88-95; and Kohen et al., Food and Agriculture Immunology, 12 (2000), 193-201. Use of anti-idiotypic antibodies in treatment of diseases or against parasites is known in the art; see, e.g., in Sacks et al., J. Medicine, 155 (1982), 1108-1119.

“Similarity” between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide. An amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, M. O., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D.C. (1978), and in Argos, EMBO J. 8 (1989), 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: -Ala, Pro, Gly, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu.

For the general parameters, the “Max Target Sequences” box may be set to 100, the “Short queries” box may be ticked, the “Expect threshold” box may be set to 10 and the “Word Size” box may be set to 28. For the scoring parameters the “Match/mismatch Scores” may be set to 1,−2 and the “Gap Costs” box may be set to linear. For the Filters and Masking parameters, the “Low complexity regions” box may not be ticked, the “Species-specific repeats” box may not be ticked, the “Mask for lookup table only” box may be ticked, and the “Mask lower case letters” box may not be ticked. BLAST protein searches are performed with the BLASTp program. For the general parameters, the “Max Target Sequences” box may be set to 100, the “Short queries” box may be ticked, the “Expect threshold” box may be set to 10 and the “Word Size” box may be set to “3”. For the scoring parameters the “Matrix” box may be set to “BLOSUM62”, the “Gap Costs” Box may be set to “Existence: 11 Extension:1”, the “Compositional adjustments” box may be set to “Conditional compositional score matrix adjustment”. For the Filters and Masking parameters the “Low complexity regions” box may not be ticked, the “Mask for lookup table only” box may not be ticked and the “Mask lower case letters” box may not be ticked.

The term “polynucleotide” is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).

The term “nucleic acid” refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. By “isolated” nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding an antibody contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically.

In addition, polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator. As used herein, a “coding region” is a portion of nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region.

Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g., a single vector may separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a nucleic acid encoding a binding molecule, an antibody, or fragment, variant, or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide normally may include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s).

Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are “operably associated” or “operably linked” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art.

These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins). Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or “full-length” polypeptide to produce a secreted or “mature” form of the polypeptide.

In certain embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

The term “expression” as used herein refers to a process by which a gene produces a biochemical, for example, an RNA or polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors.

Expression of a gene produces a “gene product.” As used herein, a gene product can be either a nucleic acid, e.g., small interfering RNA (siRNA), a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like. A variety of expression vector/host systems may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

To express the peptide, polypeptide or fusion protein (hereinafter referred to as “product”) in a host cell, a procedure such as the following can be used. A restriction fragment containing a DNA sequence that encodes said product may be cloned into an appropriate recombinant plasmid containing an origin of replication that functions in the host cell and an appropriate selectable marker.

The plasmid may include a promoter for inducible expression of the product (e.g., pTrc (Amann et al, Gene 69 (1988), 301 315) and pET1 Id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), 60 89). The recombinant plasmid may be introduced into the host cell by, for example, electroporation and cells containing the recombinant plasmid may be identified by selection for the marker on the plasmid. Expression of the product may be induced and detected in the host cell using an assay specific for the product. Alternatively, the expression of the product may be performed by in vitro synthesis of the protein in cell-free extracts which are also particularly suited for the incorporation of modified or unnatural amino acids for functional studies; see also infra.

The use of in vitro translation systems can have advantages over in vivo gene expression when the over-expressed product is toxic to the host cell, when the product is insoluble or forms inclusion bodies, or when the protein undergoes rapid proteolytic degradation by intracellular proteases. The most frequently used cell-free translation systems consist of extracts from rabbit reticulocytes, wheat germ and Escherichia coli. All are prepared as crude extracts containing all the macromolecular components (70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, etc.) required for translation of exogenous RNA. To ensure efficient translation, each extract must be supplemented with amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for the E. Coli lysate), and other co-factors known in the art (Mg 2+, K +, etc.).

Appropriate transcription/translation systems are commercially available, for example from Promega Corporation, Roche Diagnostics, and Ambion, i.e. Applied Biosystems (Anderson, C. Et al., Meth. 101 (1983), 635-644; Arduengo, M.

(2007), The Role of Cell- Free Rabbit Reticulocyte Expression Systems in Functional Proteomics in, Kudlicki, Katzen and Bennett eds., Cell- Free Expression Vol. Austin, Tx: Landes Bioscience, pp. 1-18; Chen and Zubay, Meth.

101 (1983), 674-90; Ezure et al., Biotechnol. 22 (2006), 1570-1577).

Host Cells:•. In respect of the present invention, host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell. Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S.

The term “prokaryotic” is meant to include all bacteria which can be transformed or transfected with a DNA or RNA molecules for the expression of an antibody of the invention or the corresponding immunoglobulin chains. Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. Typhimurium, Serratia marcescens and Bacillus subtilis. The term “eukaryotic” is meant to include yeast, higher plant, insect and preferably mammalian cells, most preferably HEK 293, NSO, CSO and CHO cells.

Depending upon the host employed in a recombinant production procedure, the antibodies or immunoglobulin chains encoded by the polynucleotide of the present invention may be glycosylated or may be non-glycosylated. Antibodies of the invention or the corresponding immunoglobulin chains may also include an initial methionine amino acid residue.

A polynucleotide of the invention can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art. Furthermore, methods for preparing fused, operably linked genes and expressing them in, e.g., mammalian cells and bacteria are well-known in the art (Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). The genetic constructs and methods described therein can be utilized for expression of the antibody of the invention or the corresponding immunoglobulin chains in eukaryotic or prokaryotic hosts. In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted polynucleotide are used in connection with the host.

The expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells. Suitable source cells for the DNA sequences and host cells for immunoglobulin expression and secretion can be obtained from a number of sources, such as the American Type Culture Collection (“Catalogue of Cell Lines and Hybridomas,” Fifth edition (1985) Rockville, Md., U.S.A., which is incorporated herein by reference). Furthermore, transgenic animals, preferably mammals, comprising cells of the invention may be used for the large scale production of the antibody of the invention. The transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth. Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention, can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see, Scopes, “ Protein Purification”, Springer Verlag, N.Y. The antibody or its corresponding immunoglobulin chain(s) of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions. The isolation and purification of the, e.g., recombinantly expressed antibodies or immunoglobulin chains of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies directed, e.g., against the constant region of the antibody of the invention.

It will be apparent to those skilled in the art that the antibodies of the invention can be further coupled to other moieties for, e.g., drug targeting and imaging applications. Such coupling may be conducted chemically after expression of the antibody or antigen to site of attachment or the coupling product may be engineered into the antibody or antigen of the invention at the DNA level. The DNAs are then expressed in a suitable host system, and the expressed proteins are collected and renatured, if necessary. Enzyme-linked immunosorbent assays (ELISAs) for various antigens include those based on colorimetry, chemiluminescence, and fluorometry. ELISAs have been successfully applied in the determination of low amounts of drugs and other antigenic components in plasma and urine samples, involve no extraction steps, and are simple to carry out. ELISAs for the detection of antibodies to protein antigens often use direct binding of short synthetic peptides to the plastic surface of a microtitre plate.

The peptides are, in general, very pure due to their synthetic nature and efficient purification methods using high-performance liquid chromatography. A drawback of short peptides is that they usually represent linear, but not conformational or discontinuous epitopes. To present conformational epitopes, either long peptides or the complete native protein is used. Direct binding of the protein antigens to the hydrophobic polystyrene support of the plate can result in partial or total denaturation of the bound protein and loss of conformational epitopes. Coating the plate with an antibody, which mediates the immobilization (capture ELISA) of the antigens, can avoid this effect. A “binding molecule” as used in the context of the present invention relates primarily to antibodies, and fragments thereof, but may also refer to other non-antibody molecules that bind to the “molecules of interest” of the present invention as enlisted in tables 1 to 14 and 28 to 31 wherein the molecules of interest are proteins, peptides, polysaccharides, lipopolyproteins and lipopolysaccharides, for example, but not limited to leukotrienes, lymphokines and cytokines, e.g., interleukins and interferons. The molecules of interest of the present invention will be defined in further detail within the description of the particular embodiments of the present invention below.

The binding molecules of the present invention include but are not limited to hormones, receptors, ligands, major histocompatibility complex (MHC) molecules, chaperones such as heat shock proteins (HSPs) as well as cell-cell adhesion molecules such as members of the cadherin, intergrin, C-type lectin and immunoglobulin (Ig) superfamilies. Thus, for the sake of clarity only and without restricting the scope of the present invention most of the following embodiments are discussed with respect to antibodies and antibody-like molecules which represent the preferred binding molecules for the development of therapeutic and diagnostic agents. The terms “antibody” and “immunoglobulin” are used interchangeably herein. An antibody or immunoglobulin is a molecule binding to a molecule of interest of the present invention as defined hereinabove and below, which comprises at least the variable domain of a heavy chain, and normally comprises at least the variable domains of a heavy chain and a light chain.

Basic immunoglobulin structures in vertebrate systems are relatively well understood; see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, ( Cold Spring Harbor Laboratory Press, 2nd ed. The terms “binds” and “recognizes” are used interchangeably in respect of the binding affinity of the binding molecules of the present invention, e.g., antibodies. Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. Both the light and heavy chains are divided into regions of structural and functional homology.

The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (V L) and heavy (V H) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.

By convention the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively. As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the V L domain and V H domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three dimensional antigen-binding site.

This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on each of the V H and V L chains. Any antibody or immunoglobulin fragment which contains sufficient structure to specifically bind to a molecule of interest of the present invention is denoted herein interchangeably as a “binding fragment” or an “immunospecific fragment.” •. In naturally occurring antibodies, an antibody comprises six hypervariable regions, sometimes called “complementarity determining regions” or “CDRs” present in each antigen-binding domain, which are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The “CDRs” are flanked by four relatively conserved “framework” regions or “FRs” which show less inter-molecular variability. The framework regions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure.

Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen.

This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined; see, “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J.

196 (1987), 901-917, which are incorporated herein by reference in their entireties. In the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” (“CDR”) to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., U.S. Of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia and Lesk, J. 196 (1987), 901-917, which are incorporated herein by reference, where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein.

The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 16 as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular hypervariable region or CDR of the human IgG subtype of antibody given the variable region amino acid sequence of the antibody. TABLE 16 CDR Definitions 1 Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-65 52-58 VH CDR3 95-102 95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VL CDR3 89-97 91-96 1Numbering of all CDR definitions in Table 16 is according to the numbering conventions set forth by Kabat et al.

Also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody or antigen-binding fragment, variant, or derivative thereof of the present invention are according to the Kabat numbering system, which however is theoretical and may not equally apply to every antibody of the present invention. For example, depending on the position of the first CDR the following CDRs might be shifted in either direction.

Antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are fragments binding to a molecule of interest of the present invention, said fragments comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. Antibodies or immunospecific fragments thereof of the present invention equivalent to the monoclonal antibodies isolated in accordance with the method of the present invention, in particular to the human monoclonal antibodies may be from any animal origin including birds and mammals.

Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be condricthoid in origin (e.g., from sharks). Optionally, the framework region of the human antibody is aligned and adopted in accordance with the pertinent human germ line variable region sequences in the database; see, e.g., Vbase (hosted by the MRC Centre for Protein Engineering (Cambridge, UK). For example, amino acids considered to potentially deviate from the true germ line sequence could be due to the PCR primer sequences incorporated during the cloning process.

Thus, in accordance with the present invention the terms “human monoclonal antibody”, “human monoclonal autoantibody”, “human antibody” and the like are used to denote binding molecule which binds a molecule of interest of the present invention, which is of human origin, i.e. Which has been isolated from a human cell such as a B cell or hybridoma thereof or the cDNA of which has been directly cloned from mRNA of a human cell, for example a human memory B cell.

A human antibody is still “human” even if amino acid substitutions are made in the antibody, e.g., to improve binding characteristics. The invention also relates to grafted antibodies (interchangeably referred to as equivalents) containing CDRs derived from the antibodies of the present invention, such as IL-17 and IL-22 antibodies, respectively. Such grafted CDRs include animalized antibodies, in which CDRs from the antibodies of the present invention have been grafted or in which a CDR containing one or more amino acid substitutions is grafted.

The CDRs can be grafted directly into a human framework or an antibody framework from animal origin as indicated above. If desired, framework changes can also be incorporated by generating framework libraries. The optimization of CDRs and/or framework sequences can be performed independently and sequentially combined or can be performed simultaneously, as described in more detail below.

To generate grafted antibodies donor CDRs of the antibodies of the present invention are grafted onto an antibody acceptor variable region framework. Methods for grafting antibodies and generating CDR variants to optimize activity have been described previously (see, e.g., international patent applications WO 98/33919; WO 00/78815; WO 01/27160). The procedure can be performed to achieve grafting of donor CDRs and affinity reacquisition in a simultaneous process. The methods similarly can be used, either alone or in combination with CDR grafting, to modify or optimize the binding affinity of a variable region. The methods for conferring donor CDR binding affinity onto an acceptor variable region are applicable to both heavy and light chain variable regions and as such can be used to simultaneously graft and optimize the binding affinity of an antibody variable region.

The donor CDRs can be altered to contain a plurality of different amino acid residue changes at all or selected positions within the donor CDRs. For example, random or biased incorporation of the twenty naturally occurring amino acid residues, or preselected subsets, can be introduced into the donor CDRs to produce a diverse population of CDR species. Inclusion of CDR variant species into the diverse population of variable regions allows for the generation of variant species that exhibit optimized binding affinity for a predetermined antigen. A range of possible changes can be made in the donor CDR positions. Some or all of the possible changes that can be selected for change can be introduced into the population of grafted donor CDRs. A single position in a CDR can be selected to introduce changes or a variety of positions having altered amino acids can be combined and screened for activity. One approach is to change all amino acid positions along a CDR by replacement at each position with, for example, all twenty naturally occurring amino acids.

The replacement of each position can occur in the context of other donor CDR amino acid positions so that a significant portion of the CDR maintains the authentic donor CDR sequence, and therefore, the binding affinity of the donor CDR. For example, an acceptor variable region framework, either a native or altered framework, can be grafted with a population of CDRs containing single position replacements at each position within the CDRs. Similarly, an acceptor variable region framework can be targeted for grafting with a population of CDRs containing more than one position changed to incorporate all twenty amino acid residues, or a subset of amino acids. One or more amino acid positions within a CDR, or within a group of CDRs to be grafted, can be altered and grafted into an acceptor variable region framework to generate a population of grafted antibodies.

It is understood that a CDR having one or more altered positions can be combined with one or more other CDRs having one or more altered positions, if desired. A population of CDR variant species having one or more altered positions can be combined with any or all of the CDRs which constitute the binding pocket of a variable region. Therefore, an acceptor variable region framework can be targeted for the simultaneous incorporation of donor CDR variant populations at one, two or all three recipient CDR locations in a heavy or light chain.

The choice of which CDR or the number of CDRs to target with amino acid position changes will depend on, for example, if a full CDR grafting into an acceptor is desired or whether the method is being performed for optimization of binding affinity. Another approach for selecting donor CDR amino acids to change for conferring donor CDR binding affinity onto an antibody acceptor variable region framework is to select known or readily identifiable CDR positions that are highly variable. For example, the variable region CDR3 is generally highly variable.

This region therefore can be selectively targeted for amino acid position changes during grafting procedures to ensure binding affinity reacquisition or augmentation, either alone or together with relevant acceptor variable framework changes. Murinized Antibodies:•. An example of antibodies generated by grafting, as described above, are murinized antibodies. As used herein, the term “murinized antibody” or “murinized immunoglobulin” refers to an antibody comprising one or more CDRs from a human antibody of the present invention; and a human framework region that contains amino acid substitutions and/or deletions and/or insertions that are based on a mouse antibody sequence. The human immunoglobulin providing the CDRs is called the “parent” or “acceptor” and the mouse antibody providing the framework changes is called the “donor”. Constant regions need not be present, but if they are, they are usually substantially identical to mouse antibody constant regions, i.e. At least about 85-90%, preferably about 95% or more identical.

Hence, in some embodiments, a full-length murinized human heavy or light chain immunoglobulin contains a mouse constant region, human CDRs, and a substantially human framework that has a number of “murinizing” amino acid substitutions. Typically, a “murinized antibody” is an antibody comprising a murinized variable light chain and/or a murinized variable heavy chain. For example, a murinized antibody would not encompass a typical chimeric antibody, e.g., because the entire variable region of a chimeric antibody is non-mouse. A modified antibody that has been “murinized” by the process of “murinization” binds to the same antigen as the parent antibody that provides the CDRs and is usually less immunogenic in mice, as compared to the parent antibody. Antibody Fragments:•. As used herein, the term “heavy chain portion” includes amino acid sequences derived from an immunoglobulin heavy chain.

A polypeptide comprising a heavy chain portion comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, a binding polypeptide for use in the invention may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain. Further, a binding polypeptide for use in the invention may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As set forth above, it will be understood by one of ordinary skill in the art that these domains (e.g., the heavy chain portions) may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule. The heavy chain portions of a binding polypeptide for use in the diagnostic and treatment methods disclosed herein may be derived from different immunoglobulin molecules.

For example, a heavy chain portion of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion can comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule. Thus, as also exemplified in the Examples, in one embodiment the constant region of the antibody of the present invention or part thereof, in particular the CH2 and/or CH3 domain but optionally also the CH1 domain is heterologous to the variable region of the native human monoclonal antibody isolated in accordance with the method of the present invention. In this context, the heterologous constant region(s) are preferably of human origin in case of therapeutic applications of the antibody of the present invention but could also be of for example rodent origin in case of animal studies; see also the Examples.

As previously indicated, the subunit structures and three dimensional configuration of the constant regions of the various immunoglobulin classes are well known. As used herein, the term “V H domain” includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CH1 domain” includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the V H domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule. As used herein the term “CH2 domain” includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat E A et al. The CH2 domain is unique in that it is not closely paired with another domain.

Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues. As used herein, the terms “linked”, “fused” or “fusion” are used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means. An “in-frame fusion” refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature).

Although the reading frame is thus made continuous throughout the fused segments, the segments may be physically or spatially separated by, for example, in-frame linker sequence. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region may be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the “fused” CDRs are co-translated as part of a continuous polypeptide. The minimum size of a peptide or polypeptide epitope for an antibody is thought to be about four to five amino acids. Peptide or polypeptide epitopes preferably contain at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids. Since a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an epitope need not be contiguous, and in some cases, may not even be on the same peptide chain. In the present invention, a peptide or polypeptide epitope recognized by antibodies of the present invention contains a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or between about 15 to about 30 contiguous or non-contiguous amino acids of a molecule of interest of the present invention as defined in Tables 1 to 14, 28-31, FIGS. 1 to 4 and in the Examples.

Binding Characteristics:•. By “binding” or “recognizing”, used interchangeably herein, it is generally meant that a binding molecule, e.g., an antibody binds to a predetermined epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D”. Unrelated epitopes are usually part of a nonspecific antigen (e.g., BSA, casein, or any other specified polypeptide), which may be used for the estimation of the binding specificity of a given binding molecule.

In this respect, term “specific binding” refers to antibody binding to a predetermined antigen with a K D that is at least twofold less than its K D for binding to a nonspecific antigen. The term “highly specific” bindingas used herein means that the relative K D of the antibody for the specific target epitope is at least 10-fold less than the K D for binding that antibody to other ligands. Where present, the term “immunological binding characteristics,” or other binding characteristics of an antibody with an antigen, in all of its grammatical forms, refers to the specificity, affinity, cross-reactivity, and other binding characteristics of an antibody. By “preferentially binding”, it is meant that the binding molecule, e.g., antibody specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope. Thus, an antibody which “preferentially binds” to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope. By way of non-limiting example, a binding molecule, e.g., an antibody may be considered to bind a first epitope preferentially if it binds said first epitope with a dissociation constant (K D) that is less than the antibody's K D for the second epitope. In another non-limiting example, an antibody may be considered to bind a first antigen preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's K D for the second epitope.

In another non-limiting example, an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's K D for the second epitope. In another non-limiting example, a binding molecule, e.g., an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an off rate (k(off)) that is less than the antibody's k(off) for the second epitope. In another non-limiting example, an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's k(off) for the second epitope.

In another non-limiting example, an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's k(off) for the second epitope. A binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative disclosed herein may be said to bind a molecule of interest of the present invention, a fragment or variant thereof with an off rate (k(off)) of less than or equal to 5×10 −2 sec −1, 10 −2 sec −1, 5×10 −3 sec −1 or 10 −3 sec −1. More preferably, an antibody of the invention may be said to bind a molecule of interest of the present invention or a fragment or variant thereof with an off rate (k(off)) less than or equal to 5×10 −4 sec −1, 10 −4 sec −1, 5×10 −5 sec −1, or 10 −5 sec −1 5×10 −6 sec −1, 10 −6 sec −1, 5×10 −7 sec −1 or 10 −7 sec −1. A binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative disclosed herein may be said to bind a molecule of interest of the present invention or a fragment or variant thereof with an on rate (k(on)) of greater than or equal to 10 3 M −1 sec −1, 5×10 3 M −1 sec −1, 10 4 M −1 sec −1 or 5×10 4 M −1 sec −1.

More preferably, an antibody of the invention may be said to bind a molecule of interest of the present invention or a fragment or variant thereof with an on rate (k(on)) greater than or equal to 10 5 M −1 sec −1, 5×10 5 M −1 sec −1, 10 6 M −1 sec −1, or 5×10 6 M −1 sec −1 or 10 7 M −1 sec −1. A binding molecule, e.g., an antibody is said to competitively inhibit binding of a reference antibody to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody to the epitope. Competitive inhibition may be determined by any method known in the art, for example, competition ELISA assays. An antibody may be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%. As used herein, the term “affinity” refers to a measure of the strength of the binding of an individual epitope with the CDR of a binding molecule, e.g., an immunoglobulin molecule; see, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988) at pages 27-28. As used herein, the term “avidity” refers to the overall stability of the complex between a population of immunoglobulins and an antigen, that is, the functional combining strength of an immunoglobulin mixture with the antigen; see, e.g., Harlow at pages 29-34.

Avidity is related to both the affinity of individual immunoglobulin molecules in the population with specific epitopes, and also the valencies of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity. The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method; see, for example, Berzofsky et al., “Antibody-Antigen Interactions” In Fundamental Immunology, Paul, W. E., Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W.H.

Freeman and Company New York, N Y (1992), and methods described therein. General techniques for measuring the affinity of an antibody for an antigen include ELISA, RIA, and surface plasmon resonance. The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions, e.g., salt concentration, pH. Thus, measurements of affinity and other antigen-binding parameters, e.g., K D, IC 50, are preferably made with standardized solutions of antibody and antigen, and a standardized buffer. Binding molecules, e.g., antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may also be described or specified in terms of their cross-reactivity. As used herein, the term “cross-reactivity” refers to the ability of an antibody, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances. Thus, an antibody is cross reactive if it binds to an epitope other than the one that induced its formation.

The cross reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, may actually fit better than the original. For example, certain antibodies have some degree of cross-reactivity, in that they bind related, but non-identical epitopes, e.g., epitopes with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a reference epitope. An antibody may be said to have little or no cross-reactivity if it does not bind epitopes with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a reference epitope. An antibody may be deemed “highly specific” for a certain epitope, if it does not bind any other analog, ortholog, or homolog of that epitope. Binding molecules, e.g., antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may also be described or specified in terms of their binding affinity to a molecule of interest of the present invention. Preferred binding affinities include those with a dissociation constant or K D less than 5×10 −2M, 10 −2M, 5×10 −3M, 10 −3M, 5×10 −4M, 10 −4 M, 5×10 −5M, 10 −5M, 5×10 −6M, 10 −6M, 5×10 −7M, 10 −7M, 5×10 −8M, 10 −8M, 5×10 −9M, 10 −9M, 5×10 −10 M, 10 −10 M, 5×10 −11M, 10 −11M, 5×10 −12M, 10 −12M, 5×10 −13M, 10 −13M, 5×10 −14 M, 10 −14 M, 5×10 −15 M, or 10 −15 M.

Typically, the antibody binds with a dissociation constant (K D) of 10 −7 M or less to its predetermined antigen. Preferably, the antibody binds its cognate antigen with a dissociation constant (K D) of 10 −9 M or less and still more preferably with a dissociation constant (K D) of 10 −11M or less. Modifications of Antibodies:•. The immunoglobulin or its encoding cDNAs may be further modified. Thus, in a further embodiment the method of the present invention comprises any one of the step(s) of producing a chimeric antibody, humanized antibody, single-chain antibody, Fab-fragment, bispecific antibody, fusion antibody, labeled antibody or an analog of any one of those.

Corresponding methods are known to the person skilled in the art and are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. When derivatives of said antibodies are obtained by the phage display technique, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to the same epitope as that of any one of the antibodies provided by the present invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J.

Methods 183 (1995), 7-13). The production of chimeric antibodies is described, for example, in international application WO89/09622. Methods for the production of humanized antibodies are described in, e.g., European application EP-A1 0 239 400 and international application WO90/07861.

Further sources of antibodies to be utilized in accordance with the present invention are so-called xenogeneic antibodies. The general principle for the production of xenogeneic antibodies such as human antibodies in mice is described in, e.g., international applications WO91/10741, WO94/02602, WO96/34096 and WO 96/33735. As discussed above, the antibody of the invention may exist in a variety of forms besides complete antibodies; including, for example, Fv, Fab and F(ab) 2, as well as in single chains; see e.g. International application WO88/09344. The antibodies of the present invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. And Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.

Modifications of the antibody of the invention include chemical and/or enzymatic derivatizations at one or more constituent amino acids, including side chain modifications, backbone modifications, and N- and C-terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like. Likewise, the present invention encompasses the production of chimeric proteins which comprise the described antibody or some fragment thereof at the amino terminus fused to heterologous molecule such as a label or a drug. Antigen binding molecules generated this way may be used for drug localization to cells expressing the appropriate surface structures of the diseased cell and tissue, respectively.

This targeting and binding to cells could be useful for the delivery of therapeutically or diagnostically active agents and gene therapy/gene delivery. Molecules/particles with an antibody of the invention would bind specifically to cells/tissues expressing the particular antigen of interest, and therefore could have diagnostic and therapeutic use. As used herein, the term “sample” refers to any biological material obtained from a subject or patient.

In one aspect, a sample can comprise blood, cerebrospinal fluid (“CSF”), or urine. In other aspects, a sample can comprise whole blood, plasma, mononuclear cells enriched from peripheral blood (PBMC) such as lymphocytes (i.e. T-cells, NK-cell or B-cells), monocytes, macrophages, dendritic cells and basophils; and cultured cells (e.g., B-cells from a subject). A sample can also include a biopsy or tissue sample including tumor tissue.

In still other aspects, a sample can comprise whole cells and/or a lysate of the cells. In one embodiment a sample comprises peripheral blood mononuclear cells (PBMC). Samples can be collected by methods known in the art. Identification and Isolation of B-Cells:•. Identification of B-cells specific for the particular molecules of interest of the present invention, as enlisted in Tables 1 to 14, and as exemplary shown for IL17 or IL-22 and molecular cloning of antibodies displaying specificity of interest as well as their recombinant expression and functional characterization can be generally performed as described in particular in Example 2. A method for identification of B-cells expressing the antibodies of the specificity of interest and molecular cloning of antibodies displaying specificity of interest as well as their recombinant expression and functional characterization is provided within this application.

As described in detail below, in one embodiment of the present invention cultures of single or oligoclonal B-cells are cultured and the supernatant of the culture, which contains antibodies produced by said B-cells is screened for presence and affinity of new antibodies specific for a molecule of interest of the present invention, such as molecules enlisted in Tables 1 to 14, and described in the Examples, or in particular molecules involved in inflammation or related to an autoimmune disease in a subject, e.g., IL17 or IL-22 therein. In another embodiment, not B-cells cultures but patient sera are first screened for the presence of autoantibodies against a molecule of interest and then those with high titer are selected for peripheral blood mononuclear cells isolation; see Example 2. The screening process comprises screening for binding on fragments, peptides or derivates of the particular molecule of interest. Subsequently the antibody for which binding is detected or the cell producing said antibody are isolated; see Example 3. Thus, a preliminary screen can be done on a panel of candidate donors, using samples containing antibody secreting cells (such as total peripheral blood or serum). In particular, mononuclear cells can be isolated from blood or lymphatic tissues using standard separation techniques for isolating peripheral blood mononuclear cells (PBMCs), such as gradient centrifugation. After and/or before this separation step, the samples of sera (or plasma), cell culture supernatants, or cells (obtained from different patients, from different tissues, and/or at different time points) can be prescreened using standard technologies for detecting the presence of antibodies and antibody-secreting cells (e.g.

ELISA, BIACORE, Western blot, FACS, SERPA, antigen arrays, neutralization of viral infection in a cell culture system, or ELISPOT assays). The literature provides several examples of these technologies showing, for example, the use of ELISPOT for characterizing the immune response in vaccinated donors (Crotty et al., Immunol Meth. 286 (2004), 111-122), the use of antigen microarrays as diagnostic tools for newly infected patients (Mezzasoma et al., Clin Chem. 48 (2002), 121-130, and other technologies for measuring antigen-specific immune responses (Kern et al., Trends Immunol.

26 (2005), 477-484). B-Cell Immortalization:•. B-cells producing antibodies may be immortalized, e.g., by infection with viruses such as the oncogenic Epstein-Barr virus (EBV; Sugimoto et al., Cancer Res. 64 (2004), 3361-3364; Counter et al., J. 68 (1994), 3410-3414) or long term repeated CD40 ligand/IL-4 stimulation (Wiesner et al., PLoS ONE. 3 (2008), e1464.). Methods of producing clones of an immortalized human B cell and a B memory lymphocyte, comprising the step of transforming human B memory lymphocytes using Epstein Barr Virus (EBV) in the presence of a polyclonal B cell activator are summarized in international application WO2004/076677.

This international application also describes methods for obtaining a nucleic acid sequence that encodes an antibody of interest, comprising the steps of preparing an immortalized B cell clone and obtaining/sequencing nucleic acid from the B cell clone that encodes the antibody of interest and further inserting the nucleic acid into or using the nucleic acid to prepare an expression host that can express the antibody of interest, culturing or sub-culturing the expression host under conditions where the antibody of interest is expressed and, optionally, purifying the antibody of interest. It goes without saying that the nucleic acid may be manipulated in between to introduce restriction sites, to change codon usage, and/or to add or optimize transcription and/or translation regulatory sequences.

All these techniques are state of the art and can be performed by the person skilled in the art without undue burden. For example, the heavy chain constant region can be exchanged for that of a different isotype or eliminated altogether. The variable regions can be linked to encode single chain Fv regions. Multiple Fv regions can be linked to confer binding ability to more than one target or chimeric heavy and light chain combinations can be employed. Once the genetic material is available, design of analogs as described above which retain both their ability to bind the desired target is straightforward. Methods for the cloning of antibody variable regions and generation of recombinant antibodies are known to the person skilled in the art and are described, for example, in Gilliland et al., Tissue Antigens 47 (1996), 1-20; Doenecke et al., Leukemia 11 (1997), 1787-1792. Diseases and Disorders:•.

Unless stated otherwise, the terms “disorder” and “disease” are used interchangeably herein. The term “autoimmune disorder” as used herein is a disease or disorder arising from and directed against an individual's own tissues or organs or a co-segregate or manifestation thereof or resulting condition therefrom. Autoimmune diseases are primarily caused by dysregulation of adaptive immune responses and autoantibodies or autoreactive T cells against self structures are formed. Nearly all autoimmune diseases have an inflammatory component, too. Autoinflammatory diseases are primarily inflammatory, and some classic autoinflammatory diseases are caused by genetic defects in innate inflammatory pathways. In autoinflammatory diseases, no autoreactive T cells or autoantibodies are found. In many of these autoimmune and autoinflammatory disorders, a number of clinical and laboratory markers may exist, including, but not limited to, hypergammaglobulinemia, high levels of autoantibodies, antigen-antibody complex deposits in tissues, benefit from corticosteroid or immunosuppressive treatments, and lymphoid cell aggregates in affected tissues.

Without being limited to a theory regarding B-cell mediated autoimmune disorder, it is believed that B cells demonstrate a pathogenic effect in human autoimmune diseases through a multitude of mechanistic pathways, including autoantibody production, immune complex formation, dendritic and T-cell activation, cytokine synthesis, direct chemokine release, and providing a nidus for ectopic neo-lymphogenesis. Each of these pathways may participate to different degrees in the pathology of autoimmune diseases. Immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) is a rare X-linked recessive disease. In 25% of males with clinical phenotype, mutations in the FOXP3 gene (forkhead box P3; member of the forkhead/winged-helix family of transcriptional regulators) could be found, however data has been presented (Owen et al., J. 2003; 88:6034-6039) suggesting that at least one additional autosomal locus possibly from the same pathway might be involved leading to a dysfunction of FOXP3 since in several cases without an identified mutation, lowered expression or low numbers of FOXP3-expressing cells were detected (Torgerson, unpublished results-GeneReviews; NCBI Bookshelf). IPEX syndrome can be taken as a prototype of a disease caused by a defect in peripheral tolerance. Mutations in the FoxP3 gene lead to suppression of regulatory T-cells and subsequent hyper activation of auto aggressive T-cells and autoantibody formations.

Clinical findings include a basic clinical triad (Powell et al., J. 100 (1982), 731-737; Ochs et al., Immunol.

203 (2005), 156-164) comprising: •. Endocrinopathy with most commonly type 1 diabetes mellitus or autoimmune thyroid disease leading to hypothyroidism or hyperthyroidism (Wildin et al., J.

39 (2002), 537-545; Gambineri et al., J. Allergy Clin. 122(2008), 1105-1112) Enteropathy with chronic watery diarrhea Dermatitis with eczema most common, nail dystrophy, erythroderma, psoriasiform lesions, and alopecia universalis also reported Without treatment, most patients die within first 12 to 24 months of life, immunosuppressive regimens and bone marrow transplantation prolong survival but not beyond the second or third decade of life in milder disease cases (Powell et al., J. 100 (1982); Kobayashi et al, J. 38 (2001), 874-876; Levy-Lahad and Wildin, J. 138 (2001), 577-580; Taddio et al., Eur.

166 (2007), 1195-1197). An important feature of the thymoma is the various systemic diseases associated with this tumor. Most of these thymoma-associated conditions are linked to autoimmunity. The most well know is the relatively rare neuromuscular disease Myasthenia gravis, which is caused by autoantibodies to the acetylcholine receptors, located at postsynaptic neuromuscular junction.

These antibodies block the effect of the neurotransmitter acetylcholine and thus will cause muscular fatigue. Close to half (30-45%) or patients with thymoma have myasthenia gravis.

Other autoimmune diseases associated with thymoma are acute pericarditis, Addison's disease (adrenocortical failure) alopecia areata, ulcerative colitis, hemolytic anemia, pernicious anemia, rheumatoid arthritis, scleroderma, systemic lupus erythematosis and thyroiditis. Labels and Diagnostics:•. Labeling agents can be coupled either directly or indirectly to the antibodies or antigens of the invention. One example of indirect coupling is by use of a spacer moiety. Furthermore, the antibodies of the present invention can comprise a further domain, said domain being linked by covalent or non-covalent bonds. The linkage can be based on genetic fusion according to the methods known in the art and described above or can be performed by, e.g., chemical cross-linking as described in, e.g., international application WO94/04686.

The additional domain present in the fusion protein comprising the antibody of the invention may preferably be linked by a flexible linker, advantageously a polypeptide linker, wherein said polypeptide linker comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of said further domain and the N-terminal end of the antibody of the invention or vice versa. The therapeutically or diagnostically active agent can be coupled to the antibody of the invention or an antigen-binding fragment thereof by various means. This includes, for example, single-chain fusion proteins comprising the variable regions of the antibody of the invention coupled by covalent methods, such as peptide linkages, to the therapeutically or diagnostically active agent.

Further examples include molecules which comprise at least an antigen-binding fragment coupled to additional molecules covalently or non-covalently include those in the following non-limiting illustrative list. Traunecker, Int. SuDP 7 (1992), 51-52, describe the bispecific reagent janusin in which the Fv region directed to CD3 is coupled to soluble CD4 or to other ligands such as OVCA and IL-7. Similarly, the variable regions of the antibody of the invention can be constructed into Fv molecules and coupled to alternative ligands such as those illustrated in the cited article. Disease 166 (1992), 198-202, described a hetero-conjugate antibody composed of OKT3 cross-linked to an antibody directed to a specific sequence in the V3 region of GP120. Such hetero-conjugate antibodies can also be constructed using at least the variable regions contained in the antibody of the invention methods.

Additional examples of specific antibodies include those described by Fanger, Cancer Treat. 68 (1993), 181-194 and by Fanger, Crit. 12 (1992), 101-124. Conjugates that are immunotoxins including conventional antibodies have been widely described in the art. The toxins may be coupled to the antibodies by conventional coupling techniques or immunotoxins containing protein toxin portions can be produced as fusion proteins. The antibodies of the present invention can be used in a corresponding way to obtain such immunotoxins. Illustrative of such immunotoxins are those described by Byers, Seminars Cell.

2 (1991), 59-70 and by Fanger, Immunol. Today 12 (1991), 51-54.

The above described fusion protein may further comprise a cleavable linker or cleavage site for proteinases. These spacer moieties, in turn, can be either insoluble or soluble (Diener et al., Science 231 (1986), 148) and can be selected to enable drug release from the antigen at the target site. Examples of therapeutic agents which can be coupled to the antibodies and antigens of the present invention for immunotherapy are chemokines, homing molecules, drugs, radioisotopes, lectins, and toxins.

The drugs with which can be conjugated to the antibodies and antigens of the present invention depend on the disease context in which the conjugated molecules are intended to be used. For example, antibodies specific for targets useful in treatment of tumor diseases can be conjugated to compounds which are classically referred to as anti-neoplastic drugs such as mitomycin C, daunorubicin, and vinblastine. In using radioisotopically conjugated antibodies or antigens of the invention for, e.g., tumor immunotherapy, certain isotopes may be more preferable than others depending on such factors as leukocyte distribution as well as stability and emission. Depending on the autoimmune response, some emitters may be preferable to others.

In general, a and B particle emitting radioisotopes are preferred in immunotherapy. Preferred are short range, high energy a emitters such as 212Bi. Examples of radioisotopes which can be bound to the antibodies or antigens of the invention for therapeutic purposes are 125I, 131I, 90Y, 67Cu, 212Bi, 212At, 211Pb, 47Sc, 109Pd and 188Re. Other therapeutic agents which can be coupled to the antibody or antigen of the invention, as well as ex vivo and in vivo therapeutic protocols, are known, or can be easily ascertained, by those of ordinary skill in the art. Non-limiting examples of suitable radionuclides for labeling are 198Au, 212Bi, 11C, 14C, 57Co, 67Cu, 18F, 67Ga, 68Ga, 3H, 197Hg, 166Ho, 111In, 113mIn, 123I, 125I, 127I, 131I, 111In, 171Lu, 15O, 13N, 32P, 33P, 203Pb, 186Re, 188Re, 105Rh, 97Ru, 35S, 153Sm and 99mTc. Other molecules suitable for labeling are a fluorescent or luminescent dye, a magnetic particle, a metal, and a molecule which may be detected through a secondary enzymatic or binding step such as an enzyme or peptide tag. Commercial fluorescent probes suitable for use as labels in the present invention are listed in the Handbook of Fluorescent Probes and Research Products, 8th Edition, the disclosure contents of which are incorporated herein by reference.

Magnetic particles suitable for use in magnetic particle-based assays (MPAs) may be selected from paramagnetic, diamagnetic, ferromagnetic, ferromagnetic and superpara-magnetic materials. General methods in molecular and cellular biochemistry useful for diagnostic purposes can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed.

(Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. Eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996).

Reagents, detection means and kits for diagnostic purposes are available from commercial vendors such as Pharmacia Diagnostics, Amersham, BioRad, Stratagene, Invitrogen, and Sigma-Aldrich as well as from the sources given any one of the references cited herein, in particular patent literature. Treatment and Drugs:•. As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development of an autoimmune and/or autoinflammatory disease.

Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the manifestation of the condition or disorder is to be prevented. Examples of “anti-rheumatic drugs” and immunosuppressive drugs include chloroquine, hydroxycloroquine, myocrisin, auranofin, sulfasalazine, methotrexate, leflunomide, etanercept, infliximab (plus oral and subcutaneous methotrexate), adalimumab etc., azathioprine, D-penicilamine, gold salts (oral), gold salts (intramuscular), minocycline, cyclosporine including cyclosporine A and topical cyclosporine, tacrolimus, mycophenolate mofetil, cyclophosphamide, staphylococcal protein A (Goodyear and Silverman, J. Med., 197 (2003), 125-39), including salts and derivatives thereof, etc. Pharmaceutically acceptable carriers and administration routes can be taken from corresponding literature known to the person skilled in the art. The pharmaceutical compositions of the present invention can be formulated according to methods well known in the art; see for example Remington: The Science and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0-683-306472, Vaccine Protocols.

2nd Edition by Robinson et al., Humana Press, Totowa, N.J., USA, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems. 2nd Edition by Taylor and Francis. (2006), ISBN: 0-8493-1630-8.

Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways.

Examples include administering a composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods. Aerosol formulations such as nasal spray formulations include purified aqueous or other solutions of the active agent with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucous membranes. Pharmaceutical compositions for oral administration, such as single domain antibody molecules (e.g., “Nanobodies™”) etc are also envisaged in the present invention. Such oral formulations may be in tablet, capsule, powder, liquid or semi-solid form. A tablet may comprise a solid carrier, such as gelatin or an adjuvant.

Formulations for rectal or vaginal administration may be presented as a suppository with a suitable carrier; see also O'Hagan et al., Nature Reviews, Drug Discovery 2(9) (2003), 727-735. Further guidance regarding formulations that are suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985) and corresponding updates. For a brief review of methods for drug delivery see Langer, Science 249 (1990), 1527-1533. Dosage Regimen:•.

The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively.

Progress can be monitored by periodic assessment. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.

Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.

Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents such as anti-tumor agents and cytotoxic drugs, depending on the intended use of the pharmaceutical composition.

In addition, co-administration or sequential administration of other agents may be desirable. A therapeutically effective dose or amount refers to that amount of the active ingredient sufficient to ameliorate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.

These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature concerning any one of the materials, methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries and databases, using for example electronic devices. For example the public database “Medline” may be utilized, which is hosted by the National Center for Biotechnology Information and/or the National Library of Medicine at the National Institutes of Health. Further databases and web addresses, such as those of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL) are known to the person skilled in the art and can also be obtained using internet search engines. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364. The above disclosure generally describes the present invention. Unless otherwise stated, a term as used herein is given the definition as provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19 850673 2.

Several documents are cited throughout the text of this specification. Full bibliographic citations may be found at the end of the specification immediately preceding the claims. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application and manufacturer's specifications, instructions, etc) are hereby expressly incorporated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention. Methods in molecular genetics and genetic engineering are described generally in the current editions of Molecular Cloning: A Laboratory Manual, (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press); DNA Cloning, Volumes I and II (Glover ed., 1985); Oligonucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1984); Transcription And Translation (Hames and Higgins eds. 1984); Culture Of Animal Cells (Freshney and Alan, Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, eds.); Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Edition (Ausubel et al., eds.); and Recombinant DNA Methodology (Wu, ed., Academic Press).

Gene Transfer Vectors For Mammalian Cells (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al., eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir and Blackwell, eds., 1986). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, and Clontech.

General techniques in cell culture and media collection are outlined in Large Scale Mammalian Cell Culture (Hu et al., Curr. 8 (1997), 148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian Cell Culture ( Curr. 2 (1991), 375); and Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251. Material and Methods Patients Selection and Disease Associations of the Invention•.

The group of 33 Finnish APECED patients studied for the autoantibodies and serving as the source for specific B-lymphocyte cloning in respect of the present invention have the following features. Of the patients, 21 were female and 12 male, the mean age being 38 yrs (females 38.3 yrs; males 37.8 yrs). The mean age at 1 st symptoms of APECED had been 6.2 yrs. The mean weight of the men was 69.75 kg and of the women 59.4 kg. These 33 patients responded to a questionnaire on their clinical symptoms.

As general symptoms, 30% of the patients experienced fever periods and 67% experienced asthenia. 30 of the patients donated blood and their sera were analyzed by ELISA as shown in FIGS.

Sera of 23 of these patients and of 7 healthy control persons (C1 to C7) were selected for protoarray screening. Organ-Specific Symptoms and Diseases•. The organ-specific symptoms and diseases in APECED patients, as a consequence of the AIRE gene defect, have been previously described and the tissue-specific autoantigens are mostly known (Betterle et al., J. 83 (1998) 1049-1055; Husebye, E. S., Perheentupa et al., J. 265 (2009), 514-529; Soderbergh et al., J.

89 (2004), 557-562; Uibo et al., J. 78 (1994), 323-328).

The present patient cohort presented with organ-specific autoimmune symptoms as follows (known autoantigen in parentheses): •. Addison's disease in 81% (adrenal gland 21 hydroxylase; 17alpha-hydroxylase; side chain cleavage enzyme (SCC)), concomitant hypoparathyroidism in 79% (parathyroid, NALP5 (NACHT leucin rich repeat protein 5) and hypothyroidism in 45% (thyroperoxydase; Thyroglobuline). Twelve of the latter 15 patients also had hypoparathyroidism and Addison's disease.

Infertility was recorded in 30% (testicular/ovarian insufficiency/atrophy in 1 male and in 10 females; 17alpha-hydroxylase and SCC), asplenia in 24%, Hypophysial/GH deficiency in 18% (mostly male; Tudor domain containing protein 6 (TDRD6)), diabetes mellitus in 12% (pancreatic GAD65; Insuline; Tyrosine phosphatase 1A2), Pernicious anemia in 12% (stomach mucosa, Intrinsic factor, gastric parietal cell as autoantigens), Autoimmune hepatitis in 9% (AntiLKM1+but different antigens: cytochrome P450 1A2, P450 2A6, P450 1A1, P450 2B6) and Nephritis in 9% (all female). Without indending of being bound to theory, it is prudent to expect that the human antibodies, naturally elicited in APECED patients as identified herein and protective against the above diseases are as follows: • • anti-interferon antibodies: protective against lupus erythematosus. • anti-IL17: protective against psoriasis and inflammatory bowel disease (IBD) • anti-IL-5, IL-7 (thymus, skin, intestine), IL-10, IL-11: protective against atopic diseases • anti-IL-10 antibodies: may be used to enhance antitumor immune response (in various types of cancer) • anti-IL-7 antibodies: protective against human T-cell acute lymphoblastic leukemias • anti-IL-28, IL-29: produced widely, esp. DC, antiviral and cytostatic, target epithelial cells and hepatocytes, upregul.

TLR 2 an 3, TLR-induced IL-12 • anti-CCL-17, CCL-22 (expressed by eosinophiles, basal cells, Th-2): protective against asthma, RA, CNS inflammation. • anti-IL-17 A/F, IL-22, also CCL-20 and anti-IL-36: protect against psoriasis Allergic Diseases•. The incidence of allergic diseases is decreased in APECED patients and we propose this is due, in part, to the autoantibodies naturally elicited in APECED patients and targeting the central inflammatory mediator pathways in allergic diseases.

In Finland, the prevalence of various allergic diseases in the general population are as follows: atopic eczema: (AE) prevalence in children 15-20% and 25-30% of the adults have experienced AE at some time during their lifetime. While in the APECED patient cohort, atopic eruption was found in only 12% of the cases, nasal allergy in 12%, ocular allergy in 15%, airway allergy/Asthma in 10% and contact allergy in 6%. The autoantibodies found in accordance with the present invention and which are directed against CCL17, CCL, 19, CCL21 and CCL22 are protective against the allergic manifestations, since e.g. CCL17 and CCL22 are known to be upregulated in AE (ref), and likewise CCL19, CCL21 in asthma: iBALT (infection/chronic infection). Microbe Infections•. Genotyping of the respective mutations in the AIRE (APECED) gene is performed as described in international application WO99/15559 in Example 2 at pages 12 to 13, the disclosure content of which is incorporated herein by reference in its entirety. In particular, for the mutation analysis the DNA samples are purified from periferal blood mononuclear cells from patients with APECED and from suspected carriers of APECED and from normal healthy controls (according to Sambrook et a/.

1989, Molecular Cloning. A Laboratory Manual. CSH Press) and subjected to PCR using primers specific for all identified exons. Invitrogen protoarray chips for seroactivity testing: The ProtoArray® Human Protein Microarray v5.0 slides (Invitrogen) were blocked and then probed with a 1:500 dilution of each serum sample diluted in PBST buffer. Arrays were then incubated 90 minutes at 4° C. With gently agitation.

After incubation, the slides were washed five times in PBST buffer. An Alexa Fluor® 647-conjugated goat anti-human IgG antibody diluted in PBST buffer was then added to each array and allowed to incubate with gentle shaking at 4° C.

For 90 minutes. After incubation, the secondary antibody was removed, arrays were washed as described above and dried by spinning Finally, arrays were scanned using an Axon GenePix 4000B fluorescent microarray scanner. Memory B Cell Culture•. Memory B cells were isolated from human peripheral blood monocytic cells derived from the peripheral blood of voluntary Finnish patients with Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED, OMIM 240300), also called autoimmune polyendocrine syndrome type 1 (APS1) with a one step protocol using phycoerythrin-conjugated mAb anti-human IgD, APC-conjugated mAbs anti-human IgM, CD3, CD56, CD8 and FITC-conjugated mAb anti human CD22 (Becton Dickinson, Basel, Switzerland).

Cell sorting was carried out using a MoFlo XDP cell sorter (Beckman Coulter). CD22-positive- and IgM-, IgD-negative B cells were then incubated with EBV containing supernatant obtained from B95-8 cells (in B cell medium containing RPMI 1640 supplemented with 10% fetal calf serum) for 3 to 5 hours (preferably 3.5 hours). Cells were seeded at 10 cells per well in IMDM medium supplemented with CpG 2006 on 30.000 irradiated feeder PBL prepared from voluntary donors. After 8-14 days of culture the conditioned medium of memory B cell culture was screened for the presence of antigen of interest-specific (e.g., IL-17, IL-22) antibodies by ELISA.

IL-17, IL-22 ELISA•. 96 well microplates (Costar, USA) were coated with human IL-17A, or IL-17F (both from BioLegend), or IL-22 (ImmunoTools). Plates were washed with PBS-T and blocked 1 h at room temperature with PBS containing 2% BSA (Sigma, Buchs, Switzerland). Patient sera, B cell conditioned medium, or recombinant antibody preparations were incubated for 2h at room temperature.

Binding of human IgG to the antigen of interest was determined using a horseradish peroxidase conjugated goat anti human Fc-gamma-specific antibody (Jackson ImmunoResearch, Europe Ltd., Cambridgeshire, UK) followed by measurement of the HRP activity using a TMB substrate solution (TMB, Sigma, Buchs, Switzerland). Example 1 Isolation of Human Peripheral Blood Mononuclear Cells (PBMC) from APECED/APS1 Patients•. As starting material for the cloning of fully human antibodies, human lymphocytes were used obtained from the peripheral blood of 23 voluntary Finnish patients with Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED, OMIM 240300), also called autoimmune polyendocrine syndrome type 1 (APS1). These volunteers were recruited for the blood donation through the Finnish APECED and Addison patient association. All patients gave their written informed consent and the study has been approved by the Medicine Ethical Review Board of the Joint Authority of Helsinki and Uusimaa hospital district. APECED is an autosomal recessive disorder caused by mutations in the AIRE (autoimmune regulator) gene, located on chromosome 21 (21q22.3) and APECED is prevalent in Finland ( 1/25,000) because of a founder effect. APECED patients present with various endocrine autoimmune dysfunctions including mainly adrenal failure and hypoparathyroidism, but also variously hypogonadism, diabetes mellitus, thyroiditis and hypophysitis.

Other main symptoms are chronic mucocutaneous candidiasis, alopecia and vitiligo (see also supra). Since a strong correlation between the antigen-specific IgG levels in the serum and the frequency of antigen-specific B cells in the memory pool of peripheral blood mononuclear cells has been reported (Bernasconi et al.

2002, Lanzavecchia et al. 2006), patient sera were first screened for the presence of autoantibodies against the proteins of interest (like IFN, IL-17, IL-22) and then those APECED cases with high titer (>1:5000) were selected for peripheral blood mononuclear cell (PBMC) isolation as follows. Heparinized peripheral blood was obtained and diluted with two volumes of 1×PBS at RT, and the cells were overlayed on Lympholyte H, centrifuged at 2000 rpm (805 rcf) at RT for 20 minutes. The cells were harvested at interphase, mixed in washing buffer fill, centrifuged at 1,500 rpm (453 rcf) for 15 min at 4° C. And resuspended by gentle flicking, with 10 ml WB. Thereinafter the cells were centrifuged at 1,000 rpm (201 rcf), 10 min at 4° C.

And washed once more with WB. The cells were then resuspended gently in appropriate volume of FBS on ice. FBS was added to adjust volume to have 20 mio/ml whereafter 1 volume of freezing medium (80% FBS (Hyclone, Thermo Scientific and 20% DMSO, #154938, Sigma) was slowly added while stirring, resuspended and aliquoted into cryovials kept on ice. The cryovials were placed in Mr. Frosty box and transferred to −80° C.

Freezer for a maximum of 5 days before further processing as described in Example 2. Alternatively the cryovials were stored in liquid nitrogen. Example 2 Molecular Cloning of Human Antibodies Specific to IL-17 or IL-22•. Memory B cells were isolated from PBMC derived from the peripheral blood of voluntary Finnish patients with APECED with a one step protocol using phycoerythrin-conjugated mAb anti-human IgD, APC-conjugated mAbs anti-human IgM, CD3, CD56, CD8 and FITC-conjugated mAb anti human CD22 (Becton Dickinson, Basel, Switzerland). Cell sorting was carried out using a MoFlo XDP cell sorter (Beckman Coulter).

CD22-positive- and IgM-, IgD-negative B cells were stimulated with EBV containing supernatant obtained from B95-8 cells (in B cell medium containing RPMI 1640 supplemented with 10% fetal calf serum). Cells were seeded in IMDM medium supplemented with CpG 2006 at 10 cells per well on 30.000 irradiated feeder PBMC prepared from voluntary donors. After 10-14 days stimulation, culture supernatants were screened for the presence of antibodies specific for the target of interest (e.g. IL17, IL-22). The screening process comprised screening for binding on fragments, peptides or derivates of the particular molecule of interest, e.g., by ELISA.

Subsequently the antibody for which binding is detected or the cell producing said antibody is isolated. Single cells obtained from IL-17/IL-22-reactive memory B cell cultures are deposited into a 96 well PCR plate, containing first strand buffer (Invitrogen, LuBioScience, Switzerland).

CDNA is prepared using Random hexamer primer (Invitrogen, LuBioScience, Switzerland). PCR amplification of immunoglobulin heavy and light chain variable regions is performed according to standard protocols (Wardemann et al., Science 301, 2003, 1374-1377).

Immunoglobulin heavy and light chain variable regions are amplified using a nested PCR approach. 1st round PCR is performed with primers specific for the IgG constant region and primer mixes specific for all signal peptides of heavy and light chain Ig variable region families (Wardemann et al., Science 301, 2003, 1374-1377). Subsequently, nested PCR is performed using primer mixes specific for the immunoglobulin J-regions and the 5′ region of framework 1 of heavy and light chain Ig variable region families. Sequence analysis is carried out to identify the individual antibody clones present in the selected B-cell culture. Subsequently, the Ig-variable heavy- and light regions of each antibody clone are cloned into expression vectors providing the constant regions of human IgG1, human Ig-Kappa or human Ig-Lambda. Upon co-transfection of the Ig-heavy- and light expression vectors into HEK 293 cells the antibody clones are produced. Identification of the antibody clone presumably responsible for the IL-17/IL-22-reactivity of the parental B cell culture is performed upon re-screening of the recombinant antibody clones in IL-17/IL-22- and control ELISA.

In order to identify and to correct primer encoded sequence mismatches in the Ig-variable region, a further PCR amplification using a semi-nested protocol is performed with 2 primer pairs specific for a conserved region of the Ig-heavy- and light chain constant regions as 3′-primers and primer mixes specific for the Ig-signal peptides as 5′-primers. PCR products are cloned into TOPO™ vector (Invitrogen, LuBioScience, Lucerne, Switzerland). Sequence determination of the complete Ig-variable region is carried out and the information is used to design specific primers for the cloning of the authentic human antibody sequence into antibody expression vectors. This approach allows the identification of the complete antibody sequence of the Ig-variable region as it occurred in the patient.

This sequence is used for recombinant production of these antibodies which are then used in the subsequent characterization steps. Example 3 Antibody Production and Purification•. Transient gene expression of human antibodies is achieved upon transfection of antibody expression vectors into 293-T human embryonic kidney cells or Chinese Hamster Ovary cells (CHO) using the Polyethylenimine Transfection method (PEI, Polyscience Warrington, USA).

After transfection cells are cultured in serum free medium (OPTI-MEM I supplemented with GlutaMAX-I Gibco). Supernatants are collected after 3-6 days of culture and IgG is purified using protein A columns (GE HealthCare, Sweden) on a fast protein liquid chromatography device (FPLC) (GE HealthCare, Sweden). Example 4 In Vitro Cell-Based Neutralizing Assays•.

The neuralizing assays are carried out on cell lines that respond to the studied cytokine, i.e. Carry the necessary receptor. The ligand binding to receptor activates a corresponding signaling pathway, translocation of transcription factors to the nucleus and upregulate responder gene transcription, translation and if applicable product secretion. The cytokine concentration used is selected from the beginning of the linear part of the dose-response curve to maximize the sensitivity of the assay. To test the neutralizing capacity of antibodies the optimal concentration of the target cytokine is preincubated with serial dilutions of serum, supernatant or purified antibody samples.

The results are expressed as titer or concentration of antibody that show the value half-way between the positive and negative controls. IL-22 Neutralizing Assay•. Serial dilutions of serum samples, culture supernatants or purified antibodies are co-incubated with 0.5 ng/ml of IL-22 in 96-well tissue culture plate at 37° C. Colo205 cell line is added after 2 hours of co-incubation at 3×10 4 cells/well in RPMI-1640 with 10% heat inactivated FBS. After incubation at 37° C.

For 16-20 h, supernatants are collected and analyzed for IL-10 production by ELISA. Results are estimated from graphs of ELISA absorbances as the serum or supernatant titer or antibody concentration yielding a value half-way between the positive and negative controls. ED50s are defined as the concentration or titer needed to halve the cytokine activity of the test sample. IL-17A Neutralizing Assay•. 1BR.3.G human skin fibroblast cells are seeded at 1×10 4 cells/well in which IL-17-A (2 ng/ml) had been pre-exposed to serially diluted serum, supernatant or antibody samples for 2 h in DMEM with 10% inactivated FBS. After incubation at 37° C.

For 16-20 h, supernatants are collected and analyzed for growth-related oncogene (GRO)-α production by ELISA. Results are estimated from graphs of ELISA absorbances as the serum or supernatant titer or antibody concentration yielding a value half-way between the positive and negative controls. ED50s are defined as the concentration or titer needed to halve the cytokine activity of the test sample. IL-17F Neutralizing Assay•.

NCTC 2544 keratinocytes are pre-treated with TNF-α (0.1 ng/ml) in DMEM with 10% inactivated FBS for 3 hours. Serial dilutions of serum samples, culture supernatants or purified antibodies are co-incubated with 10 ng/ml of IL-17F in 96-well tissue culture plate at 37° C. Keratinocytes are added after 2 hours of co-incubation at 1×10 4 cells/well. After incubation at 37° C. For 16-20 h, supernatants are collected and analyzed for growth-related oncogene (GRO)-α production by ELISA. Results are estimated from graphs of ELISA absorbances as the serum or supernatant titer or antibody concentration yielding a value half-way between the positive and negative controls.

ED50s are defined as the concentration or titer needed to halve the cytokine activity of the test sample. IL-17A/IL-17F Heterodimer Neutralizing Assay•. NCTC 2544 keratinocytes are pre-treated with TNF-α (0.1 ng/ml) in DMEM with 10% inactivated FBS for 3 hours. Serial dilutions of serum samples, culture supernatants or purified antibodies are co-incubated with 5 ng/ml of IL-17A/IL-17F heterodimer in 96-well tissue culture plate at 37° C. Keratinocytes are added after 2 hours of co-incubation at 1×10 4 cells/well. After incubation at 37° C.

For 16-20 h, supernatants are collected and analyzed for growth-related oncogene (GRO)-α production by ELISA. Results are estimated from graphs of ELISA absorbances as the serum or supernatant titer or antibody concentration yielding a value half-way between the positive and negative controls. ED50s are defined as the concentration or titer needed to halve the cytokine activity of the test sample. Example 5 Validation of Subject Antibodies 1.

Protocol for Imiquimod-Induced Psoriasis-Like Skin Inflammation in Selected Animal Models•. Mouse models were used to assay the effect of APECED-derived anti-IL-17, anti-IL-22 autoantibodies and other APECED-derived autoantibodies upon imiquimod-induced psoriasis-like skin inflammation. C57B1/6 mice were dorsal back-shaved under anaesthesia 48-72 hours prior to treatment. 24 hours prior to or post imiquimod application, mice were intraperitoneally injected with anti-IL-17, anti-IL-22 and other APECED-derived autoantibodies, with further doses at 2-day intervals.

Control mice were injected with human Ig control. Treated mice received a daily topical dose of 62.5 mg of commercially available IMQ cream (5%) (Aldara; 3M Pharmaceuticals) for 5 consecutive days. Control mice were treated with Vaseline.

Mice were scored daily using an objective scoring system based on the clinical Psoriasis Area and Severity Index (PASI). Erythema, scaling, and thickening were scored independently on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. Spleens were weighed, and lymph node-derived T cells stimulated overnight for analysis by flow cytometry. The Aire −/− mouse model, in which the Aire protein coding potential is disrupted, which would be predicted to precipitate autoimmunity, was used to investigate the potentially protective effects of anti-IL-17, anti-IFN, and other auto-antibodies derived from APECED patients upon EAE (experimental allergic encephalomyelitis) induction by MOG 35-55 CFA emulsion (immunogenic Myelin oligodendrocyte glycoprotein peptide fragment that induces an autoimmune destruction of MOG-expressing nerve cells; complete Freund's adjuvant).

For example, complete Freunds adjuvant (CFA) emulsion pre-filled syringes containing 1 mg MOG 35-55/mL emulsion and 2 mg killed mycobacterium tuberculosis H37Ra/mL emulsion were used in this respect. Aire −/− and wild-type control female mice were pre-treated 24 hours prior to the onset of EAE induction with intraperitoneal injection of antibodies (e.g., anti-IL-7 and anti-IFN antibodies), with further doses at 2-day intervals.

Control mice were injected with control immunoglobulin. 24 hours post initial antibody administration, minimally stressed (at least 7 days acclimatization, static cages, quiet environment) Aire −/− and wild-type control female mice were injected subcutaneously (upper and lower back) with MOG 35-55 CFA emulsion to instigate EAE. Within 2 hours, treated mice were injected intraperitoneally with pertussis toxin to exarcerbate EAE induction, with a further subcutaneous pertussis toxin dose administrated at 22-26 hours. EAE induction was scored at 7 days post initiation and beyond using the standard Hooke Lab clinical observation scale, scoring the severity of the EAE phenotype on a 0-5 scale. A 0 scored phenotype represents no obvious changes, ranging to a scored 5 phenotype indicating complete hind and front leg paralysis. Protocol for Inducing EAE in Mouse Models•.

A mouse model was used to investigate the effect of anti-IL-17, anti-IL-22 and other APECED-derived autoantibodies upon EAE induction by MOG 35-55 (immunogenic MOG peptide that precipitates an autoimmune destruction of MOG-expressing nerve cells) CFA emulsion. Aire −/− and wild-type control female mice were pre-treated 24 hours prior to the onset of EAE induction with intraperitoneal injection of anti-IL-17, anti-IL-22 antibodies, with further doses at 2-day intervals. Control mice were injected with Ig control. 24 hours post initial antibody administration, minimally stressed (at least 7 days acclimatization, static cages, quiet environment) Aire −/− and wild-type control female mice were injected subcutaneously (upper and lower back) with MOG 35-55 CFA emulsion to instigate EAE. Within 2 hours, treated mice were injected intraperitoneally with pertussis toxin to exarcerbate EAE induction, with a further subcutaneous pertussis toxin dose administrated at 22-26 hours. EAE induction was scored at 7 days post initiation and subsequently using the standard Hooke Lab clinical observation scale, scoring the severity of the EAE phenotype on a 0-5 scale.

A 0 scored phenotype represents no obvious changes, ranging to a scored 5 phenotype indicating complete hind and front leg paralysis. Protocol for Inducing Collagen-Induced Arthritis in Mouse Models•. A mouse model was used to investigate the effect of anti-IL-17, anti-IL-22 and other APECED-derived autoantibodies upon the induction of collagen-induced arthritis (CIA). C57B1/6 mice were pre-treated with intraperitoneally injected APECED-derived autoantibodies 24 hours prior to CIA induction, with further doses at 2-day intervals (Ig used as control). Collagen-induced arthritis was instigated with intradermal injection (2× injections at base of tail) of chicken type II collagen (prepared 1:1 with CFA) extracted from chicken sternum cartilage. A further boost of chicken type II collagen 1:1 IFA was injected intradermally at 14 days post induction. Mice were monitored and scored for arthritis every day starting 2 weeks following primary immunisation, with typical arthritis onset occurring between 3 and 6 weeks post immunisation, and was scored either by clinical monitoring (scoring of hind paw swelling using calipers), measurement of anti-collagen antibodies (ELISA using collagen coated plates), T-cell responses (T-cell proliferation in response to chicken collagen as determined using [3H] thymidine incorporation) or cytokine ELISA.

Protocol for Inducing DSS Colitis in Mouse Models•. Mouse models were used to assay the effect of anti-IL-17, anti-IL-22 and other APECED-derived autoantibodies upon the induction of DSS colitis. C57B1/6 mice were pre-treated with intraperitoneally injected APECED-derived autoantibodies 24 hours prior to DSS colitis induction, with further doses at 2-day intervals. Chronic DSS colitis was induced by cycles of drinking water containing 2% DSS for 5 days, followed by 14 days autoclaved drinking water without DSS; repeated for 3 cycles. Control mice received only autoclaved drinking water. DSS-induced colitis induction and severity was measured by histological evaluation (hematoxylin and eosin staining on paraffin sections), full thickness organ culture (with collection of supernatants at 24 hours for cytokine analysis) or direct tissue sampling for qPCR or immunoblotting. Mice subjected to DSS-induced colitis were also weighed daily to determine the degree of weight loss, as colitis is strongly associated with wasting disease.

In mice topically treated with Imiquimod to induce psoriasis-like skin inflammation (see FIG. 16 for experimental timelines and treatment scheme), APECED derived 30G1 anti-IL-22 [HD-MAB] antibodies significantly reduce Psoriasis Area and Severity Index (PASI) scores relative to control IgG antibodies.

Erythema (redness), scaling, and thickening (hardness) were scored independently on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. As can be seen in FIGS.

17, 18, 20- 22, Imiquimod treatment induces psoriasis-like skin inflammation as measured by PASI scores of Erythema (redness), scaling and skin thickening (hardness) relative to mice treated with control antibodies. Mice treated with anti-IL-22 HDMABs of the present invention, such as the exemplary 30G1 antibody, showed a decrease in both the cumulative and individual scores of measurements when compared to mice treated with control IgG (compare data indicated by solid lines (IgG+IMQ) vs. Data indicated by dotted lines (aIL22+IMQ) in FIGS. 17, 18, 20, and the back skin showing extended lessions and red skin areas (spots) of animals treated with IgG+IMQ in FIGS. 20-21 (B) with a lack or smaller extension of such lessions on backs of animals treated with 30G1 anti-IL-22 and IMQ in FIGS.

20-21 (C) and IgG+IMQ versus aIL22+IMQ entries in Tables 18 and 19 below). Furthermore, flow cytometric analysis of lymph node derived T cells showed a reduced state of effector activation in anti-IL-22 HDMAB-treated mice compared with mice treated with control IgG. APECED derived 30G1 anti-IL-22 antibody significantly reduces Psoriasis Area and Severity Index (PASI) scores relative to control IgG antibodies if administrated either pre or post IMQ-treatment. Mice treated with anti-IL-22 HDMABs of the present invention, such as the exemplary 30G1 antibody, showed a decrease of skin thickness as an indicator of skin thickness scores relative to control IgG antibodies when compared to mice treated with control IgG in both situations of prophylactic and therapeutic treatment; see skin thickness measurements shown in FIG.

38A (pre) for the prophylactic and FIG. 38B for the therapeutic treatment, 24 hours post IMQ induction. The therapeutic effect was further validated in a second experiment with an increased number of animals (see FIG.

35 for experimental timelines and treatment scheme). Mice treated with anti-IL-22 HDMABs of the present invention, such as the exemplary 30G1 antibody, showed a decrease in both the cumulative and individual scores of measurements when compared to mice treated with control IgG; see FIGS. The therapeutic effect of the mouse cross-reactive, exemplary anti-IL-22 antibody 30G1 was also tested in concern of its dose-dependency in IMQ-induced psoriasis.

Three different doses −200 μg, 20 μg and 20 μg—were administered post IMQ-induction and skin erythema and hardness of the plaques were evaluated as described hereinbefore. As can be seen in FIG.

38, treatment with exemplary anti-IL-22 antibody 30G1 shows indications of dose dependence in individual and cumulative scores, proving less effective when titrated down to 1/100 of the initial dose. Thereafter, additional screens are performed, such as optical coherence tomography (OCT) imaging for epidermal thickness measurements (Morsy et al., Arch. 302 (2010), 105-111; Phillips et al., J. 16 (2011), 040503.), RNA isolation from the animal skin and gene expression analysis in the samples for, e.g., identification of the disease-specific patterns of tissue inflammatory responses. Furthermore, FACS analysis is performed on skin single cell suspensions, whole blood or sera isolated from the animals, for sorting and/or phenotypic characterization of different cell types such as psoriatic dermal DC (dendritic cells) or peripheral blood mononuclear cell populations.

Example 7 Data Analysis of Seroactivities Identified in APECED/APS1 Patients•. TABLE 1 Summary of 372 seroreactivities from ProtoArray analysis Number of antigens proving Pathophysiological implication seropositive Autoimmune & inflammatory conditions 89 Endocrinology & metabolic disorders 14 Vascular function 34 Neurodegenerative diseases 39 Oncology, cell growth & differentiation 54 Skin, bone & mucosal medical conditions 18 Renal function, nephritis 11 Cell adhesion 24 Synapse-located proteins 26 Lipid layers 27 Protein degradation 10 Cellular counterparts of viral proteins 26 •. TABLE 7 Skin, bone and mucosal medical conditions # of Pts Description 5 sciellin (SCEL) 5 Suprabasin 5 Tyrosine-protein kinase transmembrane receptor ROR2 4 odontogenic, ameloblast asssociated (ODAM) 3 Vitamin D receptor 2 ectodysplasin A2 receptor (EDA2R) 2 Keratin 15 2 Keratinocyte growth factor 1 Defensin-5 1 Parathyroid related protein 1 Bone morphogenetic protein 3b 1 bone morphogenetic protein 7 (osteogenic protein 1) (BMP7) 1 bone morphogenetic protein 8a, mRNA (cDNA clone IMAGE: 7939588), complete cds. 1 CLCF1 1 fibroblast growth factor 10 (FGF10) 1 osteoclast stimulating factor 1 (OSTF1) 1 osteoglycin (OGN), transcript variant 1 1 Tff2 •. TABLE 24 Localization of exemplary coiled-coil protein encoding gene sequences on human chromosomes. Entrez Ensem Gene Name UniGene Gene Description bl Chr.

Enhancement of attentional processing is attained by administration of an endorphinase inhibitor or enkephalinase inhibitor and optionally, a dopamine precursor, or a serotonin precursor, a GABA precursor, or an endorphin or enkephalinase releaser, or certain herbal compounds including Rhodiola rosea extract (Pharmaline) and/or Huperzine. These components promote restoration of normal neurotransmitter function and the components combined enhance the release of dopamine at the nucleus accumbens and are non-addictive. Use of the dopamine precursors L-phenylalanine, or L-Tyrosine, the enkephalinase inhibitor D-phenylalanine, and/or the serotonin precursor -hydroxytryptophan and a natural acetylcholenesterase inhibitor and chromium salts (i.e. Picolinate, nicotinate, etc.) is especially preferred, but not limited to assist in relieving symptoms associated with brain phenylalanine deficiency. A composition for the treatment of RDS behaviors in a subject consisting essentially of a) an opiate destruction-inhibiting amount of at least one substance which inhibits the enzymatic destruction of a neuropeptidyl opiate, said substance being selected from the group consisting of amino acids, peptides, and structural analogues or derivatives thereof; b) a neurotransmitter synthesis-promoting amount of at least one neurotransmitter precursor selected from the group consisting of dopamine precursors. APPLICATION FOR UNITED STATES LETTERS PATENT for ALLELIC POLYGENE DIAGNOSIS OF REWARD DEFICIENCY SYNDROME AND TREATMENT by Kenneth Blum, David E. Comings and John L.

Ivy BACKGROUND OF THE INVENTION The government owns rights in the present invention pursuant to grant number 1-R01-DA08417 from National Institutes of Drug Abuse and Tobacco Related Research Disease Program grant 4RT-01 10. Field of the Invention This invention, in part, relates to the coupling of certain anti-craving compositions and specific genotyping of a number of genes all involved in neurotransmitter function of reward behavior. An aspect of this invention is the understanding of the involvement of how certain established neurotransmitters work in concert to activate neuropathways in the meso-limbic system of the brain leading to feelings of well being, and the development of compositions that affect these neuropathways. This invention, in part, relates to the utilization of precursor amino acids and certain herbal compounds to enhance attentional processing and memory as well increase focus in healthy individuals, as well as to enhance weight loss and control overeating.

Disclosed are various diagnostic methods of neurological disorders and behaviors utilizing genetic polymorphisms of neurotransmitter genes, and therapeutic methods of treatment of patients so identified using the compositions ofthe invention. Also disclosed are diagnostic methods for polygenic traits. Description of Related Art During the past several decades, research on the biological basis of chemical dependency has been able to establish some of the brain regions and neurotransmitters involved in reward.

In particular, it appears that the dependence on alcohol, opiates and cocaine relies on a common set of biochemical mechanisms (Cloninger, 1983; Blum, 1978; Blum, 1989). A neuronal circuit deep in the brain involving the limbic system and two regions called the nucleus accumbens and the globus pallidus appears to be critical in the expression of reward of people taking drugs (Wise and Bozarth, 1984).

It has been demonstrated that the chronic use of cocaine, morphine and alcohol results in several biochemical adaptations in the limbic dopamine system (Ortiz et a/., 1996). The mesolimbic dopamine system connects structures high in the brain, especially the orbiofrontal cortex (in the prefrontal area behind the forehead) with the amygdala in the brain's center, and with the nucleus accumbens, which has been proven in animal research to be a major site of activity in addiction. The various brain pathways involved in multiple addictions converge on certain dopaminergic receptors (DI, D2, D3, D4, D5) where the D2 site seems to be most prominent. Although each substance of abuse appears to act on different parts of the circuit, the end result is the same: dopamine is released in the nucleus accumbens and the hippocampus (Koob and Bloom, 1988). Dopamine appears to be the primary neurotransmitter of reward at these reinforcement sites.

Abnormalities in dopamine metabolism have been implicated in several behaviors, i.e. Sexual disorders (Gessa and Tagiamonte, 1975), mania (Goodwin and Jamison, 1990), schizoid behaviors (Carlsson, 1978; Snyder, 1976), ADHD (Shaywitz et al., 1976), conduct disorder or aggression (Rogeness et al., 1986; Valzelli, 1981; King, 1986), alcohol abuse (Blum et al., 1990) and stuttering. In addition, haloperidol, a DRD2 receptor antagonist, has been reported to be effective in the treatment of some stutters (Murray et al., 1977; Prins et al., 1980). While serotonergic mechanisms have been most often implicated for obsessive- compulsive behaviors, abnormalities in dopamine have also been considered (Austin et al., 1991; Delgado et al., 1990). Abnormal circuits involving the thalamus, basal ganglion and frontal lobes have been implicated in obsessive-compulsive disorder (Baxter et al., 1992; Rauch et al., 1994; Modell et al., 1989) and dopamine is a major neurotransmitter especially in the striatum and frontal lobes. Defects in central noradrenergic mechanisms have been frequently implicated in the etiology of attention-deficit hyperactivity disorder.

A significant increase in plasma noradreneline (NA) in ADHD children with reading and other cognitive disabilities compared to ADHD children without cognitive disabilities has been demonstrated (Halperin et al., YEAR). They proposed that the ADHD + cognitive disabilities was associated with NA dysregulation affecting the parietal/temporal lobe attention centers. Since these brain areas are in proximity to auditory and linguistic processing regions, this could account for the comorbid cognitive disabilities. From a clinical perspective, the significant improvement in symptoms that often occurs following treatment with clonidine (Hunt et al, 1985; Comings et al, 1990) implies a role of NA in at least some ADHD. Clonidine is a presynaptic a 2-noradrenergic receptor agonist that results in the inhibition of release of noradrenaline into the synapse (Starke et al.

It has been proposed that NA and the locus coeruleus (LC) play a role in arousal and vigilance, critical aspects of attention (Aston-Jones et al, 1984). It has been proposed that stress tolerance and good performance on tasks were related to low basal or tonic levels of catecholamines and to higher acute releases during mental stress (Dienstbier, 1989). The opposite may occur in ADHD, with an increased baseline tonic stimulation of NA and a decreased release of catecholamines during stress (Pliszka et al, 1996). To test the hypothesis that NA defects are involved in ADHD, a number of studies of CSF, plasma and urinary excretion of the NA metabolite (3-methoxy-4-hydroxyphenylglycol (MHPG) have been performed. Some show that ADHD patients have lower rates of MHPG excretion than controls (Oades, 1987; Shekim et al, 1983; Shekim, Dekirmenjian, and Chapel, 1997; Yu-cum and Yu-feng, 1984) while others show no change (Rapoport et al, 1978; Zametkin et al, 1985) or an increase in NA turnover (Khan and Dekirmenjian, 1981).

Epinephrine levels have been reported to be significantly lower (Hanna et al, 1996; Klinteberg and Magnusson, 1989; Pliszka et al, 1994), or to show a blunted response to glucose ingestion (Girardi et al, 1995) in ADHD subjects compared to controls. Norepinephrine is converted to epinephrine (adrenaline) by phenylethanolamine N-methyl-transferase coded by the PNMT gene. A model of ADHD based on failure of epinephrine to tonically inhibit NA neurons in the locus coeruleus. D-amphetamine and desipramine, both of which are commonly used in the treatment of ADHD, lead to a significant decrease in the excretion of MHPG has been proposed (Mefford and Potter, 1989; Shekim et al, 1979). However, methylphenidate (Ritalin) the most commonly prescribed medication for the treatment of ADHD does not result in a decrease in MHPG excretion (Zametkin et al, 1985) and other medications that reduce MHPG excretion, such as fenfluramine (Donnelly et al, 1989), are not effective in the treatment of ADHD. These observations are consistent with the presence of several types of ADHD and an involvement with multiple neurotransmitters and genes. It has been proposed that NA and adrenergic α2-receptors played a role in some forms of ADHD through a dysregulation at the LC of the posterior cortical attention system (Posner and Peterson, 1990; Pliszka 1996) of the parietal/temporal lobes, and that a second form of ADHD was due to dopaminergic defects that primarily affected the prefrontal lobe attentional system which was associated with impulsivity and disorders of executive dysfunction.

Several dopaminergic genes, such as the dopamine D 2 receptor (DRD2) (Comings et al, 1991 ), dopamine D 4 receptor (DRD4) (Lahoste et al, 1996), and dopamine transporter (DAT]) (Cook et al, 1995; Comings et al. 1996; Gill et al, 1997; Waldmaqn et al, 1996) genes have been found to be associated with ADHD. It has been reported that boys with ADHD and reading disabilities had significantly higher plasma MHPG levels than boys with ADHD only (Haperin et al, 1993; Halperin et al, 1997). In the latter study, they also demonstrated a significant negative correlation between plasma MHPG levels and the WISC-R verbal IQ, and the reading, spelling and arithmetic problems assessed by the WRAT-R (Wide-Range Achievement Test-Revised). This distinction was consistent with prior studies of others suggesting that ADHD with cognitive disabilities was a distinct subtype of ADHD (August and Garfinkel, 1989; McGee et al, 1989; Pennington et al, 1993).

It has also been suggested that the type of attention deficit associated with parietal lobe defects tends to be a selective attention deficit (Posner and Peterson, 1990; Dyk an et al, 1979; Richards et l, 1990). It has been proposed that ADHD + cognitive disorders was due to a dysregulation of NA metabolism of the LC involving adrenergic α2 receptors, and primarily affected the posterior attention system ofthe parietal cortex (Halperin et al, YEAR) Since these brain areas are in proximity to auditory and linguistic processing regions, this could account for the comorbid cognitive disabilities. It would be a mistake to assume that these are pure forms since ADHD is a polygenic disorder (Comings et al, 1996), and most individuals are likely to have inherited genes for both types. Studies in primates show that NA and defects in adrenergic α2 receptors also play a role in prefrontal lobe cognitive defects (Arnsten, 1997). Various studies have indicated the involvement of the dopamine receptor in addictive behaviors. Cocaine patients show a drop in those neuronal activity levels that is consistent with a lessened ability to receive dopamine (Volkow et al, 1993). Neurons with D2 dopamine receptors were shown to become 25% smaller, and lost much of their ability to receive small amounts of dopamine from nearby neurons in morphine addicted rats (Nestler et al, 1996).

Decreases in D2 receptors observed in opiate-dependent subjects have been suggested to indicate that the subjects had low D2 receptors prior to when they began abusing drugs, and that this reduction may have made them more vulnerable to drug self-administration (Wang et al, 1997). Although the system of neurotransmitters involved in the biology of reward is complex, at least three other neurotransmitters are known to be involved at several sites in the brain: serotonin in the hypothalamus, the enkephalins (opioid peptides) in the ventral tegmental area and the nucleus accumbens, and the inhibitory neurotransmitter GABA in the Substantia nigra, ventral tegmental area and the nucleus accumbens (Stein and Belluzzi, 1986; Blum and Kozlowski, 1990). Interestingly, the glucose receptor is an important link between the serotonergic system and the opioid peptides in the hypothalamus. An alternative reward pathway involves the release of norepinephrine in the hippocampus from neuronal fibers that originate in the locus coeruleus. There is evidence that the opoidergic and dopaminergic systems are anatomically and functionally interconnected, suggesting a role for the endogenous opioidergic system in mediating the effects of ethanol and other drugs on brain dopaminergic pathways associated with reward.

Dopamine antagonists and lesions of the dopaminergic pathways in the brain affect pre-proenkephalin A activity (Morris et al, 1988; Normand et al, 1988). Behavioral, pharmacological and neurochemical studies implicate the opioidergic and dopaminergic systems in the reinforcing effects of ethanol and other drugs of abuse (Blum et al, 1976a, b; Blum et al, 1982a; Blum et al, 1977; Blum et al, 1973; Koob and Bloom, 1988; Weiss et al, 1993). Animal studies show that opiate receptor agonists increase preference for ethanol, whereas antagonists of these receptors reduce ethanol consumption (Blum et al, 1975; Le et al, 1993). Further, studies on animals and human alcoholics suggest the effectiveness of the opiate receptor antagonist in reducing the positive reinforcing effects of alcohol consumption (O'Malley, 1992; Swift et al, 1994; Blum et al, 1975; Volpicelli et al, 1992). Moreover, ethanol-induced increase of brain dopamine levels in animals is blocked by both opiate receptor antagonists naloxone and naltrexone (Widdowson and Holman, 1992; Benjamin et al, 1993).

A recent review by Gianoukalis and de Waele (1994) support the role of endogenous opioids and drugs of abuse {i.e. In a normal person, these neurotransmitters work together in a cascade of excitation or inhibition between complex stimuli and complex responses, leading to a feeling of well being, the ultimate reward.

In the cascade theory of reward, a disruption of these intercellular interactions results in negative emotions. Genetic anomalies, including certain polymorphisms, prolonged stress or longer term abuse of psychoactive drugs (including glucose) can lead to a self-sustaining pattern of abnormal cravings in both animals and human beings (Blum, 1991). Pharmacological actions (bromocryptine, bupropion and N-propylnor- apomorphine) are partly determined by the individual's dopamine D2 genotype.

Al carriers of the DRD2 gene are pharmacologically more responsive to D2 agonists. One study has already shown that the direct microinjection of the D2 agonist N- propylnor-apomorphine into the rat nucleus accumbens significantly suppresses the animal's symptoms after withdrawal from opiates, while dopamine per se suppresses alcohol withdrawal symptoms (Harris and Aston-Jones, 1994; Blum et al, 1976b).

In this regard, there is evidence for dopamine/endogenous opioid peptide interactions in the nucleus accumbens and elsewhere in the brain, and it may be that overstimulation of the opioid peptide system by exogenous opiates leads to decreases in dopamine function (Pothos et al, 1991). When compared to normal non-alcohol preferring rats, alcohol preferring rats have fewer serotonin neurons in the hypothalamus, higher levels of enkephalins in the hypothalamus (because less is released), more GABA neurons in the nucleus accumbens and a lower density of D2 receptors in certain areas of the limbic system (McBride et al, 1995; Smith et al, 1997; and McBride et al, 1997). Clinical trials have demonstrated that when amino-acid precursors of certain neurotransmitters (serotonin and dopamine) and D-phenylalanine, a substance that promotes enkephalin activity by inhibiting enzymatic cleavage (U.S. 4,761,429 and 5,189,064) are administered to probands with either SUD or carbohydrate bingeing, was found to reduce craving, reduce incidence of stress, reduce relapse rates, and also increase the likelihood of recovery. A number of laboratories have pursued the association between certain genes and various behavioral disorders, including linkage of the dopamine D2 receptor alleles with a number of impulsive-compulsive-addictive behaviors. Little is known about the resultant expression of polymorphisms linked to either the DAT, 10/10 allele and the DβH B, allele except studies showing increased dopamine transporter sites in Tourette's Disorder patients by SPECT scanning techniques (Malison et al, 1995, Tiihonen et al, 1995). ADHD, Tourette syndrome, conduct disorder, ODD, dyslexia, learning disorders, stuttering, drug dependence and alcoholism all show a male predominance.

The molecular genetic studies of the DRD2, Z)βH, DAT (Comings et al, 1996a) and clinical genetic studies (Comings, 1994b; 1994c; 1995b; Bierderman et al, 1991; Comings and Comings, 1987), indicate these are etiologically related spectrum disorders. Defects in neurotransmitters has been advocated as involved in alcoholism (Blum, 1991). Studies of genes involved in neurological pathways are described below.

Androgent Receptor gene Specific mutations of the AR gene have been reported to cause a wide range of types of androgen insensitivity syndromes (Gottlieb et al, 1977). In addition, two sets of polymorphic tricnucleotide repeat sequences, CAG (Edwards et al, 1992) and GGC (Sleddens et al, 1993; Sleddens et al, 1992), resulting in polyamino acid tracts in the protein, are present in the first exon of the AR gene. When highly expanded, from 43 to 65 times, the CAG trinucleotide repeat has been shown to cause X-linked spinal muscular atrophy (La Spada et al, 1991). The repeat length in the normal population is 11 to 31 times (Edwards et al, 1992).

The non-highly expanded alleles of micro- and minisatellites present in the normal population, might play a direct role in the regulation of genes. This was based on the observation that most short tandem repeats are associated with the formation of Z-DNA (Schroth et al, 1992), and Z-DNA has repeatedly been implicated in various aspects of gene regulation (Rich et al, 1984; Hamada et al, 1982; Wolff et al, 1996). Since the amount of Z-DNA formed is highly sensitive to the length of the repeats (Schroth et al, 1992), it was suggested that the size of the repeat alleles could themselves be related to phenotypic effects (Comings, 1997). Some (Olweus et al, 1988; Mattsson et al, 1980; Schiavi et al, 1984; Kreuz and Rose, 1972) but not all (Bradford and McClean, 1984; Schaal et al, 1998) studies suggest a correlation between aggressive behavior and plasma testosterone levels. Aggressive conduct disorder is often a comorbid condition in subjects with TS and ADHD (Comings, 1995; Stewart et al, 1981; Biederman and Sprich, 1991) and there is a high degree of comorbidity between TS and ADHD (Comings and Comings, 1984, 1990; Knell and Comings, 1993). Dopamine Dj Receptor Gene (DRDl) Sequencing of the DRDl gene in controls and in patients with schizophrenia, manic-depressive disorder and alcoholism, has failed to identify exon mutations that produce an effect on the phenotype and linkage studies in schizophrenia and TD (Jensen, 1993k; Gelertner et al, 1993k). The D, receptors in frontal cortex may play a role in memory (Comings et al, 1997k; Williams et al, 1995k).

The opposing effect of the Dj and D 2 receptor agonists on cocaine seeking behavior in rats have been reported (Self et al, 1996). TD probands, smokers, and pathological gamblers, were consistent with negative heterosis, in that the most consistent difference was a relative decrease in the frequency of 12 heterozygotes and an increase in 11 and 22 homozygotes of the Dde 1 polymorphism (Comings et al.

By contrast, positive heterosis was present at the DRD2 gene, with quantitative scores being highest for 12 heterozygotes and lowest for 11 and 22 homozygotes. While the results for ADHD at the DRDl locus alone was not significant, there was a significant additive effect of examining the presence of negative heterosis at the DRDl gene and/or positive heterosis at the DRD2 gene (Comings et al, 1997k). Dopamine D2 Receptor Gene (DRD2) Previous studies have shown a significantly increased prevalence of the D2A1 allele in individuals with ADHD, TS,CD and SUD (Comings et al, 1991). Since each of these disorders is characterized by a poor response to stress and many criteria for the diagnosis of PTSD have many symptoms in common with ADHD and The National Vietnam Veterans Readjustment Study (Kulka et al, 1990) reported a significant correlation between PTSD and a history of childhood symptoms consistent with ADHD and CD. Subjects on an addiction treatment unit who had been exposed to severe combat conditions in Vietnam were screened for posttraumatic disorder (PTSD), and showed an correlation of individuals with PTSD as carrying the D2A1 allele (Comings, et al, 1996k). An association between ADHD and the Taq Al allele of the DRD2 gene has been detected (Comings et al, 1991k).

The stimulant methlphenidate increased regional blood flow while in others it decreased blood flow. The changes in frontal, temporal and cerebellar metabolism were related to the density of D2 receptors - the higher the density the greater the increases in blood flow. Methylphenidate decreased the relative metabolic activity in the basal ganglia. These results are consistent with the idea that genetic defects in dopamine metabolism, resulting in a dopaminergic state in the limbic system and frontal lobes result in a compensatory increase in dopaminergic activity in the basal ganglia, and that methylphenidate reverses these through a combination of enhancing brain dopamine activity in dopaminergic through its inhibition of the dopamine transporter (Volkow et al, 1996k), with a secondary decrease in dopaminergic activity in the basal ganglia and decrease in basal ganglia blood flow. These studies are also consistent with the results of others (Castellanos et al, 1996k). Showing a positive correlation between the response to methylphenidate and CSF levels of HVA, a metabolite of dopamine whose levels in the CSF are related to D2 receptor density (Jonsson et al, 1996k).

Methylphenedate consistently increased cerebellar metabolism, despite the paucity of D2 receptors in this structure (Volkow et al, 1996k; Hall et al, 1994k). This is consistent with the increasing evidence that the cerebellum play an important role in attention, learning and memory (Leiner et al, 1989k). An association between the Al genotype and regional blood flow has been reported. The Taql D2 Al carriers showed a significantly lower relative glucose metabolism in the putamen, nucleus accumbens, frontal and temporal gyri and medial prefrontal, occipito-temporal and orbital cortices than those with the A22 genotype (Nobel et al, 1997). The Taql D2 Al carriers had a significantly decreased dopamine D2 receptor B max in the basal ganglia (Noble et al, 1991k). Enkephalin increases blood flow in similar regions as methylphenidate and may therefore involve a dopaminergic mechanism (Blum et al, 1985k).

A significant decrease in dopamine D2 receptor density was measured in individuals with detachment, social isolation, and lack of intimate friendships (Farde et al, 1997k). Though the DRD2 gene polymorphisms have been associated with a number of psychological disorders, but no association was found between certain psychopathy in incarcerated drug users (Smith et al, 1993). The reports of an association between alcoholism and the DRD2 allele have been quite variable, an association between the D2AI allele and polysubstance/drug abuse has been found (Smith, et al, 1992, Noble, et al, 1993, O'Hara, et al, 1993, Comings, et al, 1994, U.S. 5,210,016, U.S. After the first association of the DRD2 Al and severe alcoholism (Blum et al, 1990), several groups were unable to replicate the observation. Two possible reasons were suggested: first, inadequate screening of controls for alcohol, drug and tobacco abuse; and second, sampling errors in terms of characterization of alcoholics for chronicity and severity of disease (Blum et al, 1997; Bolos et al, 1990; Gelertner et al, 1991; Schwab et al, 1991; Turner et al, 1992; Cook et al, 1992; Goldman et al, 1992; Goldman et al, 1993; Suarez et al, 1994). Dopamine plays a role as a modulator of many different behaviors (LeMoal and Simon, 1991), and numerous studies have reported a significant association between alleles of the DRD2 gene and cocaine addiction, polysubstance abuse, smoking, attention deficit hyperactivity disorder (ADHD), Tourette syndrome, visual- perceptual disorders, conduct disorder, posttraumatic stress disorder, pathological gambling, and compulsive eating (Blum et al, 1995; Blum et al, 1996).

Despite these associations, sequencing studies have failed to find any mutations in the exons that could explain these findings. These findings could be explained if the D 2A1 allele was in linkage disequilibrium with an unknown non-exon mutation that played a role in the regulation of DRD2 function (Comings et al, 1991).

Additionally the severity of alcoholism and the type of controls used have been reported as important determinants of DRD2A1 allele association with alcoholism (Noble et al, 1994; Geijer et a/., 1994; Parsian et al, 1991; Blum et al, 1992; Blum et al, 1990; Lawford et al, 1997). Sib pair linkage analyses conducted in families multiply affected by alcoholism, using both the Taql 'A' RFLP and a microsatellite repeat polymorphism at the DRD2 locus, indicated a significant correlation with this locus and the liability to develop heavy drinking. A corresponding mutation in the DRD2 gene has not been found, the effect may arise from a closely linked locus-outside the DRD2 gene itself (Cook et al, 1996). A single point mutations in exon 8 of the DRD2 gene in alcohol- dependent patients has been demonstrated (Finch et al, 1995), while others report no structural mutations in the coding regions ofthe DRD2 gene (Gejman et al, 1993).

The DRD 2 gene A, allele has been found to associate with a number of behaviors including severe alcoholism, polysubstance dependence, crack/cocaine addiction, tobacco smoking, pathological gambling, lack of a major depressive episode, and carbohydrate bingeing or generalized to DSM- IV substance use disorder (Blum et al, 1996e; Blum et al, 1995b; Comings et al, 1996c). The MCMI-II assessed schizoid/avoidant cluster compared to other Axis II diagnostic clusters (antisocial, narcissistic, paranoid) significantly correlated with alcohol abuse scales (Corbisiero et al, 1991).

Clusters of patients with MCMI-II elevations that indicated schizoid and avoidant qualities tended to stay in treatment fewer days and relapsed earlier (Fals-Stewart, 1992). High scores of schizoid/avoidant cluster correlated with inpatient male alcoholics (Matano et al, 1994) and cocaine dependent patients (Kranzler and Satel, 1994). Schizoid/avoidant behaviors including low levels of sensation were found to consume more alcohol and to have higher MAST scores than patients with high levels of sensation (Ohannessian and Hesselbrock, 1995); and avoidant personality is significantly correlated in subjects with severe binge eating disorder (Yanovski et al, 1993). Molecular heterosis at the dopamine receptor genes was indicated in healthy individuals and alcoholics. Cerebrospinal fluid levels of monoamine metabolites consisting of HVA for dopamine, 5 -HI A A for serotonin, and MHPG for norepinephrine levels were compared in healthy volunteers to the DRD2 Taql A1A2 and B1B2 genotypes. The results indicate a statistically significant difference in the means ofthe 1,1+1,2 homozygotes vs. The 1,2, but not when analyzing the 1,1+1,2 vs.

The 2,2 genotypes and for 1 vs. The 2 alleles.

The Taql B1B2 polymorphism gave virtually identical results (Jonsson et al, 1996). In contrast, CSF HVA levels and the DRD2 Taql Al I 2 polymorphism were examined in Finnish and American alcoholics, and no association was found when examining the 1 vs. 2 alleles, and not the 1,1+1,2 vs. The 2,2 genotypes (Goldman et al, 1992). Heterosis at the DRD2 gene was indicated by comparison of the CSF levels of HVA, to the DRD2 genotype using Taql polymorphism (Jonsson et al, 1996k).

In a profile for the inattention score of TD subjects, the 12 heterozygotes showed the highest inattention score subjects who were 12 heterozygotes had the lowest levels of CSF HVA (Jonsson et al, 1996). The highest levels of HVA were seen in the 11 homozygotes, with the 22 homozygotes being intermediate. Some studies, but not all, showed a significantly lower level of CSF HVA in children (Shaywitz et al, 1979k) with ADHD and TD (Cohen et al, 1979k). A significant correlation was found between electrophysiological abnormalities and the DRD2 Al allele (Blum et al, 1994k). These abnormalities are seen in ADHD subjects as well as children of alcoholics (Comings et al, 1991k; Noble et al, 1994k). A positive association of the Taq Al of the DRD2 gene to Attention Deficit Disorder (ADHD) and Tourette's was reported (Comings et al, 1991; Comings et al, 1996a), while others found no association with ADHD probands (Sunohara et al, 1996). ADHD probands showed a significant association with the 48bp variant of the D4 gene, but not the DRD2, DRD3 or the serotonin transporter genes.

The 7-fold repeat allele of the DRD4 occurred significantly more frequently in that children with ADHD. There is evidence for an association of the 7 repeat allele of the D4 receptor gene and novelty seeking (characterized as impulsive, exploratory, fickle, excitable, quick tempered and extravagant) (Epstein et al, 1996; Benjamin et al, 1996). The DRD2 Al allele in cocaine dependent probands was associated with the opposite: low novelty seeking, characterized by reflective, rigid, stoic, slow-tempered, avoidant, as well as having enhanced withdrawal depression (Compton et al, 1996). Molecular genetic studies have found an association of the D 2 dopamine receptor (DRD2) Al allele with alcoholism and drug abuse (Blum et al, 1990). Reduced central dopaminergic function has been suggested in subjects who carry the Al allele (Al +) compared with those who do not (AT) (Nobel et al 1997). The genes responsible for alcoholism are unknown, although the many studies to date indicate a significant role for the DRD2 gene in more severe cases (Noble, 1993; Blum et al, 1995).

The DRD2 gene has been associated with the compulsive behavior {Comings and Comings, 1987b) and addictive, impulsive behaviors, including compulsive eating, gambling and smoking. (Self et al, 1996; Ogilvie et al, 1996; Blum et al, 1995b; Blum et al, 1996e).

These behaviors have previously been reported to be associated with the DRD2 gene (Comings et al, 1993a; Noble et al, 1994d; Blum et al, 1996a; Comings et al. 1996c; Noble et al, 1994c; Noble, 1993; Comings et al, 1996e) in subjects distinct from the TS group. Dopamine D2 receptor availability was significantly lower in alcoholics than in nonalcoholics, and was not correlated with days since last alcohol use (Volkow et al, 1997). The ratio DRD2 receptor to transporter availability was significantly higher in nonalcoholics than in alcoholics. Alcoholics showed significant reductions in D2 receptors (postsynaptic marker) but not in DA transporter availability tpresynaptic marker) when compared with nonalcoholics. Because D2 receptors in striatum are mainly localized in GABA cells, these results provide evidence of GABAergic involvement in the dopaminergic abnormalities seen in alcoholics.

Dopamine D 3 Receptor Gene (DRD3) Knockout mice missing the DRD3 gene are considerably more active than their litter mates with normal DRD3 genes (Williams et al, 1995k). Negative heterosis has been reported for schizophrenia at the DRD3 locus (Crocq et al, 1992k) have observed a significant decrease in DRD3 Mscl 12 heterozygosity in TD (Comings et al, 1993j) and pathological gambling (Comings et al, 1996). Dopamine D 4 Receptor Gene (DRD4) In the Dopamine D 4 receptor gene (DRD4), a 48 bp and 16 amino-acid repeat polymorphism within the DNA coding for the third cytoplasmic loop responsible for binding to guanine -nucleotide proteins (Van Tol et al, 1992k; Lichter et al, 1993k) has been reported. This DNA region is repeated 2 to 1 1 times, with the most common alleles being the 2, 4, and 7 repeat.

The 7 allele demonstrates a blunted response to dopamine in regards to intracellular adenyl cyclase inhibition (Asghari et al, 1995k). Two independent studies of normal subjects have shown an association between the presence of the 7 allele and novelty seeking, a trait associated with impulsivity (Benjamin et al, 1996k; Ebstein et al, 1996k). One study failed to find such an association (Malhotra et al, 1996).

A study ADHD children compared to controls, reported that more ADHD children carried at least one 7 allele compared to controls (LaHoste et al, 1996k). An association between the 7 allele of the DRD4 gene in TD has been reported (Grice et al, 1996k). Other work in this area is not equivocal (Spielman et al, 1993k).

Dopamine Transporter Gene (DAT1) The DAT1 gene marker frequencies at the vesicular transporter locus showed substantial heterogeneity in different Caucasian-Americans originating from different European countries, but no association with substance abuse was evident (Uhl et al, 1993; Persico et al, 1993). Distributions of the DAT1 VNTR alleles do not distinguish any substance user or control sample for psychostimulant abusers (Persico et al, 1996), however an association was observed with Japanese alcoholics (Muramatsu and Higuchi, 1995). The DAT1 gene has also been implicated as having a role in compulsive and addictive disorders. Since one of the modes of action of cocaine is to inhibit dopamine transporter function (Ritz, et al, 1992; Ritz, et al, 1990), it has been implicated in the biology of drug addiction, as well as other disorders including Parkinson's disease (Uhl, 1990) and Tourette's syndrome (Singer, et al, 1991). Increased dopamine transporter sites in Tourette's syndrome was demonstrated using SPECT scanning techniques (Malison et al, 1995), and dopamine transporter receptor sites were significantly increased in violent alcoholics compared to nonviolent alcoholics (Tiihonen et al, 1995).

Studying postmortem samples of TS subjects, reported an increased number of dopamine uptake sites in the striatum suggesting either a greater number of DAT1 molecules or an increased number of dopamine nerve terminals (Singer et al, 1991). It is the site of action of methylphenidate (Volkow et al, 1995k) and dexedrine, compounds widely used in the treatment of ADHD. These stimulants inhibit the transport process resulting in an increase in synaptic dopamine. A significant increase in the level of dopamine transporter protein in the striatum of TD subjects vs. Controls has been reported (Maison et al, 1995k). Studies of the DATl knockout mice, which are very hyperactive in confined spaces, showed a five fold increase in brain dopamine levels, down regulation of D 2 receptors, uncoupling of D 2 receptor function, and a 57% decrease in body size (Giros et al, 1996k).

It is not known whether the less common DATl repeat alleles are associated with an increase or decrease in the number of DATl molecules. An association between the 10 allele of the DATl gene was reported in cases of ADHD/ ADD (Cook et al, 1995k), and behavioral variables in Tourette Disorder (TD) (Comings et al, 1996). The significant increase in subjects with autism is consistent with studies suggesting that TS and autism are genetically related and involve similar sets of genes (Burd et al, 1987; Comings and Comings, 1991b; Sverd, 1991). Significant increased prevalence of the nine-repeat allele VNTR polymorphism in the 3' untranslated region of the DATl gene was seen in 93 alcoholics displaying withdrawal seizures or delirium, compared with 93 ethnically matched non-alcoholic controls (Sander et al, 1997). The 5' UTR 40 bp repeat polymorphism in the DATl was examined in subjects with drug abuse and found no significant difference in the frequency of any ofthe 3' UTR repeat alleles compared to normal controls (Persico et al, 1993).

The 9/10 genotype has been found to associate with 'pathological violent' adolescents; and the 9/9 genotype is associated in alcohol dependence with withdrawal seizures or delirium. An association between the 9 allele of the 40 bp repeat of the DATl gene with cocaine induced paranoia has been reported (Gelernter et al, 1994a).

Dopamine-β-Hydroxylase DβH is one of the major enzymes for dopamine metabolism and catalyzes the conversion of dopamine to norepinephrine (NE). In study animals, the inhibition of DβH activity results in a decrease in norepinephrine levels which releases the inhibition of tyrosine hydroxylase resulting in the excessive production of dopamine. The later is associated with hyperactivity, aggression, self- stimulation, and stereotypic movements (Randrup and Scheel-Kruger, 1966; Shekin et al, 1983k). Studies of blood enzyme levels of DβH have implicated a role of this enzyme in sensation seeking (Kuperman et al, 1988k; Comings et al, 1996), ADHD and conduct disorder (Rogeness et al, 1982k; Rogeness et al, 1989k). Disturbances in dopamine-Beta-hydroxylase (DβH) activity have previously been associated with childhood CD and alcoholism (Pliszka et al, 1991). It has been proposed that externalizing disorders such as CD were associated with a decrease in noradrenergic function and an increase in dopaminergic function, a pair of conditions that would be uniquely brought about by a DβH deficiency (Quay 1986). Others reported an increased frequency of the diagnosis of CD in emotionally disturbed boys with low plasma DBD levels.

However, an outpatient study by Bowden et al, 1988 found that low DβH levels were much more likely in ADHD children who also had CD than ADHD children without CD (Rogeness et al, 1987; Pliszka et al, 1988; Bowden et al, 1988; Comings et al, 1996). In contrast, in outpatient studies at a juvenile detention center an association between CD and plasma DβH was not found. Umberkomen et al (1981) have shown a correlation between low DβH levels and sensation-seeking behaviors. Examination of CSF DβH levels in patients with a variety of psychiatric disorders including major depression, bipolar affective disorder and schizophrenia found that the only significant correlation was between low CSF DβH and bipolar affective disorder (Lerner et al, 1978).

Linkage studies between the DβH locus and schizophrenia (Aschauer and Meszaros, 1994), alcoholism, depression, manic- depression and Tourette's syndrome (Comings, et al, 1986) have been negative. However, some sib pair analyses suggest a weak linkage between the ABO blood group and DβH, and some psychiatric disorders such as depression and alcoholism (Wilson et al, 1992). Linkage studies between the DβH locus and schizophrenia, alcoholism, depression, manic-depression, and Tourette syndrome have been negative (Aschauer and Meszaros, 1994; Comings et al, 1986). No association was found between the D HTaqlBl allele and pathological SAB (Blum et al, 1997). The Taql B1/B2 polymorphism was reported to be associated with controls screened to exclude drug, alcohol, and tobacco abuse. However, the BI allele of dopamine-beta-hydroxylase gene also associated TD probands, and ADHD probands (Comings et al. Cannabinoid Receptors (CB1) While the association of cannabinoid receptors with the reward pathways may be primary, it is more likely that the effect is secondary through the modulating effect of anandaide and cannabinoid receptors on dopamine metabolism.

This is consistent with the similarity between the results with CB1 receptors and the DRD2 receptors. Like the CB1 gene the association of genetic variants of the DRD2 gene with polysubstance abuse has been more reproducible (OΗara et al, 1993; Smith et al, 1992; Noble et al, 1993; Comings et al, 1994) than the association with alcoholism per se. One interpretation of these observations is that the dopaminergic-cannabinoid reward pathways are activated more by drugs, especially cocaine and amphetamines, than by alcohol (DiChiara and Imperato. Activation of the mesolimbic dopamine system is known to trigger a relapse to cocaine seeking behavior in animal models of drug dependence. This priming effect is enhanced by dopamine D 2 agonists but inhibited by dopamine D, agonists (Self et al 1996).

In this regard, the ability of anandamide to cause a decrease in the ratio of D, and D 2 receptors in the striatum (Romero et al, 1995) may be the link that accounts for the role of CB1 variants in drug dependence. Monamine Oxidase The E.4H1 polymorphism, associated with a T-»C variant at position 1460, and the EcoKV polymorphism, associated with a T- G variant at position 941, of the MAO-A cDNA has been examined (Hotamisligil and Breakefield, 1994). Since both involved substitutions in the third base of a codon, they were not associated with amino acid substitutions. They examined 40 cell lines of known MAO-A activity.

All lines that carried the Fnu4Hl C variant also carried the EcoRV G variant. When the sample was divided into two groups on the basis of lower vs higher MAO-A activity, the less common Fn 4Hl C or + allele (the inventors' 2 allele), present in 25% of the cell lines, was significantly (R = 0.028) associated with the higher activity group. (1994) reported a significant increase in the more common MAO A Fn 4Hl T or 1 allele (Lin et al, 1994), associated with lower MAO levels (Lin et al, 1994) in manic depression, while Craddock et al. (1995) and Nothen et al ( 995) were unable to confirm this.

Vanyukov et al. (1993) examined the MAO A gene in 23 male and 34 female alcoholics compared to 31 male and 78 female controls, using a CA repeat polymorphism (Black et al.

There was a trend in males (R = 0.17) but not in females (R = 0.8) for an association between higher molecular weight alleles (>115 bp) in young substance abusers, and a marginal association of the >115 bp alleles with age of onset (R = 0.03). Tivol et al (1996) have recently sequenced the exons of 40 control males who showed a >100-fold variation in MAO A enzyme activity. There was remarkable conservation of the coding sequence.

Only five polymorphisms were found. Of these, four involved the third codon position with no change in the amino acid sequence. The other was a neutral lys — arg substitution.

Nicotine Receptor Genes The gene for the CHRNA4 gene is located on chromosome 20ql3.2-13.3 (Steinlein et al, 1994) and consists of 6 exons over 17 kb of genomic DNA (Steinlein et al, 1996). A Ser248Phe missense mutation in the transmembrane domain 2 of the CHRNA4 gene was found to be associated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) in one extended Australian pedigree (Steinlein et al, 1995). An insertion of three nucleotides (GCT) into the coding region for the C-terminal end of the M2 domain was found in a Norwegian pedigree with autosomal dominant nocturnal frontal lobe epilepsy (Steinlein et al, 1997b). Two other disorders of brain function, benign familial neonatal convulsions (Leppert et al, 1989; Malafosse et al, 1992) and low-voltage EEG (Steinlein et al, 1992) have also been linked to the region of the CHRNA4 locus. D20S19, a highly polymoφhic locus, is in tight linkage with the genes for all three of these disorders (Steinlein et al, 1996). A highly polymoφhic dinucleotide VNTR polymoφhism located in the first intron of the CHRNA4 gene was reported by Weiland and Steinlein (Weiland and Steinlein, 1996). Single base pair polymoφhisms have also been reported (Steinlein, 1995; Phillips and Mulley, 1997; Guipponi et al, 1997; Steinlein et al, 1997a).

Using three single base pair polymoφhisms Steilein et al. (1997a) found no association between the CHRNA4 gene and panic disorder.

Using the Ser248Phe missense mutation associated with ADNFLE and four silent polymoφhisms, Steinlein et al, Ser248Phe missense mutation reported a modest increase in the frequency of the T allele of the Cfol 595 polymoφhism in common idiopathic generalized epilepsies of childhood (.085) versus controls (.027). Micro/minisatellite polymorphisms Studies of behavioral phenotypes associated with micro/minisatellite polymoφhisms at different neuropsychiatric candidate genes have found a significant association between the shorter or longer alleles with various quantitative behavioral traits and mini- or microsatellites at the following genes: MAOA, MAOB, HTR1A, DATl, DRD4, HRAS, HTT, OB, CNR1, GABRA3, GABRB3, FRAJXA, and NO (Comings et al, 1996k; Comings et al, 19961; Comings et al, 1996m; Johnson et al.

1997; Comings et al, 1998; Gade et al, 1997). Significant phenotypic behavioral effects with specific size alleles of the same polymoφhisms ofthe DATl (Cook, 1995; Gelernter et al, 1994), DRD4 (Benjamin et al, 1996; Ebstein et /., 1996; Grice et al.

1996; Lahoste et al, 1996), HRAS (Herault et al, 1993; Eggers et al, 1995; Thelu et al, 1993), H7T (Ogilvie et al, 1996; Lesch et al, 1996), INS (Bennett et al, 1955; Kennedy et al, 1995; Pugliese et al, 1997; Vafiadis et al, 1997) and DBH (Wei et al, 1997) genes. These studies do not rule out the presence of an important role of single base pair changes in a subset of these length variants, (see below and Grice et al, 1996; Lichter et al, 1993; Krontiris et al, 1985). The is evidence for an involvement of long triplet repeats in a variety of neurological disorders (Caskey et al, 1992) including fragile-X syndrome, Huntington's disease (Huntington's Disease Collaborative Research Group, 1993), myotonia dystrophica, Kennedy's disease, Friedreich's ataxia (Campuzano et al, 1996), and others (Caskey et al, 1992).

At least five of these disorders involve intronic GAG repeats producing polyglutamine tracts in the amino acid sequence of the respective gene products. Obesity Related Genes Previous studies have failed to identify any mutations of the human OB gene in several hundred obese individuals (Ezzel, 1995; Hamilton et al, 1995; Considine et al, 1996b). However, prior studies (Comings, 1996b; Comings et al, 1996c) have suggested that the mutations involved in polygenic disorders may be outside the exons and that the polymoφhic dinucleotide repeats may themselves play a role in regulating the expression of the genes to which they are close to (Krontiris et al, 1993; Green and Krontiris, 1993; Trepicchio and Krontiris, 1992; Trepicchio and Krontiris, 1993; Bennett et al, 1955; Kennedy et al. Taql polymorhisms of the apolipoprotein gene {APOE-D) was found to associate with obese subjects and between the APO-D and fasting-insulin. This work suggests that that the APO-D polymoφhism may be a genetic marker for both obesity and hyperinsulinemia (Vijayaraghavan et al, 1994). Serotonin Genes Functional variants of this gene could account for the observed simultaneous increase or decreases of both serotonin and tryptophan in various disorders.

Four different polymoφhisms of the human TD02 gene have been identified. Association studies show a significant association of one or more of these polymoφhisms and TS, ADHD, and drug dependence. The intron 6G-T variant was significantly associated with platelet serotonin levels (Comings et al, 1996a). Multiple Gene Analysis Combined examination of the dopamine D4 receptor gene {DRD4), cannabinoid receptor gene (CNRl) and the GABAB3 receptor gene (GABRB3) explained 25% of the variance of the trait of IV drug use (Saucier et al, 1996; Comings et al, 1997; Johnson et al, 1997).

It was observed that testing for both the OB and the DRD2 gene explained 22.8%) ofthe variance of body mass index, demonstrating that polygenic factors influence body weight, while the association with psychiatric symptoms, determined by examination alone, accounted for a smaller percent (Comings et al, 1996b). Individual polymoφhisms at three dopaminergic genes: Taql Al of the dopamine D2 receptor (DRD2),TaqlBl of the dopamine B- hydroxylase (DβH), and the 10/10 genotype of the 40bp repeat of the dopamine transporter {DATl) genes, were shown to have significant association with TS, ADHD, and CD (Comings, 1996). The Role of Neurotransmitters and Amino Acid Precursors In addition to the genes thought to be involved in neurological disorders, neurotransmitters and pharmaceuticals have been studied for their roles in creating or alleviating certain psychological traits.

In humans, it has been suggested that meso-prefrontal dopaminergic activity is involved in human cognition (Weinberger et al, 1988). In patients with Parkinson's disease and possibly in patients with schizophrenia, prefrontal activation during a cognitive task and with clinical signs of dopaminergic function (Weinberger et al, 1988k). Brain chemical turnover in animals have demonstrated changes in neurotransmitter levels following precursor amino acid loading, or systemic and direct central nervous system delivery (Blum et al, 1996a; Blum et al, 1996b). Animal studies implicate NE and dopamine in a wide range of attention-related behaviors involving search and exploratory activity, distractibility, response rate, discriminability and the switching of attention. Overall, the animal and human studies indicate a role for dopamine and NE in the early and late processing of information, respectively (Sara et al, 1994k).

Several neurotransmitters, specifically, dopamine, serotonin, norepinephrine, GABA, glutamine, and opioid peptides which play vital roles in brain functioning and in mood regulation, can be dramatically influenced by the circulating levels of their precursor amino acid nutrients (Wurtman, 1981k). Data suggest that amino acid precursors and enkephalinase inhibitors provide a substantive effect on recovery from alcohol, cocaine, and food addictions (Blum et al, 1987a; Blum et al, 1987b; Blum et al, 1987c; Blum et al, 1989b; Blum et al, 1990; (Strandburg et al, 1996. One important function ofthe catecholamine innervation of the cerebral cortex may be the control of attention. Of particular interest are the catecholamine projections to the cerebral cortex from the reticular formation, namely dopamine neurons in the ventral tegmentum of the midbrain and the NE neurons of the locus coeruleus in the upper pons.

Both acetylcholinergic (ACH) and dopaminergic systems (DA) have been found to be crucial for the maintenance of accurate cognitive performance. A series of studies, examining those aspects of cognitive function, revealed by the radial-arm maze, found that these two neurotransmitter systems interact in a complex fashion (Levin et al, 1995). Choice accuracy deficit induced by blockade of either muscarinic- or nicotinic-ACH receptors. The choice accuracy deficit induced by blockade of muscarinic receptors with scopolamine can be reversed by the dopamine receptor blocker, haloperidol. The specific DAD1 blocker SCH23390 also has this effect, whereas the specific D 2 blocker raclopride did not.

This effect is seen with the D 2 antagonist raclopride, but not with the Dj antagonist SCH23390. The D 2 receptor was indicated in nicotinic actions on cognitive function by the finding that the selective D 2 agonist LY 1771555 reverses the choice accuracy deficit caused by mecamylamine. The effectiveness of these selective DA treatments in reversing cognitive deficits was due to ACH under-activation (Levin et al. Accumulating evidence suggests that serotonin may modulate cholinergic function in several regions of the mammalian brain and that these serotonergic/cholinergic interactions affect cognition. It is concluded that not all mnesic perturbations induced by concurrent manipulations of the serotonergic and cholinergic systems can be attributed to a serotonergic modification of the cholinergic system. The cognitive faculties of an organism arise from interactions among several neurotransmitters such as DA within brain structures such as, for instance, the hippocampus or the cortex, but also from influences on memory of other general functions that may involve cerebral substrates different from those classically related to mnesic functions (e.g., attention, arousal, sensory accuracy, etc.) (Cassel et al, 1995k).

Additionally, it has been determined that extrinsic modulation of hippocampal theta depends on the co-activation of cholinergic and GABA-ergic medial septal inputs. Cholinergic projections provide the afferent excitatory drive for hippocampal theta-on cells and septal GABA-ergic projections act to reduce the overall level of inhibition by inhibiting hippocampal GABA-ergic interneurons (hippocampal theta- off cells). Both activities must be present for the generation of hippocampal theta field and cellular activities. The balance between the cholinergic and GABA-ergic systems may determine whether hippocampal synchrony (theta) or asynchrony occurs (Smythe et α/., 1992). Other neurotransmitters like Norepinephrine (NE) may also play a role in learning and memory. Neuromodulatory properties of NE suggest that the coeruleo- cortical (LC) NE projection should play an important role in attention and memory processes. For example, the gating and tuning action of NE released in target sensory systems would promote selective attention to relevant stimuli at the critical moment of change (Sara et al, 1994).

Other research suggest that one consequence of LC activation during stress or physiological challengers may be to increase or maintain arousal via release of both DA and NE (Page et al, 1994). It has been reported that discharge of NE LC neurons in behaving rats and monkeys suggest a role for the LC system in regulating attentional state or vigilance (Aston- Jones et al, 1991k).

Additional research on studies of NE in the dentate gyrus support a role for the LC in promoting both short- and long-term enhancement of responses to complex sensory inputs and are consistent with a role for the LC in memorial as well as attentional processes (Harley, 1988k). NE applied exogenously or released endogenously can initiate both a short- and a long-term potentiation (LTP) of the dentate gyrus. Studies dealing with the effects of the neurokinin substance P (SP) and its N- and C-terminal fragments on memory, reinforcement, and brain metabolism. It was shown that Sp, when applied peripherally, promotes memory and is reinforcing. Most important, however, is the finding that these effects seemed to be encoded by different SP sequences, since the N-terminal SP1-7 enhanced memory, whereas C-hepta- and hexapeptidsequences of SP proved to be reinforcing in a dose equimolar to SP. These differential behavioral effects were paralleled by selective and site-specific changes in DA activity, as both SP and its C-, but not N-terminus, increased DA in the nucleus accumbens (Nac), but not in the neo-striatum. These results show that the reinforcing action of peripheral administered SP may be mediated by its C-terminal sequence, and that this effect could be related to DA activity in the NAC (Huston et al, 1991 k).

In terms of dopaminergic activity, previous research has shown that bromocriptine, a D 2 dopamine receptor agonist, can have a beneficial effect on visual- spatial working memory functions in normal human subjects (Kimberg et al, 1997). This form of memory, in which some aspect of a stimulus is maintained over a short interval of time, has also been found to be closely tied to prefrontal cortical function in both lesion and single unit recording studies with monkeys and in neuro-imaging studies in humans (Goldman et al, 1987k; Jonidas et al, 1993). A selective positive effect of bromocryptine, in reducing release rates in alcoholics as a function of dopamine D 2 receptor genotype (Lawford, et al, 1995) has also been reported. In addition, it has been demonstrated a direct effect of dopamine antagonists on delay period activity of neurons in monkeys performing memory tasks (Williams et al, 1995k). Phentermine, a dopamine releaser, has been implicated in weight loss (Weintramb et fl/., 1992). Moreover, pharmacological manipulation of brain dopamine concentration effects visual-spatial working memory in humans and in animals, the later effects localized to the prefrontal cortex.

However, the effects of dopamine agonists on humans are still poorly understood. It has been hypothesized that bromocriptine would have an effect on cognitive functions associated with the prefrontal cortex via its effects on cortical dopamine receptors and on sub-cortical receptors in areas that project to the neocortex (Kimberg et al, 1997). They found that the effects of bromocriptine on young normal subjects depended on the subject's working memory capacity. High-capacity subjects performed more poorly on the drug, while low- capacity subjects improved.

These results demonstrate an empirical link between a dopamine-mediated working memory system and higher cognitive function in humans. It has been shown that the DRD2 Al allele is also associated with visual- spacial memory deficits as well (Berman et al, 1995k). A double-blind study demonstrated that a D 2 agonist bromocryptine or a placebo administered to alcoholics who were carriers of the Al allele (Al/Nl and A1/A2 genotypes), or who only carried the A2 allele (A2/A2) reduced craving and anxiety among the Al carriers who were treated with bromocryptine.

The attrition rate was highest among the Al carriers who were treated with placebo. The bromocryptine effect on the Al carriers was much more robust as one approached the six wk period of treatment. Dopamine D 2 agonist bromocryptine can improve higher- level cognitive functions. Studies using sophisticated techniques in animals, including microdialysis measurements, have demonstrated changes in neurotransmitter output following precursor amino acid loading (Hernandez et al, 1988). In addition, behavioral changes have been demonstrated in animals following systemic and direct central nervous system delivery of precursor amino acids (Blum et al, 1972). While certain L-amino acids are neurotransmitter and neuromodulator precursors, their racemates, the D-amino acids also have biological activity.

In particular, D-phenylalanine, D- leucine. Other D-amino acids as well as certain metabolites {e.g., hydrocinnamic acid) decrease the degradation of opioid peptides which are central to regulation of mood and behavior (Blum et al, 1977; Delia Bella et al, 1980). In some individuals scientists have described a phenylalanine deficiency (PHD) (Lou, 1994k). In this regard, phenylalanine and tyrosine constitute the two initial steps in the biosynthesis of dopamine, which, in its turn, is the metabolic precursor of NE. The extracellular phenylalanine concentration influences brain function in PHD by decreased dopamine synthesis. It has been shown to induce EEG slowing and has prolonged the performance time on neuropsychological tests.

The tyrosine concentration in the CNS is reduced in PHD, possibly implying an insufficient substrate of tyrosine for catecholamine synthesis due to competition inhibition, for instance across the blood brain barrier. In experimental studies it has been shown that the synthesis and release of dopamine can be influenced by an increase in the availability of tyrosine. In PHD an extra dietary intake of three doses of tyrosine (160 mg/kg/24 h) induced a shortening of reaction time and decreased variability and in a double-blind, crossover study a similar dose has been reported to induce an improvement on psychological tests, while lower doses failed. A combination of precursor amino acids having enkephalinase inhibition activity may be used for the treatment of cocaine dependence (U. It is known that acute use of cocaine can improve certain aspects of brain electrophysiological dysfunction (Maurer et al, 1988k).

Chronic cocaine abuse alters attentional processing (Noldy et al, 1990k). It is known that acute use of cocaine can improve certain aspects of brain electrophysiological dysfunction (Jonsson et al, 1996). However, paradoxically, chronic cocaine abuse alters attentional processing (Braverman and Blum, 1996). Although still controversial, attentional processing has been shown to be dependent on biogenic amine regulation (Lyoo et al, 1996). Obesity and Neurological Functions Obesity generally is defined as being 20% or more over ideal body weight.

Numerous methods of weight reduction have been attempted including hypocaloric balanced diets, 'fad' diets, behavior modification, drugs {i.e. D-phenflouramine, phenteramine, etc.), surgery, total starvation, jaw wiring, and combinations of these methods. Most of these are short- term approaches to the problem and have been only transiently effective and some can even pose serious danger (Lockwood and Amatruda, 1984). Even if weight loss is demonstrated in the short-term, the weight usually is regained following discontinuation of the weight-loss regiment. Despite the fact that about 28% of the American population is obese, obesity is widely perceived as a food-addiction, a self- imposed condition with cosmetic rather than health indications (Krai et al, 1989; Weintraub and Bray, 1989). An understanding is emerging from recent studies of some of the causes of obesity and the difficulties of treating this condition.

Studies of twins among the Pima Indians have substantiated a strong genetic basis for obesity (Bouchard, 1989; Stunkard et al, 1990). Obesity is a heterogeneous and prevalent disorder which has both genetic and environmental components. The relationship between macro selection of various foods and familial substance use disorder (SUD) has been documented throughout the literature and neurochemical studies have supported the commonality of reinforcement through dopaminergic systems by alcohol, nicotine, cocaine, and carbohydrates (Nobel, 1998; DiChiara, 1988). In this regard, both obesity and SUD can be considered appetitive compulsions.

Some genes such as the dopamine D2 receptor (DRD2), and dopamine transporter (DATl) genes may be a risk factor not only for obesity (Noble et al, 1994; Comings et al, 1993; Blum et al, 1995a) but also for SUDs in general and other psychiatric disorders (Noble et al, 1994; Smith et al, 1992; Comings, 1994; Blum et al, 1995b; Comings et al, 1996; Cook et al, 1995). Additionally, the cloning and sequencing of the mouse ob gene and its human OB homologue raised hopes that defects in this gene may play a significant role in the cause of obesity in man and that Leptin, its gene product, would be useful in treatment (Zhang et al, 1994; Peileymounter et al, 1995).

While genetic effects can act alone, in most cases the genetic profile only sets the stage defining the opportunity for a genetic-environmental interaction {i.e. Dramatic increase in weight when coupled with increased food). For persons with such a genetic risk profile, obesity is a life-long condition requiring long term therapy as in other chronic diseases. The specific causes of uncontrollable ingestive behavior for alcohol, drugs, and food (in particular, carbohydrates) are incompletely understood. Nevertheless, it is clear that these appetitive compulsive behaviors are a product of genetic predisposition and environmental insult factors. Numerous studies have implicated the interaction of opiates, opioid peptides, CCK-8, glycogen, DA, and insulin in glucose utilization and selective intake of carbohydrates (Morley and Levine.

1988; Moore et al, 1982; Morley et al, 1985; Riviere and Bueno, 1987). The primary neurotransmitters involved in eating behavior include the monoamines dopamine (DA), norepinephrine (Ne), epinephrine (EPI), and serotonin (5-HT); the inhibitory neurotransmitter gamma-aminobutyric (GABA); and a variety of neuropeptides such as the pancreatic polypeptides, opioid peptides, hormone-releasing factors, and various gut-brain peptides (for reviews see Cooper et al, 1988; Gosnell, 1987; Bouchard, 1994). There is extensive evidence for the role of a number of brain monoamines and neuropeptides in the control of normal eating behavior operating in concert at the mesolimbic reward system (Leibowitz and Hor, 1982).

Analyses of cerebrospinal fluid in both humans and animals indicate specific disturbances in brain neurochemical function in association with abnormal eating patterns (Kaye et al, 1985; Kaye et al, 1984). A study of overeaters demonstrated that study subjects taking a variant of PHENCAL™, which is a dietary supplement containing amino acid precursors, lost an average of 27 lbs in 90 days compared to 10 lbs lost in the control group (Blum, 1990). Finding that PHENCAL™ or other similar neuronutrients, (Blum et al, 1988c; Blum and Trachtenberg, 1988; Cold, 1996) with alcoholics, polydrug abusers, heroin abusers, and cocaine-dependent individuals facilitates recovery and further indicates a common mode of treatment for addiction to these diverse substances (Blum et al, 1996; Blum et al, 1997). The Role of Nicotine Nicotine also releases dopamine, and nicotine has been found to improve memory performance in a variety of tests in rats, monkeys, and humans (DiChiara et al, 1988). Nicotine in a dose dependent fashion reduced incorrect responding on discrimination behavior in rats (Geller et al, 1970). This effect was similar to chlordiazepoxide but could not be mimicked by the stimulant caffeine (Geller et al, 1970). Nicotine, in the form of gum or skin patches (Sanberg et al, 1988; McConville et al, 1992; Sanberg et al, 1997) has been shown to be effective in the treatment of tics in some subjects with Tourette syndrome (TS), and cigarette smoking has been reported to enhance attention, arousal, learning and memory (Wesnes and Warburton, 1984; Warburton, 1992; Balfour and Fagerstrδm, 1996) and to improve the symptoms of ADHD (Coger et al, 1996; Conners et al, 1996; Levin et al, 1996).

It has been reported a placebo-controlled double-blind study to determine the effect of using nicotine in the treatment of adults with ADHD (Levime et al, 1996; Conners et al, 1996). Of the 17 subjects, 6 were smokers and 1 1 were nonsmokers. All meet DSM-IV criteria for adult ADHD. The drug was delivered via a transdermal patch at a dosage of 7 mg/day for nonsmokers and 21 mg/day for smokers.

Active and placebo patches were given in a counter-balanced order approximately 1 wk apart. Nicotine caused a significant overall improvement on the Clinical Global Impressions (CGI) scale. This effect was significant even when only the nonsmokers were considered, which indicated that it was not due merely to relief of withdrawal from regular smoking. Nicotine caused significantly increased vigor as measured by the Profile of Mood States (POMS) test, and an overall significant reduction in reaction time on Continuous Performance Test. There was also a significant reduction in indices of inattention.

Nicotine improved accuracy of time estimation and lowered variability of time-estimation response curves. Since smoking is significantly more common in adults with ADHD than those without ADHD (Conners et al, 1996). Interactions of nicotinic systems with dopamine systems may be important for this effect. A series of studies of nicotinic agonist and antagonist interactions with dopamine systems was conducted using rats in a win-shift working memory task in the radial-arm maze (Levin and Rose, 1995k). The working memory deficit caused by the nicotinic antagonist mecamylamine was potentiated by the D1/D2 DA antagonists haloperidol and the specific D 2 antagonist raclopride. In contrast, the mecamylamine- induced deficit was reversed by co-administration of the D2/D3 agonist quinpirole. Nicotine also has significant interactions with dopamine drugs with regard to working memory performance in the radial-arm maze.

The dopamine agonist pergolide did not by itself improve radial-arm choice accuracy. Nicotine was effective in reversing this deficit. When given together with nicotine, the D2/D3 agonist quinpirole improved RAM choice accuracy relative to either drug alone. Acute local infusion of mecamylamine to the midbrain dopamine nuclei effectively impairs working memory function in the radial-arm (Noble et al, 1998). The Role of Chromium Salts (CrP and CrN) Trivalent chromium is a mineral essential for normal insulin function (Jeejeehboy et al, 1977; Schwartz et al, 1959). Some but not all previous research suggests that chromium supplementation may favorably alter risk factors for coronary artery disease (CAD) and non-insulin- dependent diabetes mellitus (NIDDM)( Abraham et al, 1992; Anderson et al, 1991; Donaldson et al, 1985; Glinsmann et al, 1966; Kaats et al, 1991; Levine et al, 1968; Page et al, 1991; Press et al, 1990; Roeback et al, 1991). Chromium is thought to cause these changes via its potentiating effect on insulin (Offenbacher et al, 1988).

Animal studies have supported the contention that CrP can lower insulin resistance and improve body composition (Liarn et al. 1993), one human study found positive changes in body composition with CrP supplements (Hasten et al, 1992), another reported positive, although not statistically significant changes in body composition (Hallmark et al, 1993), and a third failed to find any positive changes in body composition with CrP supplementation (Clancey et al, 1994). CrP supplementation has been indicated to improve body composition, particularly in the reduction of excess body fat (Page et al, 1992). However, previous work observing concurrent chromium supplementation and exercise training has been restricted to effects on body weight and composition, with conflicting results (Clancy et al, 1994; Evans et al, 1989; Evans et al, 1993; Hallmark et al, 1996; Hasten et al, 1992). While there still is controversy regarding the effects of chromium salts (picolinate and nicotinate) on body composition and weight loss in general (Abraham et al, 1992; Anderson, 1995; Hallmark et al, 1993; Clancy et al, 1994; Bulbulian et al, 1996), some reports seem to support the positive change in body composition in humans (Kaats et al, 1996).

In contrast, (Grant, et al, 1997; Bulbulian et al, 1996) reported weight gain with chromium picolinate with or without exercise in humans, while showing positive effects for the nicotinate salt in the same population (Kaats et al, 1992). Chromium Picolinate (CrP) is the most heavily used, studied and promoted chromium compound, but in vitro work suggests that chromium nicotinate may be also viable in the area of weight loss and changes in body composition. Previous research has shown chromium picolinate supplementation decreasing fat mass and increasing fat- free mass (Kaats et al, 1991; Page et al, 1991). Pervious studies of exercise training have shown increases in fat free mass as well (Stefanick, 1993). Although studies with young men (Evans, 1989) and women (Hasten et al, 1992) suggest that combining exercise training with chromium picolinate supplementation increases the body composition changes that occur with exercise training, this finding has not been confirmed (Clancy et al, 1994; Hallmark et al, 1996). It has been reported that the nicotinate salt (CrN) may be even more important than the picolinate salt (Grant et al, 1997).

Nutritional Supplements in Treatment of Behavioral Disorders Perturbation of neurotransmitter actions may underlay a variety of psychiatric and behavioral disorders (Blum et al, 1996c; Persico and Uhl, 1997; Noble et al, 1991). Specifically, anomalous regulation of dopamine, serotonin, norepinephrine, gammaminobutyric acid (GABA), glutamine, and the opioid peptides are thought to play crucial roles in the addictive disorders, particularly those involving alcohol and cocaine abuse (Pohjalainen et al, 1996). Consequently, these observations have provided momentum to the idea that ingestion of selected nutrients could affect mood and therefore behavior in humans. While nutritional strategies have been employed in the past (Grandy et al, 1989), demonstrations of effectiveness have been decidedly limited. A substantive effect of a combination of amino acid precursors and enkephalinase inhibitors on recovery from certain RDS behaviors including alcohol, cocaine, and overeating have been indicated (Noble et al, 1993; Noble et al, 1994; Blum et al, 1994; Balldin et al, 1993; Duffy et al, 1994; American Psychiatric Association Task Force, 1991, U.S. Polygenic Analysis of Genes involved in Psychiatric and Other Polygenic Traits It has been hypothesized that psychiatric behaviors share genes in common and that once the dopamine-serotonin and other neurotransmitter balance is upset, the resulting brain dysfunction can result in a wide range of different behaviors (Comings, 1990a; Comings and Comings, 1991a; Winokur et al, 1970; Comings, 1994b; Comings, 1995b).

Others have supported the proposal that personality traits may have distinct neurochemical and genetic substrates mediated by genetic variability in dopamine transmission as well as other neurotransmitters (Cloninger, 1983; Benjamin, et al, 1996, Epstein et al, 1996, Cloninger, 1991). The molecular genetic studies of the DRD2, DβH, DAT (Comings et al. 1996a), and clinical genetic studies (Comings 1994b; Comings 1994c; Comings 1995b; Biederman et al, 1991; Comings and Comings 1987), indicate ADHD, Tourette's syndrome, conduct disorder, ODD. Dyslexia, learning disorders, stuttering, drug dependence and alcoholism are etiologically related spectrum disorders, with male predominance. In the past two decades a large proportion ofthe genes for these disorders have been identified, localized, cloned, and sequenced. As the number of such genes remaining to be identified has decreased there has been an increased interest in the more common polygenic disorders. It has often been suggested that the genes involved in these disorders will be far more difficult to identify.

This difficulty is well illustrated by the psychiatric disorders. Despite large numbers of linkage studies of manic-depressive disorder, schizophrenia, Tourette syndrome, panic disorder, autism, and others, with the possible exception of bipolar disorder (Risch and Botstein, 1996), there have been few replicated findings. Many of the efforts to find the genes in complex disorders have simply attempted to force feed the single-gene single-disease model into service for polygenic disorders by using lod score analysis, other family based forms of linkage analyses, or the haplotype-relative risk technique (Falk and Rubinstein, 1987). Presently the most popular method used to identify the genes in complex disorders consists of whole genome screening of affected sib pairs. Non-parametric approaches to linkage (Weeks and Lange, 1988) are better suited to complex inheritance (but see Greenberg et al, 1996). However, when a given gene accounts for less than 8% of the variance, a large number of parent-child sets or sib- pairs must be examined (Carey and Williamson, 1991).

There has been an increased recognition that only association studies may have the power to identify genes with small contributions to the percent of variance of a given polygenic trait (Risch and Merikangas. 1996; Collins et al, 1997). Association studies, comparing the frequency of the mutant candidate genes in severely affected probands to totally unrelated, ethnically matched controls that are free of the disease, can identify these small effects (Weeks and Lathrop, 1995; Comings, 1996; Owen and McGuffin, 1993). The additive effect of the DRD2, DβH and DAT genes (Comings et al, 1996j), the DRDl and DRD2 genes (Comings et al, 1997a), the OB and DRD2 genes (Comings et al, 1996d), and other gene combinations genes in TS, ADHD, conduct disorders, stuttering, and related behaviors has been examined. In TS syndrome it has been found that identifying a role of three dopaminergic genes (DRD2, DβH and DATl) was best determined by an examination of a relatively large number of TS subjects, their relatives and controls, suggesting that TS and related disorders are polygenically inherited and that each gene contributes only a small percent ofthe variance of any behavior score (Comings et al, 1996a; Comings 1996b; Comings et al, 1996d; Comings 1996c). Most psychiatric disorders are polygenic (Comings, 1996b) and that each gene accounts for less than 10%>and usually less than 5% of the variance of a given behavioral variable. In both studies, the strength ofthe associations was increased by the examination of the additive effect of more than one gene.

One of the major impediments to the wider use of association studies is the lack of availability of suitable polymoφhisms at or near the many candidate genes that have been cloned and sequenced (Comings, 1994). However, even when this technique or classical linkage techniques are used, positive findings from one group of investigators are often not replicated in subsequent studies (Egeland et al, 1987; Kelsoe et al, 1989; Blum et al, 1990; Bolos et al, 1990). This technique can also produce false positives due to population stratification, however, this can be minimized using the haplotype relative risk procedure (Falk and Rubinstein, 1987) with large numbers of subjects (Comings, 1995). The small size of these effects, and the difficulties in replication have led to a feeling of pessimism about whether it will be possible to identify the genes involved in polygenic disorders (Moldin, 1997). SUMMARY OF THE INVENTION In the United States alone there are 18 million alcoholics, 28 million children of alcoholics, 6 million cocaine addicts, 14.9 million people who abuse other substances.

25 million people addicted to nicotine, 54 million people who are at least 20% overweight, 3.5 million school-age children with ADHD or Tourette's syndrome, and about 3.7 million compulsive gamblers. The inventors believe that genotyping humans for the alleles of the DRD2 gene as well as other genes related to psychological disorders in the present invention is indeed the first step toward rational treatment for a devastating problem in society. The invention first provides a composition for the treatment of Reward Deficiency Syndrome (RDS) behaviors in a subject. In certain aspects, this composition includes at least one of the following components: an opiate destruction- inhibiting amount of at least one substance which inhibits the enzymatic destruction of a neuropeptidyl opiate, the substance being either amino acids, peptides. Alcohol Abuse Drug Abuse IV Drug User Hallucinogen Marijuana Amphetamine Cocaine Yrs. Severity Severity Yrs.

Rating Rating Total R.46.46.32.38.26.24.41 R z.21.21.14.07.06.17 P.0002.0001.002.003.013.007 5 / >5 genotype C = Family Cohesion from the FES *** ^1 STEP 4. The fourth step is to identify one or more polymoφhisms associated with each gene. These can be single base pair restriction fragment length polymoφhisms (RFLPs), or dinucleotide, trinucleotide, or other repeat polymoφhisms, such as well as variable tandem repeats, or any other marker of a gene locus. Such polymoφhisms and methods of detection may be readily available in previously published or unpublished bodies of work, as previously described above for identifying candidate genes, in addition to the polymoφhisms disclosed herein. Alternatively, if a gene is suspected of contributing to a polygenic trait of interest, but no polymoφhism is currently available for use in the MAA technique after a review of the literature and genetic databases, one may perform genetic assays to determine polymoφhisms in a gene that may be used in the MAA technique. Such assays are commonly used and described in the literature', in addition to the techniques described herein. Methods for genetic screening to accurately detect mutations in genomic DNA, cDNA or RNA samples may be employed, depending on the specific situation.

The present invention concerns the detection, diagnosis, prognosis and treatment of RDS diseases, and the detection, diagnosis, and prognosis of polygenic traits using the MAA technique. Markers of alleles that contribute additively or subtractively to a polygentic trait, in the form of nucleic acid sequences isolated from an individual, and methods of identifying and detecting new markers to be used in MAA assays, are disclosed.

These markers are indicators of a polygenic trait being assayed, and are diagnostic of the potential for an individual to exhibit a particular trait. Those skilled in the art will realize that the nucleic acid sequences disclosed herein, as well as those available through public databases, such as found at the National Center for Biotechnology Information, the published scientific literature, may be used in the MAN technique, and thus will find utility in a variety of applications in the detection, diagnosis, prognosis and treatment or RDS or other polygenic traits.

Examples of such applications within the scope of the present invention comprise amplification of one or more markers of a polygenic trait, using specific primers, detection of markers of a polygenic trait, such by hybridization with oligonucleotide or nucleic acid probes, incoφoration of isolated nucleic acids into vectors, and expression of RNA from the vectors. The requirement to test for multiple genes in behavioral disorders and other polygenic traits is feasible and requires no new technology. The polymoφhisms and variants involved are to two types, 1) single base pair changes producing restriction fragment length polymoφhisms (RFLPs), and 2) short tandem repeat polymoφhisms (STRs) [ especially di-and trinucleotide repeats]. The methods for large scale testing for these are different for each type.

Applied Biosystems, a division of Perkin-Elmer Coφoration, has developed a new technology and instrumentation that allows the rapid testing for PCR™ based single pair RFLP type genetic polymoφhisms. This instrument, Applied Biosystems Prism 7200 sequence Detection System (TaqMan) allows for multiple gene testing. This approach uses standard primers to electrophorese the section of DNA containing the restriction endonuclease polymoφhism site. The unique aspect of this technology is that two short oligmers are then designed, one exactly matching one of the alleles, the other matching the other allele. A fluorescent dye is attached to one end of each, and a quenching dye is attached to the other end. If the match is perfect, when the DNA polymerase reaches the hybridized oligmer, it is digested into nucleotides as the polymerase passes. This releasers the quencher and the dye now fluoresces maximally.

However, if the oligmer does not match, instead of the nuclease digestion, the oligomer is pushed off the site and the quenching persists. Dual wavelength reading of the plate allows distinction between 11,12,22 genotypes. The entire process of reading the results on 96 samples requires less than fifteen min and the results are fed into a computer for analysis and storage. This technology, aided by a computerized workstation to set up to PCR™ reactions, allows hundreds of different RFLPs to be examined in one day.

The same computerized workstation used above is used to set up the PCR™ reactions for the STRs. The difference is that for the STRs the primers themselves are labeled with different fluorescent dyes.

The accuracy necessary to identify alleles differing by only two bp is obtained from the Applied Biosystems 373 DNA sequencer which allows the sample labeled with a second dye. Each is detected by laser scanning at a different wavelength. For example, one PCR™ primer is labeled with fluorescent HEX Amidite (Applied Biosystems, Foster City, CA) or other fluorescent dye.

Two μl of the 10 fold diluted PCR™ product is then added to 2.5 μl deionized formamide and 0.5 μl of ROX 500 standard, denatured for 2 min at 92 C and loaded on 6% denaturing polyacrylamide gel in an AB 373 DNA sequencer. The gel is electrophoresed for 5 h at a constant 25W.

The gel is laser scanned and analyzed using the internal ROX 500 standards present in each lane. The peaks are recognized by Genotyper( version 1.1) based on the color fragments sized by base pair length. Historically, a number of different methods have been used to detect point mutations, including denaturing gradient gel electrophoresis ('DGGE'), restriction enzyme polymoφhism analysis, chemical and enzymatic cleavage methods, and others. The more common procedures currently in use include direct sequencing of target regions amplified by PCR™ (see below) and single-strand conformation polymoφhism analysis ('SSCP'). Another method of screening for point mutations is based on RNase cleavage of base pair mismatches in RNA DNA and RNA/RNA heteroduplexes. As used herein, the term 'mismatch' is defined as a region of one or more unpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule.

This definition thus includes mismatches due to insertion deletion mutations, as well as single and multiple base point mutations. 4,946,773 describes an RNase A mismatch cleavage assay that involves annealing single-stranded DNA or RNA test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNase A. After the RNase cleavage reaction, the RNase is inactivated by proteolytic digestion and organic extraction, and the cleavage products are denatured by heating and analyzed by electrophoresis on denaturing polyacrylamide gels. For the detection of mismatches, the single-stranded products of the RNase A treatment, electrophoretically separated according to size, are compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as positive. Currently available RNase mismatch cleavage assays, including those performed according to U.S. 4,946,773, require the use of radiolabeled RNA probes.

Myers and Maniatis in U.S. 4,946,773 describe the detection of base pair mismatches using RNase A.

Other investigators have described the use of an E. Coli enzyme, RNase I, in mismatch assays. Because it has broader cleavage specificity than RNase A, RNase I would be a desirable enzyme to employ in the detection of base pair mismatches if components can be found to decrease the extent of non-specific cleavage and increase the frequency of cleavage of mismatches.

The use of RNase I for mismatch detection is described in literature from Promega Biotech. Promega markets a kit containing RNase I that is shown in their literature to cleave three out of four known mismatches, provided the enzyme level is sufficiently high. The RNase protection assay was first used to detect and map the ends of specific mRNA targets in solution. The assay relies on being able to easily generate high specific activity radiolabeled RNA probes complementary to the mRNA of interest by in vitro transcription. Originally, the templates for in vitro transcription were recombinant plasmids containing bacteriophage promoters.

The probes are mixed with total cellular RNA samples to permit hybridization to their complementary targets, then the mixture is treated with RNase to degrade excess unhybridized probe. Also, as originally intended, the RNase used is specific for single-stranded RNA, so that hybridized double-stranded probe is protected from degradation. After inactivation and removal of the RNase, the protected probe (which is proportional in amount to the amount of target mRNA that was present) is recovered and analyzed on a polyacrylamide gel. The RNase Protection assay was adapted for detection of single base mutations. In this type of RNase A mismatch cleavage assay, radiolabeled RNA probes transcribed in vitro from wild-type sequences, are hybridized to complementary target regions derived from test samples. The test target generally comprises DNA (either genomic DNA or DNA amplified by cloning in plasmids or by PCR™), although RNA targets (endogenous mRNA) have occasionally been used.

If single nucleotide (or greater) sequence differences occur between the hybridized probe and target, the resulting disruption in Watson-Crick hydrogen bonding at that position ('mismatch') can be recognized and cleaved in some cases by single-strand specific ribonuclease. To date, RNase A has been used almost exclusively for cleavage of single-base mismatches, although RNase I has recently been shown as useful also for mismatch cleavage. There are recent descriptions of using the MutS protein and other DNA-repair enzymes for detection of single-base mismatches.

Additional methods for detection of nucleic acids, and mutations are described herein. Nucleic Acids As described herein, an aspect of the present disclosure is 29 previously known genes whose allelic polymoφhisms are markers of polygenic traits, including markers for such polygenic traits as ADHD, oppositional defiant disorder, conduct disorder, learning disorders, alcohol, cholesterol, and LDL. In one embodiment, the nucleic acid sequences disclosed herein will find utility as hybridization probes or amplification primers. These nucleic acids may be used, for example, in diagnostic evaluation of tissue samples or employed to clone full length cDNAs or genomic clones corresponding thereto.

In certain embodiments, these probes and primers consist of oligonucleotide fragments. Such fragments should be of sufficient length to provide specific hybridization to a RNA or DNA tissue sample. The sequences typically will be 10-20 nucleotides, but may be longer. Longer sequences, e.g., 40, 50, 100, 500 and even up to full length, are preferred for certain embodiments. Nucleic acid molecules having contiguous stretches of about 10, 15, 17, 20, 30, 40, 50, 60, 75 or 100 or 500 nucleotides from a sequence selected from any gene that may be used in the diagnostic or treatment methods disclosed herein are contemplated.

Molecules that are complementary to the above mentioned sequences and that bind to these sequences under high stringency conditions also are contemplated. These probes will be useful in a variety of hybridization embodiments, such as Southern and Northern blotting.

In some cases, it is contemplated that probes may be used that hybridize to multiple target sequences without compromising their ability to effectively diagnose a polygenic trait. Various probes and primers can be designed around the disclosed nucleotide sequences, or the sequences surrounding a polymoφhism useful as a marker, be it a gene disclosed herein or a gene latter added the set of 29 genes described herein. It is contemplated that other genes may be used to create new sets for examination of different polygenic traits, and the use of any other genes, or preferably gene polymoφhisms, in the MAA technique is encompassed as part of the invention. Primers may be of any length but, typically, are 10-20 bases in length.

By assigning numeric values to a sequence, for example, the first residue is 1, the second residue is 2, etc., an algorithm defining all primers can be proposed: n to n + y where n is an integer from 1 to the last number of the sequence and y is the length of the primer minus one (9 to 19), where n + y does not exceed the last number ofthe sequence. Thus, for a 10-mer, the probes correspond to bases 1 to 10, 2 to 11, 3 to 12. For a 15-mer, the probes correspond to bases 1 to 15, 2 to 16, 3 to 17. For a 20-mer, the probes correspond to bases 1 to 20, 2 to 21, 3 to 22.

In certain embodiments, it is contemplated that multiple probes may be used for hybridization to a single sample. The use of a hybridization probe of between 14 and 100 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 20 bases in length are generally preferred, in order to increase stability and selectivity ofthe hybrid, and thereby improve the quality and degree of particular hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having stretches of 20 to 30 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.

Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of genes or RNAs or to provide primers for amplification of DNA or RNA from tissues. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C. Such high stringency conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating specific genes or detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide. For certain applications, for example, substitution of amino acids by site- directed mutagenesis, it is appreciated that lower stringency conditions are required.

Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results. In other embodiments, hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2, 10 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C. Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl 2, at temperatures ranging from approximately 40°C to about 72°C. In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization.

N wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected. In preferred embodiments, one may desire to employ a fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known which can be employed to provide a detection means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples. In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization, as in PCR™, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase.

In embodiments involving a solid phase, the test DΝA (or RΝA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface to remove non-specifically bound probe molecules, hybridization is detected, or even quantified, by means ofthe label. Amplification and PCR™.

Nucleic acid used as a template for amplification is isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al, 1989). The nucleic acid may be genomic DNA or fractionated or whole cell RNA.

Where RNA is used, it may be desired to convert the RNA to a complementary DNA. In one embodiment, the RNA is whole cell RNA and is used directly as the template for amplification.

Pairs of primers that selectively hybridize to nucleic acids corresponding to genes of a polygenic trait are contacted with the isolated nucleic acid under conditions that permit selective hybridization. The term 'primer', as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty or thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred.

Once hybridized, the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as 'cycles,' are conducted until a sufficient amount of amplification product is produced.

Next, the amplification product is detected. In certain applications, the detection may be performed by visual means.

Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incoφorated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax technology). A number of template dependent processes are available to amplify the marker sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR™) which is described in detail in U.S. 4,683,195, 4,683,202 and 4.800,159, each incoφorated herein by reference in entirety. Briefly, in PCR™, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence.

An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated. A reverse transcriptase PCR™ amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDN A are well known and described in Sambrook et al, 1989. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases.

These methods are described in WO 90/07641, filed December 21, 1990, incoφorated herein by reference. Polymerase chain reaction methodologies are well known in the art.

Gharana Mogudu Hd Mp4 Video Songs Free Download here. Another method for amplification is the ligase chain reaction ('LCR'), disclosed in EPA No. 320 308, incoφorated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR™, bound ligated units dissociate from the target and then serve as 'target sequences' for ligation of excess probe pairs. Patent 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence. Qbeta Replicase, described in PCT Application No.

PCT/US87/00880, incoφorated herein by reference, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence that can then be detected. An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha-thio]-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention.

Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e. Nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA.

Target specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion.

The original template is annealed to another cycling probe and the reaction is repeated. Still another amplification methods described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, each of which is incoφorated herein by reference in its entirety, may be used in accordance with the present invention.

In the former application, 'modified' primers are used in a PCR™-like, template- and enzyme-dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence ofthe target sequence.

Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Gingeras et al, PCT Application WO 88/10315, incoφorated herein by reference). In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.

The double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNA's are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences. Davey et al, EPA No. 329 822 (incoφorated herein by reference in its entirety) disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ('ssRNA'), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).

The resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large 'Klenow' fragment of E. Coli DNA polymerase I), resulting in a double-stranded DNA ('dsDNA') molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle.

Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA. Miller et al, PCT Application WO 89/06700 (incoφorated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ('ssDNA') followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e. New templates are not produced from the resultant RNA transcripts. Other amplification methods include 'RACE' and 'one-sided PCR™' (Frohman, 1990, incoφorated herein by reference). Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting 'di-oligonucleotide', thereby amplifying the di-oligonucleotide, may also be used in the amplification step of the present invention.

Following any amplification, it may be desirable to separate the amplification product from the template and the excess primer for the puφose of determining whether specific amplification has occurred. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al, 1989). Alternatively, chromatographic techniques may be employed to effect separation. There are many kinds of chromatography which may be used in the present invention: adsoφtion, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography. Amplification products must be visualized in order to confirm amplification of the marker sequences. One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.

Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation. In one embodiment, visualization is achieved indirectly. Following separation of amplification products, a labeled, nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety. In one embodiment, detection is by Southern blotting and hybridization with a labeled probe.

The techniques involved in Southern blotting are well known to those of skill in the art and can be found in many standard books on molecular protocols. See Sambrook et al, 1989. Briefly, amplification products are separated by gel electrophoresis. The gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the nucleic acid and non-covalent binding. Subsequently, the membrane is incubated with a chromophore-conjugated probe that is capable of hybridizing with a target amplification product.

Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices. One example of the foregoing is described in U.S.

5,279,721, incoφorated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention. All the essential materials and reagents required for detecting gene markers of one or more polygenic traits in a biological sample may be assembled together in a kit. This generally will comprise preselected primers for specific markers. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker primer pair. Preferred pairs of primers for amplifying nucleic acids are selected to amplify the sequences specified in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28.

SEQ ID NO:29. SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34. In another embodiment, such kits will comprise hybridization probes specific for genes involved in polygenic traits corresponding to the sequences specified in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20. SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25.

SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30. SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34. The inventors contemplate that any primers known to be effective to hybridize to an polymoφhic allele that is suspected of being diagnostic in the methods disclosed herein, particularly the MAN technique, may be used in such a kit. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker hybridization probe.

DΝA segments encoding a specific gene may be introduced into recombinant host cells and employed for expressing a specific structural or regulatory protein. Alternatively, through the application of genetic engineering techniques, subportions or derivatives of selected genes may be employed.

Upstream regions containing regulatory regions such as promoter regions may be isolated and subsequently employed for expression ofthe selected gene. It will be understood that this invention is not limited to the particular probes disclosed herein and particularly is intended to encompass at least nucleic acid sequences that are hybridizable to the disclosed sequences or are functional sequence analogs of these sequences. For example, a partial sequence may be used to identify a structurally-related gene or the full length genomic or cDΝA clone from which it is derived. Those of skill in the art are well aware of the methods for generating cDΝA and genomic libraries which can be used as a target for the above-described probes (Sambrook et al, 1989). For applications in which the nucleic acid segments of the present invention are incoφorated into vectors, such as plasmids, cosmids or viruses, these segments may be combined with other DNA sequences, such as promoters, polyadenylation signals, restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. Novel Methodology to Detect multiple Genes in one DNA Sample It is expected that since a number of genes and their polymoφhic loci would be required to diagnose RDS and related behaviors, the inventors propose a Multiple-Gene Screen (GENESCREEN™).

This could utilize novel DNA technology, such as the Gene Chip developed by Affymetrix. In summary, a glass chip is coated with light sensitive chemical compounds. Turnigy Trackstar 80A Turbo Manual.

These chemicals contain light-sensitive protecting groups that prevent the binding of DNA bases to the chip and to each other. When light is shone on the chemicals, however, the protecting groups are inactivated and a chemical coupling reaction can occur. Through the use of 'masks' that allow light to shine on certain regions of the Chip, but not others, DNA bases can be bound to selected areas of the Chip. Each new base that is added has a protecting group attached so that the process can be repeated to couple one base to another.

In this way, a large number of DNA probes of different sequences can be synthesized simultaneously on a single 1 /2-inch chip. It takes only 80 chemical steps to construct any set of up to 400,000 probes that are up to 20 DNA bases long. It is not possible to build as many probes with as many different DNA sequences in a reasonable timeframe utilizing conventional DNA synthesis machines.

These machines build probes in series rather than in the massively parallel manner employed by Affymetrix. In this rapid DNA analysis, sample DNA is first labeled with a fluorescent tag and then added to the probe array on the Chip. If the sample finds a complementary probe on the Chip, it will bind; if it does not find a complementary strand, it will wash off the Chip (segments of DNA that have complementary bases are said themselves to be complementary: the fragments ATTTGCGC (SEQ ID NO:l) will bind, for example to a complementary fragment with the sequence TNAACGCG (SEQ ID ΝO:2). The sequence and location of each probe is known, so the scanner can determine to which probe the sample has bound.

Because the sequence of the probe on the Chip is known, the sequence of the sample DNA is also known, since its sequence will be complementary. The use of gene chips does not require the copying of messenger RNA into cDNAs and can quantitatively detect 1 messenger rNAs and cDNAs.

However, for the analysis proposed in this present invention other methods which depend on DNA machines might be quite adequate for commercialization. For example, Genotying by mass spectrometry is contemplated. As an alternative to using DNA chip technology to genotype many genes at a time, Sequenom (San Diego, CA) has adopted matrix-assisted laser desoφtion/ionization-time-of-flight mass spectrometry (MALDI-TOF) for mass genotyping of single-base pair and short tandem repeat polymoφhisms (Little et al, 1997; Braun, Little, Kδster, 1997; Braun et al, 1997). This is accomplished by the following steps. First, PCR™ amplification of the region of the polymoφhism with biotin attached to one of the primers is conducted (Jurinke et al, 1997). Second, immobilization of the amplified DNA to strepavidin beads occurs (Jurinke et al, 1997) Third, hybridization of a primer adjacent to the polymoφhism site is done (Braun, Little, Kδster, 1997). Fourth, extension with DNA polymerase past the polymoφhic site in the presence of dNTPs and ddNTPs which are not present in the deoxyform form is done.

When suitably designed according to the sequence, this results in the addition of only a few additional bases (Braun, Little, Kδster, 1997). Fifth, the DNA is then processed to remove unused nucleotides and salts. Sixth, the short primer + polymoφhic site is removed by denaturation and transferred to silicon wafers using a piezoelectric pipette (O'Donnell et al, 1997). Seventh, the mass of the primer + polymoφhic site is then determined by delayed extraction MALDI-TOF mass spectrometry (Li et al, 1996; Tang et al, 1995).

Single base pair and tandem repeat variations in sequence are easily determined by their mass. This final step is very rapid, requiring only 5 sec per assay. All of these steps are robotically automated.

This technology has the potential of performing up to 20,000 genotypings per day. This technology is rapid, extremely accurate, and adaptable to any polymoφhism. It has a significant advantage over chip technology in that it is much more accurate, can identify both single base pair and short tandem repeat polymoφhisms, and adding or removing polymoφhisms to be tested can be done in a few sec at trivial cost. Polygene Kit(s)-GeneScreen Testing Kits In terms of the various genes proposed in this application the following Table 2 details the potential gene-disorder kit based on the GENECHIP™ concept. A component of this embodiment is to first genotype the patient and then based on his/her genotype provide the appropriate cocktail. In terms of the D2 anomaly the inventors have developed an appropriate cocktail which is described herein. Table 5 summarizes the uses of compositions of the present invention as improved with specific genotypes.

Tables 6-16 list the preferred components of these compositions that are useful for the treatment of various disorders. Also, see Tables 17-19 for a brief schematic of how certain elements effect reward induced by stimulants (cocaine, etc.), opiates and sedative-hypnotics. TABLE 5 Polygenic Diagnosis and Anti-Craving Compositions: Targeted Prevention and Treatment Selected Composition of RDS Behavior (Targeted Gene (SI)/Alleles Treatment Treated Effect of Treatment Study Type Associated for Improved Response Alcotrol™ Substance Use Disorder Anti-craving, reduced anxiety, DBPC- DI (Increased frequency of Dde — special emphasis on reduced-relapse, reduced against Inpatient homozygosity of the Al allele) sedative-hypnotic abuse medical advice rates (AMA), D 2 (Taq Al, BI, exon ' haplotypes, (i.e. Alcohol, opiates, improved physical and BESS Cl) barbiturates) scores DBPC- DATl VNTR (10/10) Outpatient CNRl (homozygosity VNTR for. Selected Composition of RDS Behavior (Targeted Gene (SI)7AlleIes Treatment Treated Effect of Treatment Study Type Associated for Improved Response DBH - Taql Bl allele Alcotrol™ 2 COMT - 108 Valine allele Continued TDO2 - intron 6 (G→A) and/or (G→T) D 4 VNTR 2 or 7 o Cocotrol™ Substance Use Disorder Same as for Alcotrol™ plus DBPC- D 2 Taql Al, BI, Cl or exon ID — special emphasis on reduction of cocaine dreams.