ES2726539T3 - Method to provide monoclonal autoantibodies with desired specificity - Google Patents

Abstract

An anti-interleukin-17F (IL-17F) human monoclonal antibody, or antigen-binding fragment thereof, in which the antibody recognizes a conformational epitope in IL-17F and is capable of neutralizing the biological activity of IL-17F , and the antibody or antigen binding fragment thereof, comprises in its variable heavy chain (VH) and in its variable light chain (VL), the amino acid sequence of the VH and VL chain of SEQ ID NO: 15 and 17, respectively, or of SEQ ID NO: 23 and 25, respectively.

Description

DESCRIPTION

Method to provide monoclonal autoantibodies with desired specificity

Field of the Invention

The present invention relates to new human monoclonal antibodies, as well as fragments, derivatives and variants thereof, which recognize interleukin 17 (IL-17). Also, the present invention relates to antibodies for use in immunotherapy of autoimmune and auto-inflammatory disorders, as well as malignant tumors. More specifically, the present invention relates to monoclonal autoantibodies isolated from B lymphocytes from affected subjects with impaired tolerance and loss of central and / or peripheral autotolerance, typically due to a mutation in a gene involved in immune regulation.

Background of the invention

Inappropriate immune system responses can cause stressful symptoms in the body involved. Exaggerated immune responses against foreign substances or physical states, which generally have no significant effect on the health of an animal or a human being, can cause allergies with symptoms ranging from mild reactions, such as skin irritations, to situations that endanger life, such as an anaphylactic shock or various types of vasculitis. Immune responses against endogenous antigens can cause autoimmune disorders such as systemic lupus erythematosus, autoimmune idiopathic hemolytic anemia, pernicious anemia, type 1 diabetes mellitus, blistered skin diseases and different types of arthritis.

Immune responses occur in a coordinated manner, involving several cells and requiring communication by signaling molecules such as cytokines between the cells involved. This communication may be influenced or inhibited, for example, by the interception of the signals or blocking of the respective receivers.

Cytokines are secreted soluble proteins, peptides and glycoproteins that act as humoral regulators at concentrations of nanomolars to picomolars that behave like classical hormones in the sense that they act at the systemic level and that, in 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, that is, there is no single organ or cellular source for these mediators, since they are expressed in virtually all cells involved in innate immunity and adaptive, such as epithelial cells, macrophages, dendritic cells (CD), natural cytolytic lymphocytes (NK, natural English killer) and especially T lymphocytes, being among the most prominent, auxiliary T lymphocytes (Th, from English T helper). At this time, the subclasses of Th1, Th2, Th17 lymphocytes and induced regulatory T lymphocytes (iTreg) have been defined depending on the specific spectrum of the expressed cytokines and the respective immune responses modulated by them.

In this regard, the main products of Th1 lymphocyte cytokines are IFN-gamma, alpha lymphotoxin and interleukin 2 (IL-2). Th1 lymphocytes act as mediators in immune responses against intracellular pathogens and are responsible for some autoimmune diseases (Mosmann et al., Annu. Rev. Immunol.

7 (1989), 145-173; Paul and Seder, Cell 76 (1994), (241-251). Th2 lymphocytes mainly produce IL-4, IL-5, IL-9, IL-10, IL-13, IL-25 and anfirregulin. Th2 lymphocytes act as mediators in 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, for example, Mosmann et al. (1989) and Paul and Seder, (1994), cited above.

Some lymphocytes, such as subsets of gamma delta T lymphocytes and natural cytolytic lymphocytes (NKT) produce Th1 and Th2 cytokines. The main products of induced regulatory T lymphocytes (iTreg) are IL-10, IL-35, and TGF-beta. ITreg lymphocytes are involved in immunotolerance, in homeostasis of lymphocytes and in the regulation of immune responses.

The main cytokine products of Th17 lymphocytes are IL-17 and IL-22; Th17 lymphocytes act as mediators in immune responses against bacteria and extracellular fungi and are also involved in inflammation and autoimmune diseases such as psoriasis (Zhu and Paul, Blood 112 (2008), 1557-1569; Shevach, Immunity 25 (2006 ), (195-201) This spectrum of pathophysiological activities of cytokine production is very parallel with the subsets of gamma delta T lymphocytes.

Depending on their respective functions, cytokines can be classified into three functional categories: regulation of innate immune responses, regulation of adaptive immune responses and stimulation of hematopoiesis. Due to their pleiotropic activities within these three categories, for example, with regard to activation, proliferation, differentiation, recruitment or other physiological cellular responses, for example, the secretion of proteins characteristic of inflammation by the target cells , Have been found Alterations in cell signaling mediated by aberrantly regulated cytokine production, as a cause of many disorders associated with defective immune responses, for example, inflammation and cancer. The cytokines IL-17A and IL-17F, IL-22, IL-12, IL-23 and the subtypes of IFN-alpha and IFN-omega are central to inflammation and autoimmune diseases. Except for IL-12, IL-23 and IFN, these cytokines are expressed by the subset of Th17 effector T lymphocytes, which have well-described functions in various autoimmune and allergic disorders and in tumor immunology.

Unregulated Th17 responses are associated with chronic inflammation and serious immunopathological conditions. Studies in animal models and tissues of affected patients have confirmed the participation of the Th17 pathway in chronic inflammatory diseases such as acne vulgaris; arthritis, such as gouty arthritis, systemic lupus erythematosus (SLE), osteoarthritis, psoriatic arthritis, rheumatoid arthritis (RA); asthma; celiac disease; Crohn's disease (CD); chronic prostatitis; dermatitis (for example, atopic dermatitis); type 1 diabetes mellitus; glomerulonephritis; hypersensitivity; myocarditis; multiple sclerosis; inflammatory bowel diseases; pelvic inflammatory disease; polymyositis; psoriasis (PS); sarcoidosis; vasculitis; interstitial cystitis or inflammation caused due to reperfusion injury or transplant rejection. IL-17A induces the production of proinflammatory cytokines, chemokines and matrix metalloproteins. This causes the infiltration of inflammatory cells and the destruction of the extracellular matrix. In addition to IL-17A, the related IL-17F cytokine, which can be heterodimerized with IL-17A, has recently been implicated in the pathogenesis of RA and CD.

IL-22, a third cytokine related to Th-17 (Zenewicz and Flavell, Eur. J. Immunol. 38 (2008), 3265-3268), is a cytokine that acts primarily on epithelial cells. In the skin, it acts as a mediator in keratinocyte proliferation and epidermal hyperplasia and plays a central role in inflammatory diseases, such as psoriasis. IL-22 is a representative product of Th17 lymphocytes, other immune cells also secrete it at functionally significant levels, especially cytolytic lymphocytes (NK) that express NKp44 / NKp46 and lymphoid tissue inducing cells after stimulation with IL-23 . Interleukin-22 (IL-22) is a member of the IL-10 anti-inflammatory cytokine family that acts as a mediator in epithelial immunity. IL-22 expression is enhanced in the inflamed colon mucosa in individuals with inflammatory bowel disease. It should be noted that, for communication between immune cells, IL-22 is not necessary. It acts mainly on epithelial cells and, for example, on hepatocytes, where it favors antimicrobial defense, regeneration and protection against damage and induces acute phase reactants and some chemokines (Wolk et al., Semin. Immunopathol. 32 (2010 ), (17-31).

IL-23 is a key cytokine in the promotion of chronic inflammation, secreted by activated inflammatory cells such as macrophages and dendritic cells (for a review see Langrish et al., Immunological Reviews 202 (2004), 96-105; Ferraccioli and Zizzo , Discov. Med. 60 (2011), 413-24; and also Langrish et al., J. Exp. Med. 201 (2005), 233-40; Vaknin-Dembinsky et al., J. Neuroimmunol. 195 (2008 ), 140-145; Melis et al., Ann. Rheum. Dis. 69 (2010), (618-23). Initially, IL-23 was found to have a role in supporting lymphocyte expansion and maintenance. Th17 (Aggarwal et al., J. Biol. Chem. 278 (2003), 1910-1914; Weaver et al., Annu. Rev. Immunol. 25 (2007), (821-852). However, it was shown that had multiple effects on the immune response, including restriction of activity (intestinal accumulation) of positive regulatory T lymphocytes for Foxp3 (Barnes and Powrie, Immunity 31 (2009), 401-411; Izcue et al., Immunity 28 (200 8), 559-570; Ahern. Immunity 33 (2010), 279-88) and that induced the expression of Th17 type cytokines from sources that did not come from T lymphocytes (Buonocore et al., Nature 464 (2010), 1371-1375; Morrison et al., Immunology 133 (2011), 397-408). It contributes to inflammation, for example, in psoriasis and in inflammatory bowel diseases (see the publications cited above and Duvallet et al., Ann. Med. 43 (2011), 503-511). IL-23 is composed of two subunits, p19 and p40; The last subunit is shared with IL-12.

IL-17A, IL-22, oncostatin M, TNF-alpha and IL-1 alpha, are potent cytokines produced by immune cells and / or infiltrating keratinocytes of the skin and induce skin inflammation (Boniface et al., J. Immunol. 178 (2007), 4615-4622; Nograles et al., Br. J. Dermatol. 159 (2008), 1092-1102; Guilloteau et al., J. Immunol. 184 (2010), 5263-5270 ).

Recent therapeutic approaches for chronic inflammatory diseases, such as severe RA and PS, involve the blocking of tumor necrosis factor a (TNF-a). Although in some cases this method is extremely effective, many patients are 'non-responders'. Likewise, sustained neutralization of TNF-a can improve susceptibility to microbial infections, highlighting the need for alternative and more specific therapeutic approaches. So far, modulation of the function and / or differentiation of Th-17 lymphocytes has been more successful with monoclonal antibodies against IL-12 p40, which target both IL-12 (a heterodimer of p35 and p40) and a IL-23 (a heterodimer of p19 and p40, a growth factor for Th17 lymphocytes). These antibodies, ustekinumab and ABT-874, which target IL-12 p40, have been successful in chronic plaque psoriasis, moderate to severe, and in CD (Miossec et al., N. Engl. J. Med 361 (2009), 888-898, Steinman, Nat. Immunol. 11 (2010), 41 44).

The most feasible way to control the biological effects of Th17 lymphocytes and cells with related properties, for example, IL-17 producing gamma delta T cells, would be to direct them and a selection of the effector cytokines they produce. This new approach would allow a blockade of IL-22 on PS, and IL-17A (e IL17F) in RA and EC, leaving the other non-pathogenic groups intact to fight pathogens and, as in the case of IL-22, to exercise their tissue repair and antimicrobial functions. Also, in some initial responders, the efficacy of anti-TNF pharmacotherapy decreases over time. Therefore, severe cases that do not respond to monotherapy may require combination therapy with two or more biological drugs. Monoclonal antibodies against IL-17A have been developed for clinical application. Phase 2 clinical trials of a monoclonal antibody against IL-17A (AIN457) for PS, EC and psoriatic arthritis are under way. So far, IL-22 inhibitors have been tested only in preclinical models of autoimmune diseases (Miossec et al., N. Engl. J. Med. 361 (2009), 888-898, Steinman, Nat. Immunol. 11 ( 2010), 41-44).

Due to immune responses against foreign antibodies, such as mouse antibodies in humans (HAMA response; Schroff et al., Cancer Res. 45 (1985), 879-885; Shawler et al., J. Immunol. 135 (1985) , 1530 1535), mainly humanized versions of antibodies are used in current 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 obtaining such antibodies was to transplant the complementarity determining regions (CDR ) into a completely human structure, a process known as antibody humanization (Jones et al., Nature 321 (1986), 522- 525). This approach is often complicated by the fact that the mouse CDR does not easily transfer to a human variable domain structure, resulting in a lower affinity of the humanized antibody over its precursor murine antibody. Therefore, additional and elaborate mutagenesis experiments are often required to increase the affinity of antibodies designed in this way. Another approach to obtain humanized antibodies is to immunize mice whose innate antibody genes have had to be replaced with human antibody genes and isolate the antibodies produced by these animals. However, this method still requires immunization with an antigen, which is not possible with all antigens due to the toxicity of some of them. Also, this method is limited to the production of transgenic mice of a specific strain. Another method is to use human antibody libraries, such as phage display, as described, for example, in international application WO 2005/007699, for the generation of antibodies specific for IL-13. In this case, by inserting a human antibody gene into the phage population bacteriophages are designed that have human scFv / Fab fragments on their surface. Unfortunately, this method has several disadvantages, including the limitation of protein sequence size for polyvalent presentation, the need for protein secretion, i.e. scFv / Fab fragments of antibodies, bacteria, size limits from the library, the limited number of possible antibodies produced and tested, a reduced proportion of antibodies with somatic hypermutations produced by natural immunization and that all phage-encoded proteins are fusion proteins, which may limit the activity or accessibility for binding of some proteins. Similarly, European patent application EP 0 616 640 A1 describes the production of autoantibodies from repertoires of antibody segments presented in phages. In this regard, phage libraries (phage libraries) are generated from non-immunized human beings (see for example, Example 1 on page 16, lines 43-51; Example 2 on page 17, paragraph [0158], lines 57-58). However, the methods described in this patent application also have the general disadvantages, mentioned above, of antibodies generated from phage libraries, compared to antibodies produced and matured in a mammal, that is, in the human body.

WO2009 / 136286 discloses completely human antibodies against IL-17F that were obtained by immunization of transgenic mice expressing human antibodies with human IL-17F. Exemplary 15E6FK antibody recognizes and neutralizes all IL-17F, IL-17A and the IL-17A / F heterodimer. In view of the above, there is still a need for new and additional compounds such as high specificity binding molecules, which are tolerable in humans, either for monotherapy or combinatorial approaches.

The embodiments of the present invention, as characterized in the claims, are disclosed in the description and illustrated in the examples below, solve this problem.

Summary of the invention

The present invention relates to embodiments as characterized in the claims and, in general, to antibodies of affected human beings with impaired tolerance and loss of central and / or peripheral autotolerance, which may be due to or associated with a genesis of destabilized or poorly regulated self-tolerance, preferably caused by a monogenic autoimmune disorder. A particularly suitable source for autoantibodies in accordance with the present invention are humans who have a disorder associated with a mutation in the AIRE ( Autoimmune Regulator) gene such as autoimmune polyocrinopathy syndrome type 1 ( APS1, from the English autoimmune poliendocrinopathy syndrome type 1) (Peterson et al., Nat. Rev. Immunol. 8 (2008), 948-957).

The present invention is generally based on the surprising findings that patients with type 1 autoimmune polendocrinopathy syndrome (APS1) provide a source of monoclonal autoantibodies against proteins that, alone or in combination, are responsible for and / or are associated with autoimmune or auto-inflammatory diseases in which the respective cytokines or other molecules, as indicated above, are strongly involved in the initiation and maintenance of pathogenic immune responses, such as systemic lupus erythematosus (SLE), arthritis, type 1 diabetes mellitus (T1DM , from English Type 1 Diabetes mellitus) or others, as indicated in detail above and later in this document.

The present invention relates to high affinity neutralizing monoclonal antibodies against IL-17F. Therefore, human monoclonal antibodies (mAb or MAB ) against IL-17F are provided, which will be described in detail below, from the naturally matured humoral immune response of a predetermined subject, which is considered to be they are safe and effective therapeutic agents for disorders in which cytokines are involved.

In particular, anti-IL-17F antibodies are provided, as well as antigen binding fragments thereof, which demonstrate immunological binding characteristics and / or biological properties as indicated for the antibodies illustrated in the Examples. When present, the term "immunological binding characteristics" or other binding characteristics of an antibody with an antigen, in all its grammatical forms, refers to the specificity, affinity, cross-reactivity, and other binding characteristics of an antibody.

Naturally, the present invention extends to antibody producing cell lines and recombinant cells. The present invention also relates to pharmaceutical compositions, diagnostic assays and kits comprising isolated antibodies according to the present invention and to therapeutic methods based on them.

Brief description of the drawings

Figure 1: Neutralizing capabilities in vivo of the recombinant antibodies of the present invention. TO:

Neutralizing capabilities against the IL17A / IL-17F heterodimer. The IC50 of the recombinant antibody 24D3 is 6 ng / ml, the antibody 17E3 being 12 ng / ml, using 10 ng / ml of IL17A / IL-17F heterodimer. Recombinant 9A2 antibody is not neutralizing at the concentrations tested. B: Neutralizing capabilities against IL17F. The IC50 of the 24D3 recombinant antibody is 12 ng / ml, the 17E3 antibody being 15 ng / ml and the 9A2 antibody 45 ng / ml using 10 ng / ml of IL17F.

Figure 2: Amino acid sequences of the variable region, ie, heavy chain and kappa / lambda light chain (VH, VL) of the IL-17A and IL17F specific human antibodies of the present invention. A: IL-17F specific 9A2 antibody: IgG1, kappa (VH3; G1m17; VK1, Km3). B:

IL-17F and IL-17A specific 17E3 antibody: IgG1, kappa (VH1,

Figure 3: G1m17; VK1, Km3). C: IL-17F specific 24D3 antibody: IgG1, lambda (VH3, G1m17; VL3). The structural regions (FR ) and the complementarity determining regions (CDRs) are indicated underlining the CDRs. Due to the cloning strategy, the amino acid sequence at the N-terminus of the heavy chain and light chain may possibly contain primer-induced alterations in FR1, which however do not substantially affect the biological activity of the antibody. To provide a consensus human antibody, the nucleotide and amino acid sequences of the original clone are aligned and adjusted according to the relevant sequences of the human germline variable region of the databases; see, for example, Vbase (http://vbase.mrc-cpe.cam.ac.uk) offered by the MRC Center for Protein Engineering (Cambridge, United Kingdom). In vivo study of psoriasiform lesion with anti-IL22 and anti-IL17F imiquimod. Experimental protocol and treatment schedule with antibodies and imiquimod (IMQ) of C57BL / 6J mice. Shaving age 9 weeks of life, Control not treated DOB: at 10 weeks. A: Tables indicating the composition of the treatment of the four groups of animals. B: Amounts of the control and anti-IL22 antibody applied to animals. C:

Experimental schedule The respective experiments were performed with the 24D3 anti-IL-17F antibody. Vas = Vaseline

Figure 4: Combined results of two independent CytoEar experiments - measurements of ear thickness over time show a reduction in the ear inflammation induced phenotype by IL17F, by injections of antibody 24D3, aIL-17, exemplary of the present invention. (A) Outline of the results. (B) Control experiments - PBS injection. (C) Induction of inflammation by injections of IL17A. (D) Induction of inflammation by injections of IL17F - reduced induction due to injections of 24D3. (E) shows combined graphs of Figs. B and C. (F) shows combined graphs of Figs. B and D. A significant difference (Day 8) in ear thickness was observed between groups C and F in mice that received IL-17F and treated with IgG or 24D3 (D, F). (D and F) A ⁿ bidirectional OVA ns from Day 0 to Day 7 ** p <0.01 on Day 8. Figures GL show the data of the corresponding figures AF after normalization to PBS. (J and L) Bidirectional ANOVA: ns on Day 7; * p <0.1 on Days 3-5; ** p <0.01 on Day 8; *** p <0.001 on Days 1 and 2.

Figure 5: CytoEar Experiment - Control of the body weight of the animals tested to determine the reduction of the ear inflammation phenotype by treatment with the exemplary antibody 24D3 anti-IL-17 of the present invention. (A) Scheme of experimental results. (B) Preliminary data suggest a possible treatment effect based on a difference in weight observed in groups C and F.

Figure 6: Epitopic mapping. Differential binding of the MAB IL-17 copies of the present invention to different binding sites. Cross-competition experiments show that antibodies 17E3 and 24D3, anti-IL-17, compete with each other but not against antibody 9A2, indicating a different binding site for the latter. The 9A5 antibody is a MAGEA3 specific antibody that is not reactive with IL-17 and is used herein as a negative control.

Figure 7: Results of PepStar ™ analysis of the binding regions of the MABs to their respective IL17F antigens. The 20-unit peptide binding (15 amino acid overlap) applied in the microarray covering the IL-17F, including all known variants, is shown, showing the full-length IL-17F in the last column. The low signal intensity observed with respect to binding with the full length antigen does not necessarily correlate with an absence of binding, since the low signal intensity or the absence of signal can also occur due to the orientation and use of surface lysines to immobilize the antigen. However, the observed binding to full-length IL-17F suggests a preferential binding of exemplary antibodies 24D3 (A), 9A2 (B) and 17E3 (C) to conformational epitopes. The X-axis represents, from left to right: Peptides numbered from 1 to 23 and full-length IL-17F

Figure 8: Results of ProteOn XPR36 based analysis of antibody affinities. Injection of IL-22 and IL-17F, respectively 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 (current buffer only). In front of identifiers A1-A6 are the prefixes L1 to L6 (L1A1, etc.). The data was referenced using the regions between the applications of the detector plate.

Detailed description of the invention

The present invention relates, in general, to anti-IL17 antibodies as characterized in the claims, which were originally obtained by a method of isolating monoclonal autoantibodies with a desired antigenic specificity, in which a B lymphocyte, expressing the monoclonal antibody, is isolated from a sample obtained from a mammal that is affected with a tolerance or loss of impaired central and / or peripheral autotolerance, which is a disorder typically associated with a genesis of destabilized or poorly regulated autotolerance; see example 1.

As illustrated in the examples, the autoantibodies of the present invention preferably recognize only the human antigen or at least preferentially the corresponding antigen of other species such as mice. Therefore, in one embodiment of the present invention, the antibody is specific for the human antigen and preferably does not substantially recognize the corresponding antigen of another species, such as one of murine origin, for example mice in the case of a human antibody. Binding characteristics such as the specificity and affinity of the antibodies of the present invention have been tested in several experimental assays as described in detail in Examples 8 and 9. From experimental data in vivo additional indications could be obtained in the therapeutic potential assays of the exemplary antibodies of the present invention, for example, in inflammation of the skin of induced psoriasis type (Example 6) and in inflammation of the induced ear (Example 7). In this regard, the specificity against human IL-17F of the exemplary antibody 24D3, anti-IL-17, of the present invention, could be confirmed by its therapeutic effect observed in ear inflammation experiments induced by injections of human IL-17F. and described in Example 7, and by the absence of a significant effect on inflammation of the psoriasis-like skin induced by IMQ, as shown in Example 6 (no significant effect on inflammation mediated by murine IL-17F) . In comparison, an exemplary anti-IL-22 mouse cross-reactivity antibody has shown a therapeutic effect on it, reducing the clinical symptoms of psoriasiform lesions induced by IMQ.

As has been further demonstrated for the antibodies of the present invention, they are capable of neutralizing the biological activity of their target protein. In this context, the term "neutralizer" means that the antibody of the present invention is capable of intervening with the biological activity of its target protein in a biochemical or cell-based assay, as can be assessed by performing the respective assay in the presence of the target antibody. of the present invention, wherein the biological activity of the target protein is reduced while increasing the level of the antibody of the present invention tested in comparison to the biological activity of the protein without the presence of the antibody of the present invention and in the presence of a compound, for example, a control antibody that is known to not affect the biological activity of the target protein. Said biochemical and in vitro assay can also be performed using a reference antibody that is known to be able to neutralize the biological activity of the target protein as demonstrated for the anti-IL-17 antibody of the present invention and subjecting the antibody. candidate for the test sample, in which an additive neutralizing effect resulting from the combined activity of the reference and candidate antibody can be observed or a competition of the candidate antibody and the reference antibody that can be determined by marking is observed any of the antibodies. Therefore, the antibody of the present invention obtained by the method disclosed herein is capable of neutralizing the biological activity of IL-17.

As demonstrated in the accompanying Examples 4 and 6, binding molecules, i.e. antibodies, have been identified and cloned that show particularly high in vitro neutralizing activity with low inhibitory concentrations (IC50) for the cytokine IL-17F subjected to test. In this regard, antibodies with high affinity are provided for their respective target molecules, for example IL-17, which show an ED50 at concentrations below 100 ng / ml, preferably below 10 ng / ml. For more details regarding the binding affinity of the antibodies of the present invention, see, for example, the section "Binding characteristics" below.

Examples of said binding molecules are indicated in the present invention, that is, antibodies and binding fragments thereof, characterized in that they comprise, in their variable region, that is, binding domain, at least V ^h and V ^l of the variable region comprising the amino acid sequence depicted in Figure 2 of (V ^h ) (SEQ ID NO: 15, 23) and (V ^l ) (SEQ ID NO: 17, 25) - see exemplary sequences of the CDR underlined in Fig. 2.

Therefore, this refers generally to human antibodies and antigen-binding fragments thereof, in which the antibody recognizes a conformational epitope in IL-17F and is capable of neutralizing the biological activity of IL-17F, and the antibody or antigen-binding fragment thereof, comprises in its heavy variable chain (VH, heavy variable) and in its light variable chain (VL, light variable), the amino acid sequence of the V ^h and VL chain of the S ^e Q ⁱ D NO: 15 and 17, respectively, or SEQ ID NO: 23 and 25, respectively.

To provide fully human antibodies and Fab fragments native thereto, the human antibody of the present invention preferably further comprises a constant region C ^h and / or C ¹ comprising an amino acid sequence selected from the amino acid sequences C ^h and C ¹ set forth in Table 1 (SEQ ID NO: 11, 13, 19, 21,27, 29) or an amino acid sequence derived therefrom with an identity of at least 60%.

In the case of a derived sequence, said sequence shows an identity of at least 60%, more preferably (in the following order) an identity of at least 70%, of at least 75%, of at least 80%, of at least 85 %, of at least 90%, and more preferably of 95%, of at least 96-99%, or even 100%, with a group sequence consisting of the sequences mentioned above and identified in the Sequence Listing. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of holes, and the length of each hole, which needs to be entered to obtain an optimal alignment of the two sequences. Using a mathematical algorithm, well known by the skilled artisan, the sequence comparison and the determination of the percentage of identity between two sequences can be performed. The identities referred to in this document will be determined using the BLAST programs as mentioned later in this document.

As explained above, fully human antibodies are provided for therapeutic applications. An antigen-binding fragment of the antibody can be, for example, a single chain Fv fragment (scFv ), an F (ab ') fragment, an F (ab) fragment or an F (ab') fragment ² . As mentioned in a preferred embodiment, the present invention relates to antibodies that are substantially completely human, preferably IgG that includes at least the constant heavy chain I (C ^h 1) and the corresponding light chain of the constant region, that is, y-1, Y-2, Y-3 or y-4 in combination with lambda or kappa. In a particular preferred embodiment, the nucleotide and amino acid sequences of those constant regions isolated from the target antibodies illustrated in the Examples are used as shown in Table 1 below and in SEQ ID NO: 10-13, 18 -21 and 26-29.

In accordance with the foregoing, in one embodiment, the present invention also provides a polynucleotide encoding at least the variable region of an immunoglobulin chain of the antibody or antigen-binding fragment of the present invention. Normally, 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 said antibody. The variable and constant regions of the antibodies are described below in more detail in the "IgG structure" section. 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 ^{1 region} of an antibody of the present invention as depicted in the Table. 1 below. In this regard, one skilled in the art will readily appreciate that polynucleotides encoding at least the variable domain of the light and / or heavy chain can encode the variable domain of any of the immunoglobulin chains or only one of them.

Table 1: Nucleotide and amino acid sequences of the variable and constant regions (V ^h , V ^l , C ^h ), dOL) of the IL-17A and IL17F specific antibodies of the present invention.

The person skilled in the art will readily appreciate that the variable domain of the antibody having the variable domain described above can be used for the construction of other polypeptides or antibodies of desired specificity and biological function. 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, for example, as described in European patent applications EP 0451 216 A1 and EP 0549581 A1. Also, the person skilled in the art knows that binding affinity can be improved by making amino acid substitutions in the CDRs or in the hypervariable loops (Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917) that overlap partially with the CDRs defined by Kabat.

The polynucleotide of the invention encoding the antibody described above may be, for example, synthetically produced DNA, cDNA, RNA or 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 encoding the variable region of the other immunoglobulin chain of said antibody. Said vectors may comprise additional genes such as marker genes that allow the selection of said vector in a suitable host cell and under suitable conditions.

Preferably, the polynucleotide of the invention is operably linked to expression control sequences that allow expression in prokaryotic or eukaryotic cells. The expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA. Those skilled in the art know regulatory elements that guarantee expression in eukaryotic cells, preferably in mammalian cells. They usually comprise regulatory sequences that guarantee the initiation of transcription and, optionally, poly-A signals that guarantee termination of transcription and stabilization of transcription. Additional regulatory elements may include transcriptional and translational enhancers, and / or naturally occurring heterologous or associated promoter regions.

In this regard, one skilled in the art will readily appreciate that polynucleotides that encode at least the variable domain of the light and / or heavy chain can encode the variable domains of both immunoglobulin chains or only one chain.

In the same way, for expression, said polynucleotides can be under the control of the same promoter or can be controlled separately. Possible regulatory elements that allow expression in prokaryotic host cells comprise, for example, the P ^l , lac, trp or tac promoters in E. coli, and are examples of regulatory elements that allow expression in eukaryotic host cells the AOX1 or GAL1 promoter in yeast or promoters of CMV, SV40, RSV, CMV enhancer, SV40 enhancer or globin intron in mammalian cells and other animal cells.

In addition to the elements that are responsible for the initiation of transcription, said regulatory elements may also comprise transcription termination signals, such as the SV40 poly A site or the poly A tk site, downstream of the polynucleotide. Also, depending on the expression system used, to the coding sequence of the polynucleotide of the invention, leader sequences capable of directing the polypeptide to a cellular compartment or secreting it in the medium can be added, said sequences being well known in the art. The sequence, or sequences, leader are assembled in the appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing the secretion of translated protein, or a part thereof, in the periplasmic space or extracellular medium Optionally, the heterologous sequence can encode a fusion protein that includes a C or N-terminal identification peptide that confers desired characteristics, for example, simplified stabilization or purification of the expressed recombinant product. In this context, suitable expression vectors such as Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), or pSPORT1 (GIBCO BRL) are known in the art.

Preferably, the expression control sequences will be eukaryotic promoter systems into vectors capable of transforming or transfecting eukaryotic host cells, but control sequences can also be used for prokaryotic hosts. 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, for the collection and purification of the light chains, heavy chains , light / heavy chain dimers or intact immunoglobulin antibodies, binding fragments or other forms of immunoglobulin that may appear below; see, Beychok, Cells of Immunoglobulin Synthesis, Academic Press, N.Y., (1979).

Also, the present invention relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages, conventionally used in genetic engineering, comprising 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 encoding 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, variolovaccine viruses, adeno-associated viruses, herpes viruses or bovine papillomavirus, can be used for the delivery of polynucleotides or vectors of the invention in the target cell population. Methods 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 in Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley-Interscience, N. Y. 1994). 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 (for example, the domain, or domains, heavy and / or light chain variable of the sequences encoding the immunoglobulin chains and the expression control sequences) can be transferred to the host cell by well known methods, which vary depending on the type of cell host. For example, normally, transfection with calcium chloride is used in prokaryotic cells, while for the transformation of other cell hosts, calcium phosphate treatment or electroporation can be used; see Sambrook, cited above.

With respect to the foregoing, the present invention also relates to a host cell comprising said polynucleotide or vector. Said host cell can be a prokaryotic or eukaryotic cell. The polynucleotide or vector of the invention that is present in the host cell may be integrated into the genome of the host cell or may be maintained extrachromosomal. 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 the production of the antibodies of the present invention are described in more detail below in the section "Host cells".

Binding molecules, antibodies or fragments thereof, can be used as a therapeutic product. However, in one embodiment, the antibody or antigen-binding fragment provided by the present invention is detectably labeled or bound 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. The labeled antibodies or antigen-binding fragments of the present invention can be used to detect specific targets in vivo or in vitro, including "immunochemical / immunolabel" type assays in vitro. These techniques can be used in vivo in a manner that is similar to the imaging techniques used in nuclear medicine to detect tissues, cells or other material that expresses the antigen of interest. Further on in the "markers and diagnostics" section, markers, their use in diagnostics and their coupling to the binding molecules of the present invention are described in more detail.

On the other hand, the present invention also relates to compositions comprising the antibody or antigen-binding fragment, the polynucleotide, the vector or the cell, mentioned above. In one embodiment, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, routes of administration and dosage regimen can be taken from the corresponding literature known to the person skilled in the art and are also described in more detail later in the sections "Pharmaceutical vehicles" and "Drug regimen. dosage".

In addition to cell-based and biochemical in vitro assays, the therapeutic utility of the antibody of the The present invention can be validated in appropriate animal models, such as for psoraris, IBD, candidiasis; see examples 5 and 6, and for the model of inflammatory bowel disease IBD (DSS-induced colitis (sodium dextran sulfate)) see Wirtz et al., Nature Protocols 2 (2007), 541-546; for a Psoriasis model (Imiquimod) see van der Fits et al, J Immunol 182 (2009), 5836-5845; For a multiple sclerosis model (experimental autoimmune encephalomyelitis (EAE)) see Miller, SD and Karpus, WJ, Current Protocols in Immunology. 78 (2007), 15.1.1-15.1.18 and the website http://hookelabs.com/products/eae/; for a model of rheumatoid arthritis (collagen-induced arthritis) see 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 anti-inflammatory drugs (NSAIDs), corticosteroids, antihistamines and combinations thereof. In addition or as an alternative, in a further embodiment, the pharmaceutical composition further comprises an additional agent useful for treating a disease related to inflammation, selected from the group consisting of immunosuppressive and anti-inflammatory or "anti-rheumatic" drugs.

In another embodiment, the composition is a diagnostic composition and further comprises reagents conventionally used in immunological or nucleic acid based diagnostic methods.

Likewise, the present invention provides the aforementioned antibody or antigen-binding fragment, for use in the treatment or in the prevention of the progression of an inflammatory or autoimmune disorder; for the improvement of symptoms associated with inflammation or an autoimmune disorder; to diagnose or examine a subject to determine the presence or risk of a subject developing inflammation or an autoimmune disorder.

Several routes of application can be used in this regard. In one embodiment of the present invention, the aforementioned antibody or antigen-binding fragment is provided, which is designed to be administered intravenously, intramuscularly, subcutaneously, intraperitoneally, intranasally, parenterally, or as an aerosol.

Due to the multitude of suitable molecules in the treatment of, for example, disorders associated with inflammation, the present invention also provides various methods of treating such disorders. In one embodiment, a method is provided for treating a disorder associated with an inflammation or an autoimmune disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of the aforementioned antibody or antigen-binding fragment.

In one embodiment of this method, said disorder is selected from the group consisting of common acne; arthritis, such as gouty arthritis, systemic lupus erythematosus (SLE), osteoarthritis, psoriatic arthritis, rheumatoid arthritis; asthma; celiac disease; chronic prostatitis; dermatitis; type 1 diabetes mellitus; glomerulonephritis; hypersensitivity; myocarditis; multiple sclerosis; inflammatory bowel diseases; pelvic inflammatory disease; polymyositis; psoriasis; sarcoidosis; vasculitis; APS1; interstitial cystitis or inflammation caused due to reperfusion injury or transplant rejection; see previous lines.

Treatment methods based on the use of a single monoclonal antibody specific for an epitope of a particular antigen, which is related or that causes a disease, may present several deficiencies. For example, the difficulties, and probably the inefficiency of the treatment, may be due to the multiplicity of the pathogenic mechanisms that cause a specific disorder, which requires the simultaneous addressing of several antigens. Likewise, the intrinsic diversity of the patient population must be taken into account in relation to, for example, polymorphism, heterogeneity of glycosylation or mild denaturation of a given antigen, either in different patients or in a patient, which can lead, at least, to a decrease in the binding efficiency of the monoclonal antibody used. Some of these deficiencies can be avoided, for example, by pre-treatment scans to determine if the antigen is, from an immunological point of view, relevant to the patients to whom it is intended to be treated and if there are epitope changes in particular patients. . However, such scans are often omitted due to the urgency of the treatment or the cost limitations. Therefore, the present invention further relates to methods based on the application of more than one type of a binding molecule at a time to a patient. These binding molecules can specifically bind to an antigen in different epitopes or each of the applied binding molecules specifically binds to another antigen related to the disease. In the event that the binding molecules of the present invention are directed (specifically bound) to an antigen, their binding specificity is directed to different epitopes of said antigen. The use of such cocktails is contemplated, in particular, for the treatment of patients suffering from autoimmune disorders, such as APS1, who, in view of the presence of autoantibodies against approximately 3000 endogenous antigens, are often not susceptible to receiving monotherapy with a particular antibody. In such cases, combination therapy with two or more monoclonal antibodies of the present invention with the same or different antigen specificity is expected to achieve at least some relief of symptoms.

Therefore, in one embodiment, an additional composition is provided comprising a cocktail consisting essentially in the two antibodies or antigen binding fragments thereof of the present invention that specifically bind to IL-17F.

In another embodiment, the present invention relates to a kit for the diagnosis of a disorder that is accompanied by the presence of a protein associated with the disorder as defined in any one of the claimed peptides, said kit comprising the antibody or binding fragment to antigen, polynucleotide, vector or cell mentioned above, optionally with reagents and / or instructions for use. Associated with the kits of the present invention, for example, within a package comprising the kit, there may be a notice in the form prescribed by a government agency that regulates the manufacture, use or sale of pharmaceutical agents or biological products, whose notice reflects the approval by the agency of the manufacture, use or sale for administration in humans. In addition, or as an alternative, the kit comprises reagents and / or instructions for use in appropriate diagnostic tests. The compositions, that is, the kits of the present invention, are, of course, particularly suitable for the diagnosis, prevention and treatment of a disorder that is accompanied by the presence of an inflammation-associated antigen defined above, particularly applicable for the treatment of diseases such as those mentioned above.

In another embodiment, the present invention relates to a diagnostic composition comprising any one of the antibodies, antigen binding fragments, polynucleotides, vectors or cells of the invention described above and optionally suitable for detection, such as reagents. conventionally used in immunological diagnostic methods or based on nucleic acids. The antibodies of the invention are, for example, suitable for use in immunoassays in which they can be used in liquid phase or attached to a solid phase vehicle. Examples of immunoassays that can use the antibody of the invention are competitive and non-competitive immunoassays in direct or indirect format. Examples of such immunoassays are radioimmunoassay (RIA), sandwich type (immunometric assay), flow cytometry and Western blotting assay. The antigens and antibodies of the invention can be bound to many different vehicles and used to isolate cells specifically bound to them. Examples of well-known vehicles include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agarose and magnetite. For the purposes of the present invention, the nature of the vehicle can be soluble or insoluble. There are many different markers and labeling methods known to those of ordinary skill in the art. Examples of the types of markers that can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds and bioluminescent compounds; see also the embodiments explained above in this document.

In this context, the present invention also relates to means designed specifically for this purpose. For example, a matrix based on proteins or antibodies can be used, which, for example, is loaded with antigens derived from the protein associated with the mentioned disorder and which contains the inflammation-associated antigen, to detect autoantibodies that may be present in patients suffering from autoimmune diseases, in particular, arthritis, type I diabetes or APECED / APS1, or with antibodies or equivalent antigen-binding molecules of the present invention that specifically recognize any one of those inflammation associated antigens. The design of the 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 achievements

Unless otherwise indicated, a term as used herein has the definition provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised in 2000 and reprinted in 2003, ISBN 0198506732.

It should be noted that the term "one" entity refers to one or more of that entity; for example, "an antibody" is understood to represent one or more antibodies. Therefore, the terms "a", "one (a)", one (a) or more ", and" at least one (a) "may be used interchangeably in this document.

The term "neutralizing" and "neutralizing antibody", respectively, is used as usual in the art in that it means that an antibody reduces or suppresses at least some biological activity of an antigen or a living microorganism. For example, an anti-IL-17F antibody of the present invention is a neutralizing antibody, if, in adequate amounts, it suppresses the activity of cytokine, for example, in an assay such as that described in the Examples section. Neutralization is commonly defined as the 50% inhibitory concentration (IC 50) and can be statistically evaluated based on the area under the curve (ABC) of the neutralization titration. For tumor diseases, neutralization can be evaluated, for example, by determining tumor growth inhibition. For example, it is known that for tumorigenesis interleukin-6 secretion induced by oncogenic grafts is required and that anti-IL-6 antibodies can be used for the corresponding cancer therapy; see, for example, Ancrile et al., "Genes and development" 21 (2007), 1714-1719. The neutralization of an infectious agent, such as a virus, is defined as the loss of infectivity through the reaction of the virus or its target cell, for example, blocking the virus receptor that results in the reduction of infected cells that can be readily determined in appropriate cell-based assays known to those skilled in the art. The person skilled in the art can easily determine other biological activities that can be "neutralized" by the antibody of the present invention.

The terms "fragment," "variant," "derivative," and "analog," when referring to antibodies or antibody polypeptides of the present invention, include any polypeptide that retains at least some of the antigen-binding properties of the molecule. binding, antibody or corresponding native polypeptide. The polypeptide fragments of the present invention include proteolytic fragments, as well as deletion fragments, in addition to the specific antibody fragments explained elsewhere in this document. Antibody variants and antibody polypeptides of the present invention include fragments such as those described above, and also polypeptides with modified amino acid sequences due to amino acid substitutions, deletions and insertions. Variants can occur naturally or unnaturally. Unnatural variants can be produced using mutagenesis techniques known in the art. Variant polypeptides may comprise substitutions, deletions or additions of conservative or non-conservative amino acids. Derivatives of binding molecules of the present invention, for example, antibodies and antibody polypeptides of the present invention, are polypeptides that have been modified to have additional characteristics that are not found in the native polypeptide. Examples include fusion proteins. Here, variant polypeptides may also be referred to as "polypeptide analogs." As used herein, a "derivative" of a binding molecule or a fragment thereof, an antibody or an antibody polypeptide, refers to a polypeptide in question having one or more chemically derivatized moieties by the reaction of a functional side group. Also included as "derivatives" are those peptides that contain one or more naturally occurring amino acid derivatives of the twenty conventional amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted by serine; and the ornithine can be replaced by lysine.

Polynucleotides:

The term "polynucleotide" is intended to encompass a single nucleic acid as well as several nucleic acids, and refers to an isolated nucleic acid molecule or construct, for example, messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or an unconventional bond (for example, an amide bond, such as that found in peptidonucleic acids (APN). The term "nucleic acid" refers to any one or more segments of nucleic acid, for example, DNA or RNA fragments, present in a polynucleotide "Nucleic acid or" isolated "polynucleotide means a nucleic acid molecule, DNA or RNA, which has been extracted from its natural environment. For purposes of the present invention, a recombinant polynucleotide contained in a vector encoding an antibody is considered to be isolated.Additional examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or polynucleotides in solution (partially or substantially) Purified RNA molecules isolated include RNA transcripts in vivo or in vitro polynucleotide s of the present invention. The isolated polynucleotides or nucleic acids according to the present invention further include said synthetically produced molecules. In addition, a polynucleotide or a nucleic acid can be or can include a regulatory element, such as a promoter, a ribosome binding site or a transcription terminator.

As used herein, a "coding region" is a part of nucleic acid consisting of codons translated into amino acids. Although a "termination codon" (TAG, TGA or TAA) does not translate into an amino acid, said codon can be considered part of a coding region, but any flanking sequence, for example, promoters, ribosome binding sites, terminators Transcriptional, introns, and the like are not part of a coding region. In a single polynucleotide construct, there may be two or more coding regions of the present invention, for example, in a single vector or in different polynucleotide constructs, for example, in different (different) vectors. Likewise, any vector may contain a single coding region, or it may comprise two or more coding regions, for example, a single vector may independently 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, can encode heterologous coding regions, whether fused or not fused to a nucleic acid encoding a binding molecule, an antibody, or fragment, a variant or derivatives 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 that it is DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide can usually include a promoter and / or other transcription or translation control elements operatively associated with one or more coding regions. An operative association is when a coding region for a gene product, for example, a polypeptide, is associated with one or more regulatory sequences, such that they place the expression of the gene product under the influence or control of the sequence (s). (s) regulator (s). Two DNA fragments (such as a polypeptide coding region and a promoter associated with it) are "operatively associated" or "operably linked" if induction of promoter function results in transcription of mRNA encoding the gene product desired and if the nature of the junction between the two DNA fragments does not interfere with the ability of the expression of the regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Therefore, a promoter region will be operatively associated with a nucleic acid encoding a polypeptide if the promoter is capable of transcribing that nucleic acid. The promoter can be a cell-specific promoter that directs substantial DNA transcription only in predetermined cells. Other transcription control elements, apart from a promoter, for example, enhancers, operators, repressors and transcription termination signals, can be operatively associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other regions of transcription control are disclosed herein.

Those skilled in the art know a variety of transcription control regions. These include, without limitation, transcription control regions that act in vertebrate cells, such as, but not limited to, cytomegalovirus promoter and enhancer segments (the immediate early promoter, along with intron-A), simian virus 40 (the early promoter) and retrovirus (such as, Rous sarcoma virus). Other regions of transcription control include those derived from vertebrate genes, such as actin, heat shock protein, bovine growth hormone and rabbit p 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 (eg, interferon or interleukin-inducible promoters).

Similarly, those skilled in the art know a variety of translation control elements. These include, but are not limited to, ribosome binding sites, translation start and stop codons, and picornavirus derived elements (particularly an internal ribosome entry site , or IRES ( internal ribosome entry site), also called CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA), small hairpin RNA (hRNA), small interfering RNA (siRNA) or any other RNA product.

The polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions encoding signal peptides or secretors, 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 a secretory leader sequence that is cleaved from the mature protein once the export of the growing protein chain through the reticulum has begun rough endoplasmic. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminal end of the polypeptide, which is cleaved from the full or "full length" polypeptide, to produce a secreted or "mature" polypeptide. In certain embodiments, the native signal peptide is used, for example, an immunoglobulin heavy chain or light chain signal peptide, or a functional derivative of that sequence, which retains the ability to direct secretion of the polypeptide that is operatively associated with is. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can be used. For example, the leader sequence of wild type can be replaced by the leader sequence of tissue plasminogen activator (TPA, the English tissue plasminogen activator) human or p-glucuronidase mouse.

Expression:

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 attenuation, as well as, transient expression and stable expression. This includes, without limitation, gene transcription in messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (hRNA), small interference RNA (siRNA) or any other RNA product, and translation of RNA said mRNA in polypeptide (s). If the desired final product is a biochemical, the expression includes the creation of that biochemical and any of its precursors. The expression of a gene produces a "gene product." As used herein, a gene product may be a nucleic acid, for example, a small interference RNA (siRNA), a messenger RNA produced by the transcription of a gene, or a polypeptide that is translated from a transcription. The gene products described herein further include nucleic acids with posttranscriptional modifications, for example, polyadenylation, or polypeptides with posttranslational modifications, for example, methylation, glycosylation, lipid addition, association with other protein subunits, proteolytic cleavage, and modifications. Similar.

A variety of expression / host vector systems that contain and express polynucleotide sequences can be used. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophages, plasmids, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (eg, baculovirus); plant cell systems transformed with virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (eg, Ti plasmids or pBR322); or animal cell systems.

To express the peptide, polypeptide or fusion protein (hereinafter referred to as "product" herein) in a host cell, a procedure such as the following can be used. A restriction fragment containing a DNA sequence encoding said product can be cloned into an appropriate recombinant plasmid containing an origin of replication that acts in the host cell and an appropriate selectable marker. The plasmid can include a promoter for inducible product expression (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 can be introduced into the host cell, for example, by electroporation and the cells containing the recombinant plasmid can be identified by selecting the marker in the plasmid. Product expression can be induced and detected in the host cell by a specific assay for the product.

In some embodiments, the expression of the DNA encoding the product / peptide can be optimized in the host cell. For example, the DNA may include codons for one or more amino acids that are predominant in the host cell relative to other codons for the same amino acid.

Alternatively, the expression of the product can be carried out by in vitro synthesis of the protein in acellular extracts that are also particularly suitable for the incorporation of modified or unnatural amino acids for functional studies; see also below. The use of in vitro translation systems may have advantages over gene expression in vivo when the overexpressed 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 proteases. intracellular The most commonly used acellular translation systems consist of extracts of rabbit reticulocytes, wheat germ and Escherichia coli. All of them prepared as crude extracts containing all macromolecular components (70S or 80S ribosomes, tRNA, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, etc.) necessary for the translation of exogenous RNA. To facilitate the translation to be effective, each extract must be supplemented with amino acids, energy sources (ATP, GTP), energy regeneration systems (creatine phosphate and creatine phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for the lysate of E .coli) and other cofactors known in the art (Mg2 +, K +, etc.). Appropriate transcription / translation systems are available commercially, for example from Promega Corporation, Roche Diagnostics, and Ambion, ie Applied Biosystems (Anderson, C. et al., Meth. Enzymol. 101 (1983), 635- 644; Arduengo, M. et al. (2007), The Role of Cell-Free Rabbit Reticulocyte Expression Systems in Functional Proteomics in, Kudlicki, Katzen and Bennett eds., Cell-Free Expression Vol. 2007. Austin, Tx: Landes Bioscience , pp. 1-18; Chen and Zubay, Meth. Enzymol. 101 (1983), 674-90; Ezure et al., Biotechnol. Prog. 22 (2006), 1570-1577).

Host cells:

With regard to the present invention, a 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. cerevisiae. The term "prokaryotic" is intended to include all bacteria that can be transformed or transfected with DNA or RNA molecules for the expression of an antibody of the invention or the corresponding immunoglobulin chains. Prokaryotic hosts may include gram-negative and gram-positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. It is understood that the term "eukaryotic" includes yeast, higher plant, insect and preferably mammalian cells, more preferably HEK 293, NSO, CSO and CHO cells. Depending on the host employed in a recombinant production process, the antibodies or immunoglobulin chains encoded by the polynucleotide of the present invention may or may not be glycosylated. The antibodies of the invention or the corresponding immunoglobulin chains may also include an initial moiety of the amino acid methionine. 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. Likewise, methods for preparing fused genes, operatively linked and for expressing them, for example, in mammalian cells and bacteria (Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). The genetic constructs and methods described herein can be used for the expression of the antibody of the invention or the corresponding immunoglobulin chains, in eukaryotic or prokaryotic hosts. In general, together with the host, expression vectors containing promoter sequences that facilitate efficient transcription of the inserted polynucleotide are used. The expression vector typically contains an origin of replication, a promoter and a terminator, as well as specific genes that are capable of providing phenotypic selection of the transformed cells. From various sources, suitable source cells can be obtained for DNA sequences and host cells for the expression and secretion of immunoglobulins, such as from the American Type Culture Collection ("Catalog of Cell Lines and Hybridomas," Fifth Edition (1985) Rockville, Maryland, United States). Also, for large-scale production of the antibody of the invention, transgenic animals, preferably mammals, comprising the cells of the invention can be used.

The transformed hosts can be grown in fermenters and grown in accordance with techniques known in the art to achieve optimal cell growth. Once expressed, the complete antibodies, their individual dimers, light and heavy chains, or other forms of immunoglobulin of the present invention, can be purified according to standard procedures of the art, including precipitation with ammonium sulfate, affinity columns, column chromatography, gel electrophoresis and similar procedures; see, Scopes, "Protein Purification", Springer Verlag, NY 1982). The antibody or its corresponding immunoglobulin chain (or chains) of the invention can be isolated after growth medium, cell lysates or cell membrane fractions. Isolation and purification of, for example, the antibodies or immunoglobulin chains of the invention, expressed recombinantly, can be performed 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, for example, 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 additionally coupled to other residues, for example, for drug addressing and imaging applications. Said coupling can be performed chemically after the expression of the antibody or antigen at the binding site or the coupling product can be designed in 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.

For pharmaceutical uses, substantially pure immunoglobulins are preferred, with a homogeneity of at least about 90 to 95%, with a homogeneity of 98 to 99% being even more preferred. Once purified, partially or even homogeneously, as desired, the antibodies can then be used therapeutically (even extracorporeally) or in the development and performance of test procedures.

The present invention also involves a method of producing cells capable of expressing an antibody of the invention or its corresponding immunoglobulin chain (or chains) comprising cells genetically modified with the polynucleotide or with the vector of the invention. The cells that can be obtained with the method of the invention can be used, for example, to test the interaction of the antibody of the invention with its antigen.

ELISA tests:

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, do not involve extraction stages, and are easy to perform. ELISA assays for the detection of antibodies against protein antigens often use direct binding of short synthetic peptides to the plastic surface of a microtiter plate. Peptides are, in general, very pure due to their synthetic nature and effective purification methods using high performance liquid chromatography. A drawback of short peptides is that they normally represent linear epitopes, although not conformational or discontinuous. To present conformational epitopes, long peptides or the complete native protein are used. Direct binding of protein antigens to the hydrophobic polystyrene support of the plate can cause partial or total denaturation of the bound protein and loss of conformational epitopes. Coating the plate with an antibody, which acts as a mediator in the immobilization (capture ELISA) of the antigens, can avoid this effect.

However, frequently, overexpressed recombinant proteins are insoluble and require purification under denaturation and renaturation conditions, when antibodies against conformational epitopes are analyzed. See, for example, U.S. Patent Application No. 20030044870 for a generic ELISA that uses recombinant fusion proteins as coating proteins.

Binding molecules:

A "binding molecule", as used in the context of the present invention, refers to antibodies and fragments thereof.

Antibodies:

Here, the terms "biomarker" and "immunoglobulin" are used interchangeably. An antibody or an immunoglobulin is a molecule that binds to a molecule of interest of the present invention, as defined above and later herein, which comprises at least the variable domain of a heavy chain, and usually comprises at least the variable domains of a heavy chain and a light chain. The basic structures of immunoglobulin in vertebrate systems are relatively well understood; see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.

1988). The terms "binds" and "recognizes" are used interchangeably in relation to the binding affinity of the binding molecules of the present invention, for example, antibodies.

Any antibody or immunoglobulin fragment that contains sufficient structure to bind Specifically, the molecules of interest, as defined above and later herein, are indistinctly referred to as "binding molecule,""bindingfragment," or "immunospecific fragment."

Antibodies or antigen binding fragments, immunospecific fragments, variants, or derivatives thereof, of the invention, include human monoclonal antibodies, and single chain antibodies, epitope binding fragments, for example, Fab, Fab 'and F fragments ( ab ') 2, Fd, Fv, single chain Fv (scFv), single chain antibodies, Fv fragments linked by disulfide bonds (sdFv), fragments comprising the V ^l and V ^h domain and fragments derived therefrom. Said scFv molecules are well known in the art and are described, for example, in US Patent 5,892,019. As will be explained in more detail below, the term "immunoglobulin" comprises several broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta or epsilon, ( ^and , | j, a, 8, £) with some subclasses between them (for example, Y1-Y4). It is the nature of this chain that determines the "class" of the antibody such as IgG, IgE, IgM, IgD, IgA and IgY, respectively. Immunoglobulin subclasses (isotypes) for example, IgG1, IgG2, IgG3, IgG4, IgA1, etc. They are well characterized and known to confer functional specialization. The immunoglobulin or antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. .) or subclass of immunoglobulin molecule. The modified versions of each of these classes and isotypes are readily discernible to the person skilled in the art in view of the present disclosure and, therefore, are within the scope of the present invention. All classes of immunoglobulin are clearly within the scope of the present invention, the following analysis will generally address the class of IgG immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of 53,000 to 70,000 Daltons. The four chains are typically joined by disulfide bonds in a "Y" configuration in which the light chains support the heavy chains that begin at the mouth of the "Y" and continue through the variable region. As can be seen from the classification of the exemplary anti-IL-17A and IL-17F antibodies of the present invention, listed in Table 1 above, the dominant subtypes of the antibodies of the present invention appear to be IgG1 and IgG4, possibly involving responses. of regulatory T lymphocytes and / or epithelia at their onset in these states with AIR deficit (Autoimmune Regulator). These findings are confirmed by classification of the corresponding autoantibodies found in mice with AIR deficit described by Karner et al., In Clin. Exp. Immunol. (2012); doi: 10,1111 / cei. 12024. Accordingly, in a preferred embodiment of the present invention, the human antibodies of the present invention are of the IgG type, in particular IgG1 or IgG4.

IgG structure:

Light chains are classified as kappa or lambda (k, A). Each kind of heavy chain can be linked with a light chain kappa or lambda. In general, the light and heavy chains are covalently linked to each other, and the "tail" parts of the two heavy chains are linked together by covalent or non-covalent disulfide bonds when immunoglobulins are generated by hybridomas, B lymphocytes or host cells modified by genetic engineering. In the heavy chain, the amino acid sequences are executed from an N end at the bifurcated ends of the Y configuration to the C end at the bottom of each chain.

Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in a functional way. In this sense, it will be appreciated that the variable domains of both light (V ^l ) and heavy (V ^h ) chain parts determine recognition and antigenic specificity. In contrast, the constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) give important biological properties, such as secretion, transplacental mobility, binding to the Fc receptor, complement binding and similar properties. . By convention, the numbering of the domains of the constant region increases as they are more distant from the antigen binding site or the amino terminus of the antibody. The N-terminal part is a variable region and the C-terminal part is a constant region; the CH3 and CL domains comprise, in fact, the carboxyl end 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 ¹ domain and the V ^h domain, or subset of the complementarity determining regions (CDR), 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 in each of the V ^h and V ¹ chains. Herein, any antibody or immunoglobulin fragment containing sufficient structure to specifically bind a molecule of interest of the present invention is indicated interchangeably as a "binding fragment" or an "immunospecific fragment."

In naturally occurring antibodies, an antibody comprises six hypervariable regions, sometimes referred to as "complementarity determining regions" or "CDR," which are present in each antigen-binding domain, which are short, non-contiguous sequences, which are position specifically to form the domain antigen binding as the antibody adopts its three-dimensional configuration in an aqueous environment. The "CDRs" are flanked by four relatively "structural" or "FR" regions that show less intermolecular variability. The structural regions largely adopt a sheet conformation p and the CDRs form loops that connect, and in some cases are part of, the sheet structure p. Therefore, the structural regions act forming a framework that allows the positioning of the CDRs in the correct orientation through non-covalent interactions between the chains. The antigen binding domain formed by positioned CDRs defines a surface complementary to the epitope in the immunoreactive antigen. This complementary surface promotes non-covalent binding of the antibody with its related epitope. A person skilled in the art can easily identify the amino acids comprising the CDRs and the framework regions, respectively, for any given heavy or light chain variable region, since they have been precisely defined; see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917.

In the event that there are two or more definitions of a term that is used and / or accepted in the matter, the definition of the term, as used herein, intends to include all those meanings unless explicitly stated otherwise. . A specific example is the use of the term "complementarity determining region"("CDR") which describes the combination sites of non-contiguous antigens found in the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917, where definitions include amino acid residues or subsets of overlapping amino acids when compared to each other. However, it is intended that the application of any definition referring to a CDR of an antibody or variants thereof, be within the scope of the term as defined and used herein. Appropriate amino acid residues spanning the CDRs as defined in each of the references cited above, are set forth below in Table 2 by way of comparison. The exact number of residues covered by a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which moieties comprise a particular hypervariable region or ^c D ^r of the human IgG subtype of antibody given the amino acid sequence of the antibody's variable region.

Table 2: CDR1 definitions

Kabat et al. They also defined a numbering system for variable domain sequences that is applicable to any antibody. A person skilled in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence, without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific positions of amino acid residues in an antibody or antigen-binding fragment, variant, or derivative thereof, of the present invention, are in accordance with the numbering system. of Kabat, which, however, is theoretical and may not apply equally to each antibody of the present invention. For example, depending on the position of the first CDR, the following CDRs could be changed in any direction.

In one embodiment, the antibody of the present invention is not IgM, or a derivative thereof, with a pentavalent structure. In particular, in specific applications of the present invention, especially for therapeutic use, IgMs are less useful than IgG and other corresponding bivalent antibodies or binding molecules, since often, due to their pentavalent structure and lack of maturation of affinity, IgM show nonspecific cross reactivities and a very low affinity.

In a particularly preferred embodiment, the antibody of the present invention is not a polyclonal antibody, that is, it consists substantially of a particular kind of antibody, rather than being a mixture obtained from a plasma immunoglobulin sample.

Antibody fragments:

Antibody fragments, including single chain antibodies, may comprise the variable region or regions alone or in combination with all or a part of the following: hinge region, domains CH1, CH2, and CH3. Also included in the invention are fragments that bind to a molecule of interest of the present invention, said fragments comprising any combination of region or variable regions with a hinge region, and domains CH1, CH2 and CH3. In a particularly preferred embodiment of the present invention, the antibodies are naturally occurring human monoclonal antibodies or binding fragments, derivatives and variants thereof cloned from human subjects, which specifically bind to IL-17, as defined in the claims. and is shown in the Examples. Optionally, the structural region of the human antibody is aligned and adopted in accordance with the relevant sequences of the variable region of the human germ line in the databases; see, for example, Vbase (http://vbase.mrc-cpe.cam.ac.uk/) offered by the MRC Center for Protein Engineering (Cambridge, United Kingdom). For example, amino acids that are considered to be possibly separated from the true germline sequence could be due to the PCR primer sequences that are incorporated during the cloning process. Therefore, according to the present invention, the terms "human monoclonal antibody", "human monoclonal autoantibody", "human antibody" and similar expressions are used to refer to a binding molecule that binds to a molecule of interest of the present invention, which is of human origin, that is, that it has been isolated from a human cell, such as a B lymphocyte or a hybridoma thereof, or whose cDNA has been cloned directly from mRNA of a human cell, for example, a memory B lymphocyte, human. A human antibody will continue to be considered "human" even if amino acid substitutions are made in the antibody, for example, to improve binding characteristics.

Antibodies derived from human immunoglobulin libraries or from transgenic animals for one or more human immunoglobulins and which do not express endogenous immunoglobulins, as described below and, for example, in U.S. Patent No. 5,939,598 to Kucherlapati et al. , are called human type antibodies to distinguish them from the truly human antibodies of the present invention.

For example, pairing heavy and light chains of human-type antibodies, such as synthetic and semi-synthetic antibodies, typically isolated from phage displays, does not necessarily reflect the original pairing as occurred in the original human B lymphocyte. Accordingly, Fab and scFv fragments obtained from recombinant expression libraries, as commonly used in the prior art, can be considered to be artificial with all possible associated effects on immunogenicity and stability. On the contrary, the present invention provides affinity matured antibodies isolated from selected human subjects, which are characterized by their therapeutic utility and their tolerance in man.

Antibody fragments:

As used herein, the term "heavy chain part" includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain part comprises at least one of: a CH1 domain, a hinge domain (for example, an upper, central and / or lower hinge region), a CH2 domain, a CH3 domain, or a variant or a 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 part 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 part of a hinge domain and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a part 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. Additionally, a binding polypeptide for use in the invention may lack at least a part of a CH2 domain (for example, all or part of a CH2 domain). As discussed above, a person skilled in the art will also understand that these domains (for example, heavy chain parts) can be modified, such that their amino acid sequence varies with respect to the naturally occurring immunoglobulin molecule.

In certain antibodies, or antigen-binding fragments, variants, or derivatives thereof, disclosed herein, the heavy chain portions of a polypeptide chain of a multimer are identical to those of a second polypeptide chain of the multimer. Alternatively, the monomers containing a heavy chain part of the invention are not identical. For example, each monomer may comprise a different target binding site, forming, for example, a bispecific antibody or diabody.

In another embodiment, the antibodies, or antigen-binding fragments, variants, or derivatives thereof, disclosed herein, are composed of a single polypeptide chain, such as scFv and will be expressed intracellularly (intrabodies) for possible applications. therapeutic and diagnostic in vivo.

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 part 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 part may comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule.

In another example, a heavy chain part may comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.

Therefore, as also illustrated 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 according to the method of the present invention. In this context, the heterologous constant region or regions are preferably of human origin, in the case of therapeutic applications of the antibody of the present invention, but could also be, for example, of rodent origin, in the case of animal studies. ; see also the examples.

As used herein, the term "light chain part" includes amino acid sequences derived from an immunoglobulin light chain. Preferably, the light chain part comprises at least one of a Vl or Cl domain.

As indicated above, the subunit structures and the three-dimensional configuration of the constant regions of the various classes of immunoglobulins are well known. As used herein, the expression "vh domain" includes the amino terminal variable domain of an immunoglobulin heavy chain and the expression "CH1 domain" includes the first (most amino terminal) constant region domain of a heavy chain. of immunoglobulin. The CH1 domain is adjacent to the Vh domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.

As used herein, the term "CH2 domain" includes the part of a heavy chain molecule that extends, for example, from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 a 360, Kabat numbering system; and remains 231-340, EU numbering system; see Kabat EA et al., In the cited reference). The CH2 domain is unique since it is not closely paired with another domain. Rather, between the two CH2 domains of a native and intact IgG molecule, two branched N-linked carbohydrate chains are interposed. 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 term "hinge region" includes the part of a heavy chain molecule that binds the CH1 domain with the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. The hinge regions can be subdivided into three distinct domains: upper, central and lower hinge domains; see Roux et al., J. Immunol. 161 (1998), 4083.

As used herein, the term "disulfide bond" includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or a bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond and the two heavy chains are joined by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system ( position 226 or 229, EU numbering system).

As used herein, the terms "joined", "merged" or "merged" are used interchangeably. These terms refer to the union between two or more elements or components, by any means, including chemical conjugation or recombinant means. A "phase fusion" refers to the union of two or more open reading frame polynucleotides (ORF ) to form a longer continuous ORF, in a manner that retains the correct translational reading phase of the ORFs. originals Therefore, a recombinant fusion protein is a unique protein that contains two or more segments that correspond to polypeptides encoded by the original ORFs (whose segments are not normally so bound in nature). Although the reading phase is thus continued along the merged segments, the segments may be physically or spatially separated, for example, by phase linker sequences. For example, polynucleotides encoding the CDRs of a variable region of immunoglobulin may be fused, in phase, but may be separated by a polynucleotide encoding at least one structural region of immunoglobulin or additional CDR regions, provided that the "fused" CDRs are translate together as part of a continuous polypeptide. Union Features:

The terms "binding" or "recognition", used interchangeably herein, generally mean that a binding molecule, for example, an antibody, binds to a predetermined epitope through its antigen binding domain, and that the Binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, it is said that an antibody "specifically binds" to an epitope when it binds to that epitope, through its antigen binding domain, more easily than it would bind to a random, unrelated epitope . Here, the term "specificity" is used to qualify the relative affinity by which a particular antibody binds to a specific epitope. For example, it may consider that antibody "A" has a greater specificity for a particular epitope than antibody

"B" or it can be said that the "A" antibody binds to the "C" epitope with greater specificity than that of the related "D" epitope. Unrelated epitopes are usually part of a non-specific antigen (for example, BSA (bovine serum albumin), casein, or any other specified polypeptide), which can be used to calculate the binding specificity of a given binding molecule. In this regard, the term "specific binding" refers to the binding of an antibody with a predetermined antigen with a K ^d that is at least twice less than its K ^d to bind to a non-specific antigen. The term "highly specific" binding, as used herein, means that the relative K ^d of the antibody for the specific target epitope is at least 10 times less than the K ^d to bind that antibody to other ligands.

When present, the term "immunological binding characteristics" or other binding characteristics of an antibody with an antigen, in all its grammatical forms, refers to the specificity, affinity, cross-reactivity, and other binding characteristics of an antibody.

By "preferentially binding", it is understood that the binding molecule, for example, the antibody, specifically binds to an epitope more easily than would bind a similar, homologous or similar, related epitope. Therefore, an antibody that "preferentially binds" to a given epitope would more likely bind to that epitope than to a related epitope, even though said antibody may cross-react with the related epitope.

As a non-limiting example, it can be considered that a binding molecule, for example, an antibody, preferentially binds to a first epitope if it binds to said first epitope with a dissociation constant (K ^d ) that is less than K ^d of the antibody for the second epitope. In another non-limiting example, an antibody may preferentially be bound to a first antigen if it binds to the first epitope with an affinity that is at least an order of magnitude less than the K ^d of the antibody for the second epitope. In another non-limiting example, an antibody may preferentially be bound to a first epitope if it binds to the first epitope with an affinity that is at least two orders of magnitude less than the K ^d of the antibody for the second epitope.

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

It can be said that a binding molecule, for example, an antibody or antigen binding fragment, variant or derivative disclosed herein, binds to a molecule of interest of the present invention, to a fragment or variant thereof, with a deactivation speed (k (off)) less than or equal to 5 x 10-2 sec-1, 10-2 sec-1, 5 x 10-3 sec-1 or 10-3 sec-1. More preferably, it can be said that an antibody of the invention binds to a molecule of interest of the present invention, or to a fragment or variant thereof, with a deactivation rate (k (off)) less than or equal to 5 x 10-4 sec-1, 10-4 sec-1, 5 x 10-5 sec-1 or 10-5 sec-15 x 10-6sec-1, 10 -6 sec-1,

5 x 10-7 sec-1 or 10-7 sec-1.

It can be said that a binding molecule, for example, an antibody or antigen binding fragment, variant or derivative disclosed herein, binds to a molecule of interest of the present invention or a fragment or variant thereof, with a speed (k (on)) greater than or equal to 103 M-1 sec-1, 5 x 103 M-1 sec-1, 104

M-1 sec-1 or 5 x 104 M-1 sec-1. More preferably, it can be said that an antibody of the invention binds to a molecule of interest of the present invention or to a fragment or variant thereof with a speed (k (on)) greater than or equal to 105 M-1 sec. 1.5 x 105 M-1 sec-1, 106 M-1 sec-1 or 5 x 106 M-1 sec-1 or 107 It is said that a binding molecule, for example, an antibody, competitively inhibits the binding of a reference antibody with a specific epitope, if it preferentially binds to that epitope insofar as it blocks, to some extent, the binding of the reference antibody with the epitope. Competitive inhibition can be determined by any method known in the art, for example, competition tests of the ELISA type. It can be said that an antibody competitively inhibits the binding of the reference antibody with an epitope determined 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 intensity of the binding of an individual epitope with the CDR of a binding molecule, for example, an immunoglobulin molecule; see, for example, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988) on 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 combination intensity of a mixture of immunoglobulin with the antigen; see, for example, Harlow on pages 29-34. Avidity is related to the affinity of individual immunoglobulin molecules in the population with specific epitopes and also to the valences of immunoglobulins and antigen. For example, the interaction between a Bivalent monoclonal antibody and an antigen with an extremely repetitive epitope structure, such as a polymer, would be one of high avidity. The affinity or greediness 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, WE, Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, WH Freeman and Company New York, NY (1992), and the 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 interaction between an antibody and an antigen may vary if measured under different conditions, for example, saline concentration, pH. Therefore, measurements of affinity and other antigen binding parameters, for example, K ^d , IC ⁵⁰ , are preferably performed with standardized antibody and antigen solutions and with a standardized buffer.

Binding molecules, for example, antibodies or antigen binding fragments, variants or derivatives thereof of the invention, can also be described or specified as to their cross-reactivity. As used herein, the term "cross reactivity" refers to the ability of an antibody, specific for an antigen, to react with a second antigen; a measure of the relationship between two different antigenic substances. Therefore, an antibody reacts cross-linked if it binds to an epitope different from the one that induced its formation. The cross-reactive epitope generally contains many of the same complementary structural characteristics as the inducing epitope, and in some cases, it can really fit better than the original.

For example, certain antibodies have some degree of cross-reactivity, since they bind to related but not identical epitopes, for example, 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 (calculated using methods known in the art and described in this document) with a reference epitope. It can be said that an antibody has 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 70%, less than 65%, less than 60%, less than 55% and less than 50% identity (calculated using methods known in the art and described herein) with a reference epitope. An antibody can be considered "highly specific" for a given epitope, if it does not bind to any other analogue, ortholog or homolog of that epitope.

Binding molecules, for example, antibodies or antigen binding fragments, variants or derivatives thereof of the invention, can also be described or specified as to their binding affinity with a molecule of interest of the present invention. Preferred bonding include those with a dissociation constant or K ^d less than 5 x 10 ^-2 M, 10 ^-2 M, 5 x 10 ^-3 M, 10 ^-3 M, 5 x 10 ^-4 M, 10 ^-4 M , 5 x 10 ^-5 M, 10 ^-5 M, 5 x 10 ^-6 M, 10 ^-6 M, 5 x 10 ^-7 M, 10 ^-7 M, 5 x 10 ^-8 M, 10 ^-8 M, 5 x 10 ^-9 M, 10 ^-9 M, 5 x 10 ^-10 M, 10 ^-10 M, 5 x 10 ^-11 M, 10 ^-11 M, 5 x 10 ^-12 M, 10 ^-12 M, 5 x 10 ^-13 M, 10 ^-13 M, 5 x 10 ^-14 M, 10 ^-14 M, 5 x 10 ^-15 M or 10 ^-15 M. Typically, the antibody binds to a dissociation constant ((K ^d ) of 10 ^-7 M or less with its predetermined antigen Preferably, the antibody binds to its related antigen with a dissociation constant (K ^d ) of 10 ^-9 M or less, and even more preferably with a dis constant ociation (K ^d ) 10 ^-11 M or less.

Antibody Modifications:

The immunoglobulin or its coding cDNAs can be further modified. Therefore, in a further embodiment, the method of the present invention comprises any one of the production steps of a chimeric antibody, single chain antibody, Fab 'fragment, bispecific antibody, fusion antibody, labeled antibody or an analog of any one. from those. Those skilled in the art know and describe corresponding methods, for example, in Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. As explained above, the antibody of the invention may exist in a variety of forms in addition to as complete antibodies; including, for example, Fv, Fab and F (ab) ² , as well as in single-chain forms; see, for example, international application WO88 / 09344.

The antibodies of the present invention or their corresponding immunoglobulin chain or chains, can be further modified using conventional techniques known in the art, for example, using one or more deletions, insertions, substitutions, additions and / or recombinations of amino acids and / or any other modification or modifications known in the art either alone or in combination. The person skilled in the art is well aware of methods for introducing such modifications into the DNA sequence underlying the amino acid sequence of an immunoglobulin chain; see, for example, Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY and in Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley-Interscience, NY 1994). Modifications of the antibody of the invention include chemical and / or enzymatic derivatizations in one or more constituent amino acids, including side chain modifications, main chain modifications and N and C terminal modifications, including acetylation, hydroxylation, methylation, the amidation, and the union of carbohydrate or lipid residues, cofactors, and the like. In the same way, the present invention includes the production of chimeric proteins comprising the described antibody or some fragment thereof at the amino terminus fused to a heterologous molecule, such as a label or a drug. The antigen binding molecules generated in this way they can be used for the location of the drug in cells that express the appropriate surface structures of the diseased cell and tissue, respectively. This targeting and cell binding could be useful for the delivery of agents that are therapeutic or diagnostic active and for gene therapy / gene delivery. Molecules / particles with an antibody of the invention would specifically bind to cells / tissues that express the antigen of particular interest and, therefore, could have a diagnostic and therapeutic use.

Diseases and disorders:

Unless stated otherwise, in this document, the terms "disorder" and "disease" are used interchangeably. The term "autoimmune disorder", as used herein, is a disease or disorder that originates in, and is directed against, the tissues or organs of the individual himself or a segregated co or manifestation of the same or condition resulting from the same. Autoimmune diseases are mainly caused by poor regulation of immunoadaptive responses and autoreactive autoantibodies or T lymphocytes are formed against self-structures. Almost all autoimmune diseases also have an inflammatory component. Autoinflammatory diseases are mainly inflammatory, and some classic autoinflammatory diseases are caused by genetic defects in innate inflammatory pathways. In autoinflammatory diseases, no autoreactive T lymphocytes or autoantibodies are found. In many of these autoimmune and auto-inflammatory disorders, there may be various clinical and laboratory markers, including, but not limited to, hypergammaglobulinemia, high levels of autoantibodies, complex deposits of antigen-antibody in tissues. benefits of corticosteroid or immunosuppressive treatments, and lymphoid cell aggregates in the affected tissues. Without being limited to any theory regarding autoimmune disorders mediated by B lymphocytes, it is believed that B lymphocytes demonstrate a pathogenic effect on human autoimmune diseases through a multitude of mechanistic pathways, including autoantibody production, immune complex formation, Dendritic cell and T lymphocyte activation, cytokine synthesis, direct release of chemokines, and providing a nest for ectopic neoliophogenesis. Each of these routes can participate, to different degrees, in the pathology of autoimmune diseases.

As used herein, an "autoimmune disorder" may be an organ-specific disease (that is, the immune response is specifically directed against an organ system such as the endocrine system, the hematopoietic system, the skin, the system cardiopulmonary, gastrointestinal and hepatic systems, renal system, thyroid, ears, neuromuscular system, central nervous system, etc.) or a systemic disease that can affect multiple organ systems (for example, systemic lupus erythematosus ( SLE), rheumatoid arthritis, polymyositis, autoimmune polyecrinopathy syndrome type 1 (APS1 / autoimmune polyocrinopathy with candidiasis and ectodermal dystrophy (APECED) from autoimmune polyecrypto-candidiasis-ectodermal dystrophy) etc. Preferred diseases of this type include rheumatologic disorders (such as, for example, rheumatoid arthritis, Sjogren's syndrome , scleroderma, lupus, such as SLE and lupus nephritis, polymyositis / dermatomyositis, cryoglobulinemia, antiphospholipid antibody syndrome and psoriatic arthropathy), autoimmune gastrointestinal and hepatic disorders (such as, for example, inflammatory bowel diseases (for example, ulcerative colitis and disease Crohn's), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis and celiac disease), vasculitis (such as, for example, ANCA-negative vasculitis (cytoplasmic anti-neutrophil antibodies) and ANCA-associated vasculitis, including vasculitis Churg-Strauss, Wegener's granulomatosis and microscopic polyangiitis), autoimmune neurological disorders (such as, for example, multiple sclerosis (MS), opsoclony-myoclonia syndrome, myasthenia gravis, optic neuromyelitis, Parkinson's disease, Alzheimer's disease, and polyneuropathies autoimmune ), kidney disorders (such as, for example, glomerulonephritis, Goodpasture's syndrome and Berger's disease), autoimmune dermatological disorders (such as, for example, psoriasis, atopic dermatitis, urticaria, pemphigus group diseases, bullous pemphigoid diseases, and cutaneous lupus erythematosus), haematological disorders (such as, for example, thromobocytopenic purpura, thrombotic thrombocytopenic purpura, posttransfusional purpura and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune auditory diseases (such as, for example, internal ear disease and loss of hearing), Behcet's disease, Raynaud's syndrome, organ transplantation and autoimmune endocrine disorders (such as, for example, diabetes-related autoimmune disorders such as insulin-dependent diabetes mellitus (DMID), Addison's disease, autoimmune thyroid disease (e.g. get sick ad de Graves and thyroiditis)) and diseases that affect the generation of autoimmunity, such as autoimmune polyocrinopathy syndrome type 1 (APS1) / autoimmune polyocrinopathy with candidiasis and ectodermal dystrophy (APECED) Myasthenia Grave (MG / Thymoma). Preferred diseases include, for example, RA, IBD, including Crohn's disease and ulcerative colitis, ANCA-associated vasculitis, lupus, MS, Sjogren's syndrome, Graves' disease, DMID, pernicious anemia, thyroiditis, glomerulonephritis and APS1. AR, IBD, lupus and MS are even more preferred, and RA and IBD are more preferred and RA is most preferred.

Specific examples of other autoimmune disorders, as defined herein, which in some cases include those listed above, include, but are not limited to, arthritis (acute and chronic, rheumatoid arthritis including rheumatoid arthritis of juvenile onset and stages such as rheumatoid synovitis, gout or gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, degenerative arthritis, arthritis induced by Type II collagen, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, osteoarthritis, progredient chronic arthritis, deforming arthritis, primary chronic polyarthritis, reactive arthritis, menopausal arthritis, estrogen depletion arthritis and ankylosing spondylitis / rheumatoid spondylitis), autoimmune lymphoproliferative disease, hyperproliferative inflammatory skin diseases, psoriasis such as plaque psoriasis, gout psoriasis, pustular psoriasis and nail psoriasis, atopy, including atopic diseases, such as hay fever and Job's syndrome, atopic dermatitis, allergic and toxic contact dermatitis (acute and chronic), exfoliative dermatitis, allergic dermatitis, urticaria, herpetiform dermatitis, numular dermatitis, seborrheic dermatitis, non-specific dermatitis, hyper IgM syndrome linked to the X chromosome, diseases inflammatory in allergic traoculars, hives, such as chronic allergic urticaria and chronic idiopathic urticaria, cold urticaria, including chronic autoimmune urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including local and systemic forms of sclerosis) multiple sclerosis (MS) such as spinooptic MS, progressive primary MS (PPMS) and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, ataxic sclerosis, optic neuromyelitis (NMO), inflammatory bowel disease (IBD) ) (for example, Crohn's disease, autoimmune-mediated gastrointestinal diseases, gastrointestinal inflammation, colitis such as ulcerative colitis, ulcerative colitis, microscopic colitis, collagenous colitis, chicken colitis, necrotizing enterocolitis and transmural colitis colitis, and auto intestinal inflammatory disease immune), intestinal inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including acute respiratory distress syndrome (ARDS) or adult syndrome, meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune hematological disorders, graft versus host disease, angioedema, such as hereditary angioedema, cranial nerve injury, such as meningitis, gestational herpes, diseases of the penfigoid group, pemphigus group disease, gestational herpes, scrotal pruritus, premature ovarian insufficiency autoimmune, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases, such as anaphylaxis and atopic rhinitis, various forms of asthma, chronic obstructive pulmonary disease (COPD), encephalitis such as Rasmussen encephalitis and limbic or trunk encephalitis encephalic, uveitis, such as uveitis an Terior, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, phacoantigenic uveitis, posterior uveitis or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis, such as primary GN, GN immunomediated GN membranous), idiopathic membranous GN or idiopathic membranous nephropathy, membranoproliferative or proliferative membranous GN (GNPM), including Type I and Type II, and rapidly progressive GN (GNRP), proliferative nephritis, autoimmune polyglandular endocrine insufficiency, balanitis including circumscribed plasmacellular balanitis, balanoposthitis, centrifugal annular erythema, persistent dyschromic erythema, multiform erythema, annular granuloma, crisp lichen, sclerosus and atrophic lichen, chronic simple lichen, spiny lichen, lichen planus, epidermolytic hyperkeratosis (premalignant keratosis) piod gangrenous erma, allergic conditions and responses, food allergies, drug allergies, insect allergies, rare allergic disorders, such as various forms of mastocytosis, allergic reaction, eczema including allergic or atopic eczema, steatotic eczema, dyshidrotic eczema, palmoplantar pustulosis , vesicular palmoplantar eczema, asthma, such as bronchial asthma, bronchial asthma and autoimmune asthma, conditions that involve infiltration of T lymphocytes and chronic inflammatory responses, immune reactions against foreign antigens such as ABO fetal blood groups during pregnancy, pulmonary inflammatory disease chronic, autoimmune myocarditis, leukocyte adhesion deficiency, lupus erythematosus (LE), both chronic and acute cutaneous forms (such as chronic cutaneous LE (LECC), discoid lupus erythematosus (LED), subacute cutaneous LE (LECS)) and systemic forms ( as SLE), including lupus nephritis, lupus cere bral, childhood lupus, non-renal lupus, extra-renal lupus, neonatal lupus syndrome (NNL), juvenile onset diabetes mellitus (Type I), including childhood DMID, adult onset diabetes mellitus (Type II diabetes) , autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic colitis, diabetic disorder of the large arteries, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis including lymphocytosis granulomatosis, agraulomatosis , vasculitis (including vasculitis of large vessels such as polymyalgia rheumatica and giant cell arteritis (from Takayasu)), vasculitis of intermediate vessels, such as Kawasaki disease and polyarteritis nodosa / periarteritis nodosa), immunovasculitis, CNS vasculitis , cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis, such as vasculitis itis necrotizing fibrinoid and systemic necrotizing vasculitis, ANCA-negative vasculitis and ANCA-associated vasculitis, such as Churg-Strauss syndrome (SCS), Wegener granulomatosis and microscopic polyangeitis), leukocytoclastic vasculitis, temporal arteritis, aplastic anemia, aplastic anemia autoimmune, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia, including autoimmune hemolytic anemia (AHAI), pernicious anemia, Addison's disease, anemia or pure red cell aplasia (APGR), Factor VIII deficiency, hemophilia A, autoimmune neutropenia (s), cytopenias, such as pancytopenia, leukopenia, diseases involving leukocyte diapedesis, inflammatory CNS disorders, Alzheimer's disease, Parkinson's disease, multiorganic injury syndrome such as secondary to septicemia , trauma or hemorrhage, diseases mediated by the antigen-anticu complex erpo, antiglomerular basement membrane disease, antiphospholipid antibody syndrome, motor neuritis, allergic neuritis, Beliefs disease / syndrome, Castleman syndrome, Goodpasture syndrome, Raynaud syndrome, Sjogren syndrome, Stevens-Johnson syndrome, pemphigoid or pemphigus , such as bullous pemphigoid, scarring pemphigoid (mucous membrane), cutaneous pemphigoid, vulgar pemphigus, paraneoplastic pemphigus, follicular pemphigus, mucous membrane pemphigoid and erythematous pemphigus, acquired bullous epidermolysis, ocular inflammation, preferably allergic conjunctis inflammation linear IgA bullous disease, inflammation of the induced autoimmune conjunctiva, autoimmune polyocrinopathies, Reiter's disease or syndrome, thermal injury due to an autoimmune condition, preeclampsia, an immune complex disorder such as immune complex nephritis, antibody-mediated nephritis, neuroinflammatory disorders, polyneuropathies, chronic neuropathy such as IgM neuropathies or IgM-mediated neuropathy, thrombocytopenia (such as that developed by patients with myocardial infarction, for example), including thrombotic thrombocytopenic purpura (PTT), post-transfusion purpura (PPT), heparin-induced thrombocytopenia and autoimmune or immunomediated thrombocytopenia, including, for example, idiopathic thrombocytopenic purpura (ITP), including chronic or acute ITP, scleritis such as idiopathic ceratoescleritis, episcleritis, autoimmune testicular and ovarian disease including autoimmune orchitis and oofitis oritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease (eye disease ophthalmopathy or thyroid-associated ophthalmopathy), polyglandular syndromes such as autoimmune polyglandular syndromes, for example, type I (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurological paraneoplastic syndromes such as myasthenic Lambert or Lambert myasthenic syndrome Eaton-Lambert syndrome, rigid man or rigid person syndrome, encephalomyelitis such as allergic encephalomyelitis or allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE), myasthenia gravis, such as myasthenia gravis associated with thymoma, degenerates cerebellar tion, neuromiotonia, opsoclone or myoclonus opsoclone syndrome (SOM) and sensory neuropathy, multifocal motor neuropathy, Sheehan syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or chronic active autoimmune hepatitis, pneumonitis, such as lymphoid interstitial pneumonitis (NIL), bronchiolitis obliterans (without transplantation) versus NSIP, Guillain-Barre syndrome, Berger disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, dermatosis subcorneal pustular, transient chololitic dermatitis, cirrhosis such as primary biliary cirrhosis and pneumonocirrosis, autoimmune enteropathy syndrome, celiac disease or celiac disease, celiac sprinkler (gluten enteropathy), treat treatment resistant, idiopathic, cryoglobulinemia, mixed cryoglobulinemia lateral amio trophic (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune hearing disease, such as autoimmune internal ear disease (EAIO), autoimmune hearing loss, polychondritis, such as treatment-resistant or relapsing polychondritis, alveolar pulmonary proteinosis, keratitis , such as Cogan syndrome / non-syphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, autoimmune rosacea, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, primary lymphocytosis, which includes monoclonal B lymphocytosis lymphocytosis (for example , benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, canalopathies such as epilepsy, migraine, arrhythmias, muscular disorders, deafness, blindness, periodic paralysis and CNS canlopathies, autism, inflammatory myopathy, focal glomerulosclerosis or segmental or segmental focal glomerulosclerosis (GSFS), endocrine ophthalmopathy, uveorretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine insufficiency, Schmidt syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune inflammatory neuropathy demyelinating diseases , Dressler's syndrome, alopecia areata, total alopecia, c ReST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and telangiectasia), male and female autoimmune infertility, for example, due to antibodies against sperm, mixed connective tissue disease , Chagas disease, rheumatic fever, recurrent abortion, farmer's lung disease, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, poultry farmers' pneumopathy, allergic granulomatous angiitis, benign lymphocytic angiitis , Alport syndrome, alveolitis, such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, chypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter syndrome, Caplan syndrome, Dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, fibrosing mediastinitis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythroblast fascia syndrome, eosinophilic syndrome , cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic) or Fuch cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immunodeficiency syndrome (AIDS), infection by ecovirus, septicemia (resp syndrome Systemic inflammatory tissue (SRIS), endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan syndrome , autoimmune gonadal insufficiency, Sydenham's chorea, post-streptococcal nephritis, thromboangitis obliterans, thyrotoxicosis, dorsal tabes, chorioiditis, polymyalgia of giant cells, chronic hypersensitivity pneumonitis, conjunctivitis, such as vernal catarrh, dry keratoconjunctivitis and idiopathic nephritis , minimal change nephropathy, benign familial and ischemia and reperfusion injury, reperfusion of transplanted organ, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive pulmonary disease of the respiratory tract, silicosis, thrush, aphthous stomatitis, arteriosclerotic disorders (insufficiency cerebral vascular), such as arteriosclerotic encephalopathy and arteriosclerotic retinopathy, aspermiogenesis, autoimmune hemolysis, Boek disease, cryoglobulinemia, Dupuytren's contracture, phacoanaphylactic endophthalmia, allergic enteritis, knotty erythema, idiopathic facial paralysis, chronic fatigue syndrome, chronic fatigue syndrome Hamman-Rich, sensoneural hearing loss, paroxysmal hemoglobinuria, hypogonadism, regional ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia (sympathetic ophthalmitis), neonatal ophthalmitis, optic neuritis, granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, Quervain thyroiditis, acquired splenic atrophy, non-malignant thymoma, lymphofollicular tiritis, vitiligo, toxic shock syndrome, food poisoning, conditions that involve infiltration of T lymphocytes, leukocyte adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases involving leukocyte diapitis, multiorganic lesion syndrome, diseases mediated by the antigen-antibody complex, basal antiglomerular membrane disease, autoimmune polyocrinopathies, oophoritis, primary myxedema, atrophic gastritis autoimmune, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polndocrine insufficiency, autoimmune enteropathy syndromes, including type I polyglandular syndrome, idiopathic hypoparathyroidism onset in the adult (AOIH), cardiomyopathy, such as dilated cardiomyopathy, acquired bullous epidermolysis (EAA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or non-purulent sinusitis, acute or chronic sinusitis, ethmoidal, frontal, maxillary or sphenoid sinusitis allergic sinusitis, a disorder related to eosinophils, such as eosinophilia, eosinophilia due to pulmonary infiltration, eosinophilia-myalgia syndrome, Loffler syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulloid-containing anaphylaxis, spondylophytes, , seronegative spondyloarthropathies, autoimmune polndocrine disease, sclerosing cholangitis, sclerosing, episclerotic, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of childhood, Wiskott-Aldrich syndrome, telangiectasia ataxia syndrome, angiectasia autoimmune diseases associated with collagen diseases, rheumatism, such as chronic arthritis, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and diseases that accompany vascularization, allergic hypersensitivity disorders, glomerulonephritis, reperfusion injury, ischemic reperfusion disorder, reperfusion injury of myocardium or other tissues, lymphomatous tracheobronchitis, inflammatory dermatosis, dermatosis with acute inflammatory components, multiorganic insufficiency, bullous diseases, renal cortex necrosis, Acute purulent meningitis or other inflammatory disorders of the central nervous system, inflammatory eye and orbital disorders, syndromes associated with granulocyte transfusion, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic inflammation intractable, pyelitis, endoarterial hyperplasia, peptic ulcer, valvulitis and endometriosis.

Labels and diagnostics:

The labeling agents can be directly or indirectly coupled to the antibodies or antigens of the invention. An example of indirect coupling is through the use of a spacer residue. Likewise, the antibodies of the present invention may comprise an additional 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 it can be performed by, for example, chemical crosslinking as described, for example, in 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, favorably a polypeptide linker, wherein said polypeptide linker comprises various hydrophilic amino acids, linked to peptides of sufficient length to cover the distance between the C-terminal end of said additional domain and the N-terminal end of the antibody of the invention or vice versa. The active therapeutic or diagnostic agent can be coupled to the antibody of the invention or an antigen-binding fragment thereof by various means. This includes, for example, single stranded fusion proteins that comprise the variable regions of the antibody of the invention coupled by covalent methods, such as peptide linkages, to the therapeutic and diagnostic active agent. Other examples include molecules comprising at least one antigen-binding fragment covalently or non-covalently coupled to additional molecules, including those in the following non-limiting illustrative list. Traunecker, Int. J. Cancer Surp. SuDP 7 (1992), 51-52, describes the janusin bispecific reagent in which the Fv region directed to CD3 is coupled to soluble CD4 or other ligands such as OVCA and IL-7. Similarly, the variable regions of the antibody of the invention can be constructed on Fv molecules and coupled to alternative ligands such as those illustrated in the cited article. Higgins, J. Infect. Disease 166 (1992), 198-202, describes a heteroconjugate antibody composed of OKT3 cross-linked with an antibody directed to a specific sequence in the V3 region of GP120. Such heteroconjugate antibodies can also be constructed using at least the variable regions contained in the antibody of the methods of the invention. Additional examples of specific antibodies include those described by Fanger, Cancer Treat. Res. 68 (1993), 181-194 and by Fanger, Crit. Rev. Immunol. 12 (1992), 101-124. Conjugates that are immunotoxins that include conventional antibodies have been widely described in the art. Toxins can be coupled to antibodies by conventional coupling techniques or immunotoxins can be produced that contain parts of protein toxin as fusion proteins. The antibodies of the present invention can be used in a corresponding manner to obtain said immunotoxins. Illustrative examples of such immunotoxins are described in Byers, Seminars Cell. Biol. 2 (1991), 59-70 and in Fanger, Immunol. Today 12 (1991), 51-54.

The fusion protein described above may further comprise a cleavable linker or cleavable site for proteinases. These spacer residues, in turn, can be insoluble or soluble (Diener et al., Science 231 (1986), 148) and can be selected to allow antigen drug release at the target site. Examples of therapeutic agents that can be coupled to the antibodies and antigens of the present invention for chemokine immunotherapy, targeting molecules, drugs, radioisotopes, lectins and toxins. Drugs with those that can be conjugated to the antibodies and antigens of the present invention depend on the context of the disease in which the conjugated molecules are intended to be used. For example, antibodies specific for targets useful in the treatment of tumor diseases can be conjugated with compounds that are classically referred to as antineoplastic drugs, such as mitomycin C, daunorubicin and vinblastine. In the use of radioisotopically conjugated antibodies or antigens of the invention for, for example, tumor immunotherapy, certain isotopes may be more preferable than others depending on factors such as the distribution, as well as the stability and emission, of leukocytes. Depending on the autoimmune response, some emitters may be preferable to others. In general, in immunotherapy, radioisotopes emitting particles a and p are preferred. Preferred are high-energy and short-range emitters, such as 212Bi. Examples of radioisotopes that can bind to the antibodies or antigens of the invention for therapeutic purposes 125I, 131I, 90Y, 67Cu, 212Bi, 212At, 211Pb, 47SM, 109Re and 188Re. Those of ordinary skill in the art know or can easily determine other therapeutic agents that can be coupled to the antibody or antigen of the invention, as well as therapeutic protocols ex vivo and in vivo. Non-limiting examples of radionuclides are suitable for labeling, 198Au, 212Bi, 11C, 14C, 57Co, 67Cu, 18F, 67Ga, 68Ga, 3H, 197Hg, 166Ho, 111In, 113m | n, 123I, 125I, 127I, 131I, 111In, 177Lu, 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 that can be detected through an enzymatic or secondary binding stage, such as an enzymatic or peptide tag.

Commercial fluorescent probes, suitable for use as markers in the present invention, are listed in the Handbook of Fluorescent Probes and Research Products , 8th edition. Magnetic particles suitable for use in tests based on magnetic particles (EPM) can be selected from paramagnetic, diamagnetic, ferromagnetic and superparamagnetic materials.

General methods in molecular and cellular biochemistry, useful for diagnostic purposes, can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring 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 media and kits for diagnostic purposes are available from commercial suppliers such as Pharmacia Diagnostics, Amersham, BioRad, Stratagene, Invitrogen and Sigma-Aldrich, as well as from the sources provided in any of the references cited in the This document, particularly in patent literature.

Treatment and drugs:

As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent or slow down (decrease) an unwanted physiological change or disorder, such as development of an autoimmune and / or auto-inflammatory disease. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, decreased disease grade, stabilized clinical picture (i.e. no worsening), delayed or slowed disease progression, improvement or palliation of pathology and remission (either partial or total), either detectable or undetectable. "Treatment" can also mean prolonging survival compared to expected survival if no treatment is received. People who need treatment include those who already have the condition or disorder, as well as those who are prone to have the condition or disorder or those in whom the manifestation of the condition or disorder should be prevented.

If the opposite is not indicated, the terms "drug", "medicine," or "medication" are used interchangeably herein and will include, among others, all items (A), medicines and preparations for internal or external use, and any substance or mixture of substances intended for use in the diagnosis, cure, mitigation, treatment, or prevention of the disease in humans or other animals; and articles (B), medicines and preparations (other than food) intended to influence the structure or any function of the human body or other animals; and articles (C) intended to be used as components of any article specified in clauses (A) and (B). The terms "drug," "medicine," or "medication," will include the full formula of the preparation intended for use in humans or other animals that contain one or more "agents," "compounds," "substances," or "(chemical) compositions" as and in some other context also other pharmaceutically inactive excipients such as fillers, disintegrants, lubricants, glidants, binders or that guarantee the ease of transport, disintegration, disaggregation, dissolution and biological availability of the "drug", "medicine ", or" medication "at a target location provided in the body of a human body or other animals, for example, on the skin, in the stomach or in the intestine. The terms "agent", "compound" or "substance" are used interchangeably herein and will include, in a particular context, but not limited to, pharmacologically active agents, that is, agents that induce a desired biological or pharmacological effect. or that are investigated or tested to determine the ability to induce said possible pharmacological effect by the methods of the present invention.

Examples of "anti-rheumatic drugs" and immunosuppressive drugs include chloroquine, hydroxychloroquine, myocrisine, auranofin, sulfasalazine, methotrexate, leflunomide, ethanorcept, infliximab (plus oral and subcutaneous methotrexate), adalimumab etc., azathioprine D-salts, penioprine, azathioprine salts (oral), gold salts (intramuscular), minocycline, cyclosporine including cyclosporine A and topical cyclosporine, tacrolimus, mycophenolate mofetil, cyclophosphamide, staphylococcal protein A (Goodyear and Silverman, J. Exp. Med., 197 (2003), 125-39), including salts and derivatives, etc.

Examples of "non-steroidal anti-inflammatory drugs" or "NSAIDs" include aspirin, acetylsalicylic acid, ibuprofen and ibuprofen retardant, fenoprofen, piroxicam, flurbiprofen, naproxen, ketoprofen, naproxen, tenoxicam, benorylate, diclofenac, naproxenomethane, naproxenomethane, naproxenomethane, naproxenomethane , mefenamic acid, diclofenac, fenbufen, azapropazone, acemetacin, thiaprofenic acid, indomethacin, sulindac, tolmetine, phenylbutazone, diclofenac, and diclofenac retardant, cyclooxygenase (COX) -2 inhibitors, such as GR 253035, MK966, CKX966 (CKXB); - (5- (4-methylphenyl) -3- (trifluoromethyl) -1H-pyrazol-1-yl), benzenesulfonamide and valdecoxib (BEXTRA®), and meloxicam (MOBIC®), including their salts and derivatives, etc. Preferably, they are aspirin, naproxen, ibuprofen, indomethacin or tolmetin.These NSAIDs are optionally used with an analgesic such as codenin, tramadol and / or dihydrocodinine or narcotics such as morphine.

By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, particularly a mammalian subject, for example, a human patient, for whom the diagnosis, prognosis, prevention or therapy is desired .

Pharmaceutical vehicles:

Pharmaceutically acceptable carriers and routes of administration can be taken from the corresponding literature known to the person skilled in the art. The 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 of Robinson et al., Humana Press, Totowa, New Jersey, United States, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems. 2nd edition of Taylor and Francis. (2006), ISBN: 0-8493-1630-8. Examples of pharmaceutically acceptable carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, different types of wetting agents, sterile solutions etc. Compositions comprising said vehicles can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. The administration of suitable compositions can be carried out in different ways. Examples include administering a composition containing a pharmaceutically acceptable carrier orally, intranasally, rectally, topically, intraperitoneally, intravenously, intramuscularly, subcutaneously, subdermally, transdermally, intrathecally and intracranially. Aerosol formulations, such as nasal spray formulations, include purified aqueous solutions or other active agent solutions with preservative agents and isotonic agents. Said formulations are preferably adjusted to a pH and isotonic state compatible with nasal mucous membranes. Also contemplated are pharmaceutical compositions for oral administration, such as single domain antibody molecules (eg, "nanobodies ™"), etc. Said oral formulations may be in the form of tablets, capsules, powders, liquid or semi-solid forms. A tablet may comprise a solid carrier, such as gelatin or an adjuvant. For rectal or vaginal administration, the formulations may be presented as a suppository with a suitable vehicle; see also O'Hagan et al., Nature Reviews, Drug Discovery 2 (9) (2003), 727-735. More information on formulations that are suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985) and corresponding updates. For a brief review of drug delivery methods see Langer, Science 249 (1990), 1527-1533.

Dosing regimen:

The dosage regimen will be determined by the attending physician and the clinical factors. As is well known in the medical art, dosages for any patient depends on a variety of factors, including patient size, body surface area, age, the particular compound to be administered, sex, time and route of administration, the general state of health and other drugs that are being administered at the same time. A typical dose may be, for example, in the range of 0.001 to 1000 | jg (of nucleic acid for expression or for inhibition of expression in this range); however, doses are contemplated above or below this illustrative range, especially considering the aforementioned factors. Generally, as a regular administration of the pharmaceutical composition, the regimen should be in the range of 1 jg to 10 mg units per day. If the regimen is a continuous infusion, it can also be in the range of 1 | jg to 10 mg per kilogram of body weight per minute, respectively. Progress can be checked by periodic evaluations. 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, Ringer's lactate or non-volatile oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. There may also be preservatives and other additives, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases and the like. Also, the pharmaceutical composition of the invention may comprise additional agents such as antitumor 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 active ingredient that is sufficient to improve symptoms or condition. The toxicity and therapeutic efficacy of said compounds can be terminated by conventional pharmaceutical procedures in cell cultures or in experimental animals, for example, by calculating the ED50 (therapeutically effective dose in 50% of the population) and the LD50 (lethal dose in 50 % of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50.

Preferably, in the composition, the therapeutic agent is present in an amount that is sufficient to prevent inflammation or suppression of the immune response.

These and other embodiments are disclosed and included in the description and in the examples of the present invention. More literature can be obtained on any of the materials, methods, uses and compounds to be used in accordance with the present invention, in public libraries and databases, using, for example, electronic devices. For example, the "Medline" public database, offered by the National Center for Biotechnology Information and / or the National Library of Medicine at the National Institutes of Health, can be used. Those skilled in the art are familiar with other databases and web addresses, such as those of the European Bioinformatics Institute (EBI ), which is part of the European Molecular Biology Laboratory (EMBL ) and can also be obtained using internet search engines. A general description of biotechnology patent information and a survey of relevant sources of patent information useful for retrospective search and for current knowledge is offered in Berks, TIBTECH 12 (1994), 352-364.

The above description generally describes the present invention. Unless otherwise indicated, a term, as used herein, has the definition provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised in 2000 and reprinted in the 2003, ISBN 0 198506732. Various documents are cited throughout the text of this specification. At the end of the specification, complete bibliographic citations can be found just before the claims.

Examples

Examples 1 to 9 below and corresponding Figures 1 to 8 further illustrate the invention by exemplary anti-IL-17 antibodies 17E3 and 24D3 of the present invention and anti-IL-17 9A2 antibody as the reference. Detailed descriptions of conventional methods, such as those used in this document can be found in the cited bibliography; see also 'The Merck Manual of Diagnosis and Therapy "seventh edition of Beers and Berkow (Merck & Co., Inc., 2003).

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art.

Molecular genetics and genetic engineering methods are generally described 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., NY); 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 suppliers such as BioRad, Stratagene, Invitrogen and Clontech. General techniques in cell culture and media collection are described in Large Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8, (1997), 148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2, 1991, 375); and Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251.

Materials and methods

ELISA test with IL-17

96-well microplates (Costar, United States), were coated with human IL-17A or IL-17F (both from BioLegend). The plates were washed with PBS-T and blocked for 1 hour at room temperature with PBS containing 2% BSA (Sigma, Buchs, Switzerland). Patient sera, the B-lymphocyte conditioned medium or recombinant antibody preparations, were incubated for 2 hours at room temperature. The binding of human IgG with the antigen of interest was determined using a specific goat antibody against human Fcgamma horseradish peroxidase conjugated (Jackson ImmunoResearch, Europe Ltd., Cambridgeshire, United Kingdom) followed by measurement of the activity of the HRP using a TMB substrate solution (TMB, Sigma, Buchs, Switzerland).

Example 1: Isolation of peripheral blood mononuclear cells (PBMC) from patients with APECED / APS1

As a starting material for the cloning of fully human antibodies, human lymphocytes obtained from peripheral blood of 23 Finnish voluntary patients with autoimmune polyocrinopathy with candidiasis and ectodermal dystrophy (APECED, OMIM 240300), also known as autoimmune polyecrinopathy type 1 syndrome ( APS1). These volunteers were selected for blood donation through the association of Finnish patients of APECED and Addison. All patients gave written informed consent and the study was approved by the Medicine Ethical Review Board of the Joint Authority of Helsinki and the 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 frequently in Finland (1 / 25,000) due to an effect of the initial carriers ( founder). Patients with APECED have various endocrine autoimmune dysfunctions that mainly include adrenal insufficiency and hypoparathyroidism, but also hypogonadism in various ways, diabetes mellitus, thyroiditis and hypophysitis. Other main symptoms are chronic mucocutaneous candidiasis, alopecia and vitiligo (see also above,). Since a strong correlation between serum specific antigen IgG levels and the frequency of antigen specific B lymphocytes in the peripheral blood mononuclear memory pool has been reported (Bernasconi et al. 2002, Lanzavecchia et al. 2006), the sera of the patients were examined first to detect the presence of autoantibodies against the proteins of interest (such as IFN, IL-17) and then those cases of APECED with high titer (> 1: 5000) were selected for isolation of peripheral blood mononuclear cells (PBMC ) as follows. Heparinized peripheral blood was obtained and diluted with two volumes of 1x PBS at RT, and the cells were superimposed on Lympholyte H, and centrifuged at 2000 rpm (805 rcf) at RT for 20 minutes. The cells were collected at the interface, mixed in wash buffer filling, centrifuged at 1,500 rpm (453 rcf) for 15 min at 4 ° C and resuspended with soft taps, with 10 ml of WB. The cells were then centrifuged at 1,000 rpm (201 rcf), for 10 min at 4 ° C and washed once more with WB. Then, the cells were gently resuspended in an appropriate volume of FBS on ice. FBS was added to adjust the volume to have 20 ml / ml, after which 1 volume of freezing medium (80% FBS (Hyclone, Thermo Scientific and DMSO 20%, # 154938, Sigma) was added slowly while stirred, resuspended and divided into aliquots in cryovials on ice The cryovials were placed in Mr. Frosty's box and transferred to a freezer at -80 ° C 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 specific human antibodies against IL-17

Preferably, specific antibodies against IL-17 were isolated by molecular cloning of immunoglobulin genes obtained from single-cell classified cells derived from short-term oligoclonal cultures of activated memory B cells producing antibodies of interest.

PBMC memory B lymphocytes derived from peripheral blood of voluntary Finnish patients with APECED were isolated with a single stage protocol using human anti-IgD monoclonal antibody (mAb) conjugated to phycoerythrin, CD3, CD56, c D8 anti IgM human conjugated with APC and human anti-CD22 mAb conjugated with FITC (Becton Dickinson, Basel, Switzerland). Cell sorting was carried out using a MoFlo XDP (Beckman Coulter) cell sorter. CD22 positive and IgM and IgD negative B lymphocytes were stimulated with supernatant containing Epstein-Barr virus (EBV) obtained from B95-8 cells (in the middle of B lymphocytes containing RPMI 1640 supplemented with 10 fetal calf serum %). The cells were seeded in IMDM medium supplemented with CpG 2006 at 10 cells per well in 30,000 irradiated feeder PBMCs, prepared from voluntary donors. After 10-14 days of stimulation, culture supernatants were examined for the presence of specific antibodies to the target of interest (eg, IL17). The scanning process involved scanning with respect to the binding of fragments, peptides or derivatives of the particular molecule of interest, for example, by ELISA. Subsequently, the antibody for which the binding is detected or the cell producing said antibody is isolated.

Individual cells obtained from memory B-lymphocyte cultures reactive against IL-17 are deposited in a 96-well PCR plate, containing first chain buffer (Invitrogen, LuBioScience, Switzerland). The cDNA is prepared using a random hexamic primer (Invitrogen, LuBioScience, Switzerland). PCR amplification of the variable regions of the heavy and light immunoglobulin chains was performed according to standard protocols (Wardemann et al., Science 301,2003, 1374-1377). The variable regions of the heavy and light immunoglobulin chains were amplified using a nested PCR strategy. The first round of PCR was performed with specific primers for the constant region of IgG and mixtures of specific primers for all signal peptides of the families of the variable region of Ig of the heavy and light chains (Wardemann et al., Science 301, 2003, 1374-1377). Subsequently, nested PCR was performed using mixtures of primers specific for the J regions of immunoglobulin and the 5 'region of structure 1 of the families of the Ig region of the heavy and light chains. Sequence analysis was carried out to identify clones of individual antibodies present in the selected B lymphocyte culture. Subsequently, the Ig variable regions of the heavy and light chains of each antibody clone were cloned into expression vectors that provided the constant regions of human IgG1, human Ig-Kappa or human Ig-Lambda. After co-transfection of the heavy and light chain Ig expression vectors in HEK 293 cells, antibody clones are produced. Identification of the antibody clone, presumably responsible for the reactivity of IL-17, of the culture of precursor B lymphocytes, is performed by re-scanning the recombinant antibody clones in IL-17 and in a control ELISA.

To identify and correct the pairing errors of the encoded sequence of the primer in the variable region of Ig, an additional amplification was performed by PCR using a semi-nested protocol with 2 pairs of specific primers for a conserved region of the heavy chain constant regions and Ig light as mixtures of 3 'primers and specific primers for the Ig signal peptides as 5' primers. The PCR products were cloned into the TOPO ™ vector (Invitrogen, LuBioScience, Lucerne, Switzerland). Sequence determination of the entire Ig variable region was carried out and the information was used to design specific primers for cloning of the authentic human antibody sequence in antibody expression vectors. This approach allows the identification of the complete antibody sequence of the Ig variable region as occurred in the patient. This sequence was used for the recombinant production of these antibodies which were then used in the subsequent characterization steps.

Example 3: Production and purification of antibodies

Transient gene expression of human antibodies is performed after transfection of antibody expression vectors in human embryonic kidney 293-T cells or in Chinese hamster ovary (CHO) cells using the polyethyleneimine (PEI, Polyscience transfection method) Warrington, United States). After transfection, the cells are grown in serum free medium (OPTI-MEM I supplemented with GlutaMAX-I Gibco). Supernatants are collected after 3-6 days of culture and the IgG is purified using protein A columns (GE HealthCare, Sweden) in a fast liquid protein chromatography device (FPLC ) (GE HealthCare , Sweden).

Example 4: In vitro neutralizing assays based on cells

Neutralization assays were performed on cell lines that responded to the cytokine studied, that is, carried the necessary receptor. The binding of the ligand with the receptor activates a corresponding signaling pathway, translocation of transcription factors to the nucleus and positively regulates transcription of the responding gene, 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 analyze the neutralizing capacity of the antibodies, the optimal concentration of the target cytokine was pre-incubated with serial dilutions of serum, supernatant or purified antibody samples. The results are expressed as antibody titer or concentration that shows the intermediate value between the positive and negative controls.

IL-17A neutralizing test

1BR.3.G cells from human skin fibroblasts were seeded at 1 x 104 cells / well in which IL-17-A (2 ng / ml) was previously exposed to serially diluted serum, supernatant or antibody samples during 2 hours in DMEM with 10% FBS inactivated. After incubation at 37 ° C for 16-20 hours, supernatants were collected and analyzed by ELISA assays to determine the production of a growth-related oncogene (GRO ). Based on absorbance plots of ELISA-type assays, the results are estimated as the concentration of serum antibodies or titre in the supernatant or the concentration of antibodies, which produces an intermediate value between the positive and negative controls. The ED50 values are defined as the concentration or titre necessary to reduce the cytokine activity of the test sample in half.

IL-17F neutralizing test

Keratinocytes NCTC 2544 were previously treated for 3 hours with TNF-a (0.1 ng / ml) in DMEM with 10% FBS inactivated. Serial dilutions of serum samples, culture supernatants or purified antibodies were incubated together with 10 ng / ml of IL-17F in a 96-well tissue culture plate at 37 ° C. After 2 hours of joint incubation, keratinocytes were added to 1 x 104 cells / well. After incubation at 37 ° C for 16-20 hours, supernatants were collected and analyzed by ELISA assays to determine the production of a growth-related oncogene (GRO ). From the absorbance plots of ELISA assays, the results were estimated as the concentration of serum antibodies or titer in the supernatant or the concentration of antibodies, which produces an intermediate value between the positive and negative controls. The ED50 values were defined as the concentration or titre necessary to reduce the cytokine activity of the test sample in half.

Neutralizing assay of the IL-17A / IL-17F heterodimer

Keratinocytes NCTC 2544 were previously treated for 3 hours with TNF-a (0.1 ng / ml) in DMEM with 10% FBS inactivated. Serial dilutions of serum samples, culture supernatants or purified antibodies were incubated together with 5 ng / ml of IL-17A / IL-17F heterodimer in a 96-well tissue culture plate at 37 ° C. After 2 hours of joint incubation, the keratinocytes were added to 1 x 104 cells / well. After incubation at 37 ° C for 16-20 hours, supernatants were collected and analyzed by ELISA assays to determine the production of a growth-related oncogene (GRO). Based on absorbance plots of ELISA-type assays, the results are estimated as the concentration of serum antibodies or titre in the supernatant or the concentration of antibodies, which produces an intermediate value between the positive and negative controls. The ED50 values were defined as the concentration or titre necessary to reduce the cytokine activity of the test sample in half.

Example 5: Validation of target antibodies

1. Imiquimod-induced psoriasis skin inflammation protocol in selected animal models To evaluate the effect of anti-IL-17 autoantibodies derived from APECED and other APECED derived autoantibodies on inflammation of skin induced psoriasis type by imiquimod, mouse models were used. The back of anesthetized C57B1 / 6 mice was shaved 48-72 hours before treatment. Twenty-four hours before or after the application of imiquimod, the mice received intraperitoneal injection of anti-IL-17 antibodies and other autoantibodies derived from APECED, with additional doses at 2-day intervals. Control mice received human Ig injection as a 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 petroleum jelly. Mice were scored daily using an objective scoring system based on the severity index and area of clinical psoriasis (PASI, Psoriasis Area and Severity Index). Erythema, desquamation and thickening, were scored independently on a scale of 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. Spleens were weighed and T lymphocytes derived from lymph nodes were stimulated overnight for analysis by flow cytometry.

2. Induction of EAE in Air-deficient mice, treated together with anti-IL-17 antibodies

The Aire - / _ mouse model, in which the coding potential of the Aire protein, which is predicted to precipitate autoimmunity, is interrupted, was used to investigate the possibly protective effects of anti-IL-17 autoantibodies and others autoantibodies derived from patients with APECED after induction of EAE (experimental allergic encephalomyelitis) by an emulsion of MOG35-55 (immunogenic peptide fragment of myelin glycoprotein of oligodendrocytes ( myelin oligodendrocyte glycoprotein), which induces an autoimmune destruction of nerve cells expressing MOG)); and CFA ( Freund's complete adjuvant). For example, in this regard, previously filled syringes with Freund's complete adjuvant emulsion (CFA) containing 1 mg of MOG35-55 / ml of emulsion and 2 mg of the H37Ra strain of destroyed Mycobacterium tuberculosis / ml of emulsion were used. Aire_ / 'and wild-type female mice were pretreated 24 hours before the start of EAE induction with intraperitoneal injection of antibodies (e.g., anti-IL-7 and anti-IFN antibodies), with additional doses at intervals of 2 days. Control mice received control immunoglobulin injection. Twenty-four hours after the initial administration of the antibodies, female Air_ / 'and wild-type mice minimally stressed (at least 7 days acclimatization, static cages, quiet environment) received subcutaneous injection (upper and lower dorsal part) of MOG35 emulsion -55 CFA to stimulate EAE. At 2 hours, the treated mice received intraperitoneal injection of pertussis toxin to exacerbate the induction of EAE, with an additional dose of subcutaneous pertussis toxin administered at 22-26 hours. EAE induction was scored 7 days after onset and later using the standard clinical observation scale of Hooke Lab, scoring the severity of the EAE phenotype on a scale of 0-5. A phenotype with a score of 0 represents no obvious changes, while a phenotype with a score of 5 indicates complete paralysis of the front and rear legs.

3. Protocol to induce EAE in mouse models

To investigate the effect of anti-IL-17 autoantibodies and other APECED derived autoantibodies, after induction of EAE by an emulsion of MOG35-55 (immunogenic MOG peptide that precipitates an autoimmune destruction of nerve cells expressing MOG) and CFA, a mouse model was used.

Female Air - / - and wild-type mice were pretreated 24 hours before the start of EAE induction with intraperitoneal injection of anti-IL-17 antibodies, with additional doses at 2-day intervals. As a control, control mice received injection of Ig. Twenty-four hours after initial administration of the antibodies, female Air - / - and wild-type mice minimally stressed (at least 7 days acclimatization, static cages, quiet environment) received subcutaneous injection (upper and lower dorsal part) of MOG35 emulsion -55 and CFA to cause EAE. At 2 hours, the treated mice received intraperitoneal injection of pertussis toxin to exacerbate the induction of EAE, with an additional subcutaneous dose of pertussis toxin administered at 22-26 hours. EAE induction was scored 7 days after onset and subsequently using the standard clinical observation scale of Hooke Lab, scoring the severity of the EAE phenotype on a scale of 0-5. A phenotype with a score of 0 represents no obvious changes, while a phenotype with a score of 5 indicates complete paralysis of the front and rear legs.

4. Protocol to induce collagen-induced arthritis in mouse models.

To investigate the effect of anti-IL-17 autoantibodies and other APECED derived autoantibodies on the induction of collagen-induced arthritis (AIC), a mouse model was used. Twenty-four hours before the induction of AIC, C57B1 / 6 mice were previously treated with APECED derived autoantibodies injected intraperitoneally, with additional doses at 2-day intervals (as a control Ig was used). Collagen-induced arthritis was caused by intradermal injection (2 x tail base injections) of type II collagen from chicken (prepared with CFA at a 1: 1 ratio) extracted from chicken sternum cartilage. Fourteen days after induction, an additional reinforcement of chicken type II collagen and IFA was injected intradermally at a ratio of 1: 1. Mice were monitored and scored for arthritis every day after 2 weeks after primary immunization, producing a typical onset of arthritis between 3 and 6 weeks after immunization, and scored by clinical control. (scoring the swelling of the hind legs with calipers), measuring anti-collagen antibodies (ELISA using collagen-coated plates), T-cell responses (T-cell proliferation in response to chicken collagen as determined using incorporation of [ 3H] thymidine) or an ELISA assay with cytokines.

5. Protocol to induce DSS colitis in mouse models.

To assess the effect of anti-IL-17 autoantibodies and other APECED derived autoantibodies on the induction of DSS colitis, mouse models were used. Twenty-four hours before the induction of DSS colitis, C57B1 / 6 mice were previously treated with APECED derived autoantibodies injected intraperitoneally, with additional doses at 2-day intervals. Chronic DSS colitis was induced by drinking water cycles containing 2% DSS for 5 days, followed by 14 days with autoclaved sterile drinking water without DSS; repeated for 3 cycles. Control mice received only autoclaved drinking water. The induction and severity of DSS-induced colitis was measured by histological evaluation (staining with hematoxylin and eosin in paraffin sections), full-thickness organ culture (with a collection of supernatants at 24 hours for cytokine analysis) or direct tissue sampling for qPCR (quantitative PCR) or immunoblot. Mice subjected to DSS-induced colitis were also weighed daily to determine the degree of weight loss, since colitis is highly associated with consumptive disease.

The efficacy of antibodies derived from humans against cytokines or other relevant molecules will also be evaluated to determine the effects of such antibodies, for example, anti-IL-17F on inflammation in grafted human tissue in immunodeficient mouse strains, such as It is described in Wang et al., Sci Transl Med 3 (2011): 83ra42.

Example 6: Results of imiquimod-induced psoriasis skin inflammation

In mice treated with Imiquimod topically to induce psoriasis-type skin inflammation (see Fig. 3 the chronology and experimental treatment scheme), anti-IL-22 [HD-MAB] antibodies derived from APECED reduce significantly the severity index and psoriasis area (PASI) scores in relation to control IgG antibodies.

Similarly, experiments with the exemplary human-specific 24D3 antibody, anti-IL-17, had no effect on imiquimod-induced psoriasiform lesions, validating the specificity of the 24D3 antibody against the human antigen.

Example 7: Results of the ear inflammation induced phenotype

The ear inflammation phenotype was induced in 8-week-old C57BL / 6J (TS) mice by intradermal injection of human IL-17A or IL-17F or PBS cytokines into each ear, given on alternate days on Day 1, Day 3, Day 5 and Day 7 [20 ul / ear, 500 ng / ear, 1 | jg / mouse / day]. Treatment with the exemplary 24D3 antibody, anti-IL-17 of the present invention, was analyzed in these animals for their neutralizing potential to reduce the ear inflammation induced phenotype. Every two days, for 8 days starting with the first IP the day 0, four injections of 24D3 IP or control human IgG [200 pg / IP] were administered to the animals, before induction of the inflammation of the ear. To analyze a possible therapeutic effect of the antibodies of the present invention, the body weight and ear thickness of the animals were monitored during the administration of the antibody. Also, histological stains were performed with H&E (hematoxylin and eosin; [52, 53]) of the ears. The combination of two independent experiments showed that induction of ear swelling with intradermal injection of human IL-17F was reduced in the presence of neutralizing antibody 24D3, this being significant on day 8; see Figs. 4A, D and F. The induction of inflammation of the ear with intradermal injection of human IL-17A is not affected by the treatment of 24D3, which further validates the specificity of this antibody for human IL-17F; see Figs. 4A and C and Fig. 4E. The level of inflammation of the ear after continuous intradermal injection of the control treatment with PBS, is not affected by the presence of IgG or 24D3; see Figs. 4A, B and F. The normalization of the data obtained against the values obtained in the controls treated with PBS reaches a greater significance with respect to the values which indicates the therapeutic effect of the exemplary anti-IL-17F antibody; see, in particular, Figs. 4J and L. The therapeutic effect of the exemplary anti-IL-17F antibody of the present invention is further confirmed by preliminary data obtained by controlling the weight of animals treated with 24D3, which show an almost zero or at least enormously weight loss. reduced after induction of inflammation by injections of IL-17F; see Fig. 5.

Example 8: Epitope mapping of exemplary α-IL17 antibodies

As a first mapping stage, to determine the number of different binding sites, the differential binding of MAB a-IL17 to different antigen binding sites was examined.

To this end, two approaches have been used. In the first approach, the MABs were expressed with human (hMAB) or mouse (hmMAB) Fc and the cross-competition experiments were carried out by plating antigen and detecting the binding of the hmMAB in the presence of a large excess of human MAB. The detection of hmMAB bound to the ligand was performed by a secondary antibody conjugated to HRP directed against the Fc part of the primary antibody. As can be seen in Fig. 6, the exemplary a-IL-17 antibodies, 17E3 and 24D3 of the present invention, compete with each other but not with the 9A2 antibody, indicating that the 9A2 antibody binds to another site other than of 17E3 and 24D3.

Subsequently, an attempt was made to map the MAB binding regions with their respective antigens using PepStar ™ analysis. Here, 20-unit peptides (15 amino acid overlap) were designed to cover IL-17A and IL-17F, including all known variants. the peptides and the full length antigen (as a positive control) were applied to the microarray and the peptide microarray was incubated with the primary antibody followed by a fluorescently labeled secondary antibody directed against the Fc part of the primary antibody. To avoid false negatives caused by steric hindrance, an optimized hydrophilic linker residue was inserted between the glass surface and the antigen-derived peptide sequence.

The results of the mapping of the MAB to-IL-17 are shown in Fig. 7. Full length antigens applied together were generally well recognized in the peptide microarray. Not all MAB a-IL-17 show a clear binding, however, unlike peptides, due to the orientation and use of surface lysines to immobilize antigen, low signal intensity or absence of signal is not correlates necessarily with an absence of union. The expected binding intensity that will be observed when binding to a specific peptide is in the range of 60,000. All antibodies show very weak binding patterns with peptides bound to microarrays, indicating that linear peptides derived from their specific antigen are not recognized. These data suggest that MABs against IL17 bind to conformational epitopes; for a summary of the characteristics of the antibodies see Table 3 of Example 9.

Example 9: Measurement of antibody affinity using surface plasmon resonance technology (SPR, surface p lasm on resonance)

To determine the affinity of the antibodies of the present invention, SPR measurements were made using a ProteOn ™ XPR36 instrument, in accordance with the manufacturer's instructions (BIO-RAD; Hercules, USA). First, the human anti-IgG antibody was simultaneously coupled on 5 surfaces of the 6 x 6 interaction matrix of a GLC detection microplate by EDC / NHS chemistry, no sixth antibody was immobilized on the sixth surface, although the surface was dealt with the same activation and deactivation protocol as that of the other five surfaces. Subsequently, the MABs (ligands) that were analyzed were injected into 6 channels of the microplate (in the vertical direction) and captured simultaneously at a very similar capture efficiency and percentage of activity. Subsequently, the microplate was turned 90 degrees and the analyte to be analyzed in horizontal direction was injected at different concentrations ranging from 100 nM to 1.23 nM, each association / dissociation curve representing a different concentration (A1-Red, 100 nM, A2-Cyan (33.33 nM), A3-Blue (11.11 nM), A4-Green (3.70 nM), A5-Pink (1.23 nM), A6-Orange, only current buffer) in the Fig 8. The data was referenced using the regions between applications between the flow channels. Binding experiments were repeated twice and the data was represented as an average of two independent experiments.

Injections of IL-17F showed concentration-dependent responses on surfaces with immobilized 9A2, 17E3 and 24D3 antibodies, confirming their binding specificity towards IL-17. In the dissociation phase, an "increase" could be observed in the response that seems to reflect a simultaneous association and dissociation during this phase. Possible causes of this are the aggregation of antigens on the surface of the antibody or the accumulation of antigens from the instrument tubes. Due to complex data in the dissociation phase, it is impossible to accurately determine the kd and, therefore, the kinetics, however, the affinity seems to be very high when the vast majority of the "dissociation" data is excluded, and The ka can vary from approximately 3x10E + 05 (17E3), about approximately 6x10E + 05 1 / Ms (24D3) to approximately 8x10E + 05 1 / M (9A2). There is no evidence of non-1: 1 behavior, that is, there is no evidence of a heterogeneous ligand.

Table 3: Summary of the characteristics of the exemplary a-IL-17 antibodies 9A2, 17E3 and 24D3 of the present invention. F-IL-17F A-IL17A h-human

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Claims (12)

1. A human anti-interleukin-17F monoclonal antibody (IL-17F), or antigen binding fragment thereof, in which the antibody recognizes a conformational epitope in IL-17F and is capable of neutralizing the biological activity of IL -17F, and the antibody or antigen-binding fragment thereof, comprises in its variable heavy chain (VH) and in its variable light chain (VL), the amino acid sequence of the VH and VL chain of SEQ ID NO: 15 and 17, respectively, or of SEQ ID NO: 23 and 25, respectively. 2. The antibody or antigen-binding fragment of claim 1, which is a single stranded Fv fragment (scFv), an F (ab ') fragment, an F (ab) fragment or an F (ab') 2 fragment. 3. The antibody of claim 1 or 2, which comprises a constant region Ch and / or Cl comprising an amino acid sequence selected from the amino acid sequences of Ch and Cl set forth in SEQ ID NO: 11, 13, 19, 21, 27 and 29 or of an amino acid sequence with an identity of at least 60%. 4. One or more polynucleotides encoding at least the variable region of the antibody or antigen binding fragment of any one of claims 1 to 3. 5. One or more expression vectors comprising the polynucleotide or polynucleotides of claim 4. 6. A host cell comprising the polynucleotide or polynucleotides of claim 4 or the vector or vectors of claim 5. 7. The antibody or antigen-binding fragment of any one of claims 1 to 3, which is detectably labeled or bound to a drug. 8. The antibody or antigen-binding fragment of claim 7, wherein the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorophore and a heavy metal. 9. A composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 3, 7 or 8, the polynucleotide or polynucleotides of claim 4, the vector or vectors of claim 5 or the cell of claim 6, wherein the composition preferably comprises the two antibodies comprising in its VH chain and in its VL chain, the amino acid sequences of the VH and VL chain of SEQ ID NO: 15 and 17, respectively, and of SEQ ID NO: 23 and 25, respectively. 10. The composition of claim 9, which is (a) a pharmaceutical composition and further comprising a pharmaceutically acceptable carrier, preferably comprising an additional agent useful for treating inflammation or an autoimmune disorder, selected from the group consisting of non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, antihistamines and combinations thereof, or (b) a diagnostic composition or kit and further comprising reagents for use in immunological or nucleic acid based diagnostic methods. 11. The antibody or antigen-binding fragment of any one of claims 1 to 3, 7 or 8, for use in the treatment or prevention of the progression of an inflammation or an autoimmune disorder; for the improvement of symptoms associated with inflammation or an autoimmune disorder; to diagnose or examine a subject to determine the presence or risk of a subject developing inflammation or an autoimmune disorder, preferably, wherein said antibody or antigen binding fragment is designed to be administered intravenously, intramuscularly, subcutaneously , intraperitoneal, intranasal, parenteral, or as an aerosol. 12. The antibody, or antigen binding fragment thereof, for use according to claim 11, wherein said disorder is selected from the group consisting of common acne; arthritis, such as gouty arthritis, systemic lupus erythematosus (SLE), osteoarthritis, psoriatic arthritis, rheumatoid arthritis; asthma; celiac disease; chronic prostatitis; dermatitis; type 1 diabetes mellitus; glomerulonephritis; hypersensitivity; myocarditis; multiple sclerosis; inflammatory bowel diseases; pelvic inflammatory disease; polymyositis; psoriasis; sarcoidosis; vasculitis; interstitial cystitis, or inflammation occurs due to reperfusion injury or transplant rejection.