MONOCLONAL ANTIBODIES AGAINST BAMBI AND USE THEREOF FOR TREATING INFLAMMATORY DISEASES

Abstract

The invention relates to monoclonal antibodies, in particular against a peptide of the BAMBI protein, as well as the uses thereof and methods comprising same. Preferably, the antibodies are used for the treatment of autoimmune diseases.

Claims

1. A monoclonal antibody that specifically recognizes the sequence SEQ ID NO: 1, or SEQ ID NO: 2.

2. (canceled)

3. The antibody according to claim 1, wherein the antibody comprises an amino acid heavy chain encoded by the nucleotide sequence that comprises the sequence SEQ ID NO: 3 and an amino acid light chain encoded by the nucleotide sequence that comprises the sequence SEQ ID NO: 4.

4. The antibody according to claim 1, wherein the antibody comprises an amino acid heavy chain encoded by the nucleotide sequence that comprises the sequence SEQ ID NO: 5 and an amino acid light chain encoded by the nucleotide sequence that comprises the sequence SEQ ID NO: 6.

5. (canceled)

6. A cell expressing the antibody according to claim 1.

7. An in vitro use of the antibody according to claim 1 for the inhibition of BMP and Activin Membrane Bound Inhibitor (BAMBI).

8. The antibody according to claim 1 for use as a medicament.

9. The antibody according to claim 1 for use in the treatment or prevention of autoimmune diseases.

10. The antibody for use according to claim 9 wherein the autoimmune diseases are selected from the list consisting of: autoimmune arthritis, spondyloarthritis, psoriasis, systemic lupus erythematosus, and inflammatory bowel disease, preferably wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

11. (canceled)

12. A pharmaceutical composition comprising the monoclonal antibody according to claim 1.

13. The in vitro use of the antibody according to claim 1 for drug screening.

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. A kit and/or device comprising the antibody according to claim 1.

19. (canceled)

20. The in vitro use of the kit according to claim 18 for the detection of the sequence SEQ ID NO: 1 or SEQ ID NO: 2.

21. (canceled)

22. (canceled)

23. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0067] FIGS. 1A-1C illustrate characterization of the murine anti-BAMBI (109-133) mAbs B101-37 and B143-14. FIG. 1A illustrates specificity of the anti-BAMBI mAbs B101-37 and B143-14 evaluated by Western Blot in heart cell membrane lysates from normal B6 and B6.BAMBI-KO mice. FIG. 1B illustrates recognition of human BAMBI by the mAb B101-37 evaluated by Western Blot in human heart cell membrane lysates. FIG. 1C illustrates CDR sequence of heavy and light chains of the mAbs B101-37 and B143-14. The VDJ and VJ recombination of the heavy and light chains, respectively, of both mAbs are indicated.

[0068] FIGS. 2A-2B illustrate the induction of BAMBI expression in murine and human CD4+ T lymphocytes after the activation thereof. FIG. 2A illustrates comparative analysis by means of flow cytometry of the expression of BAMBI in CD4+ T lymphocytes of normal B6 and B6.BAMBI-KO mice before, and 48 hours after in vitro activation thereof with anti-CD3 and anti-CD28 antibodies in the presence or absence of TGFβ or IL-2 (top panels). On the bottom panels, the staining with B101-37 of the CD4+ T lymphocytes activated in B6.BAMBI-KO mice is compared with that of the IgG1 isotype control in CD4+ T lymphocytes activated in normal mice. FIG. 2B illustrates induction of BAMBI in human T lymphocytes stimulated in vitro for 48 hours with anti-CD3 and anti-CD28 mAbs. The BAMBI expression was analyzed by Western Blot in plasma membrane lysates. As loading control, the N-Ras expression was compared in the same lysates.

[0069] FIGS. 3A-3C illustrates the effect of the BAMBI inhibition on the in vitro differentiation of murine and human CD4+ T lymphocytes into Treg and TH17 cells. FIG. 3A illustrates naive CD4+CD25−CD62L+CD44− T cells of normal B6 and BAMBI-KO mice were stimulated for 5 days with anti-CD3 and anti-CD28 antibodies in Treg (top panels) or TH17 (bottom panels) polarizing conditions in the presence of mAb B143-14 (IgM anti-BAMBI) or a mouse polyclonal IgM (Sigma). FIG. 3B illustrates human naive CD4+ T lymphocytes purified by magnetic separation were activated in vitro for 10 days with anti-CD3 and anti-CD28 antibodies conjugated to beads in TH0 or Treg differentiation conditions and in the presence of mBa B143-14 (IgM anti-BAMBI) or a mouse polyclonal IgM. The percentages of CD4+FoxP3+ cells are shown, analyzed by means of flow cytometry in TH0 differentiation conditions (white bars) and Treg differentiation conditions (black bars). FIG. 3C illustrates human CD4+ T lymphocytes, purified by magnetic separation, were activated in vitro for 10 days with anti-CD3 and anti-CD28 antibodies conjugated to beads in TH0 or TH17 differentiation conditions. The inhibition effect of BAMBI with the mAb B143-14 on the differentiation into TH17 cells, either IFNg producers or not, analyzed by means of flow cytometry, is shown. The statistical differences are represented as: **p<0.01.

[0070] FIGS. 4A-4D illustrate MAb B101-37 inhibits the development of arthritis in the CIA model. FIG. 4A and FIG. 4B illustrate CIA induction, normal B11RIII mice were immunized with bovine type II collagen emulsified in CFA. The different experimental groups received treatments with 2 mg/mouse/week, 0.3 mg/mouse/week of B101-37 or with 2 mg/mouse/week of irrelevant murine IgG1 (IgG1-C) during the first 4 weeks after immunization. The clinical degree of severity of each mouse (FIG. 4A) and of different radiological injuries (average±SD) associated with articular destruction at the eighth week after immunization is shown (FIG. 4B). As controls for the aforementioned experiments, the development of CIA between normal B10RIII and BAMBI-KO mice was compared. The evolution of the clinical severity of arthritis in these animals, expressed as average±SD. (FIG. 4C) and of different radiological injuries (average±SD) associated with the articular destruction at the eighth week after immunization is shown (FIG. 4D). The statistical differences are represented as: *p<0.05, **p<0.01.

[0071] FIGS. 5A-5B illustrate the effect of the treatment with B101-37 on the development of psoriatic arthritis induced by mannan injection. FIG. 5A illustrates normal B10RIII or BAMBI-KO mice treated or not treated from the beginning of the experiment with mAb B101-37 (2 mg/mouse/week) received an IP injection of 10 mg of mannan obtained from the yeast Saccharomyces cerevisiae. The evolution of the severity of the arthritis and of the percentage of the increase in ear thickness (average±SD) in the different experimental groups is shown. FIG. 5B illustrates photographs showing the macroscopic appearance of the auricle of the experimental groups described in (A). The statistical differences are represented as: *p<0.05, **p<0.01, *** p<0.001.

[0072] FIG. 6 illustrates the therapeutic effect of treatment with B101-37 in the development of chronic psoriatic arthritis. Normal B10RIII mice received a weekly injection of 10 mg of mannan (white arrows) and were treated with mAb B101-37 (2 mg/mouse/week) from the moment of the first mannan injection (preventative B101-37) or from the appearance of the first signs of the disease (therapeutic B101-37) until the end of the experiment (black arrows). As controls, mice treated with 2 mg/mouse/week of one irrelevant murine IgG1 (IgG1-C) were used. It shows the evolution of the clinical severity of the arthritis (top panel) and the ear thickness (bottom panel) as a marker of the severity of the cutaneous psoriasis. The statistical differences are represented as: *p<0.05, **p<0.01, *** p<0.001.

[0073] FIGS. 7A-7B illustrate the effect of treatment with B101-37 in the development of imiquimod-induced psoriasis. 12.5 mg of imiquimod (Aldara□) was applied for 6 days on the right ears of the B10RIII mice, BAMBI deficient or not. The different experimental groups were treated from the moment of the first application of imiquimod with a single dose of 2 mg of B101-37 or of irrelevant murine IgG1 (IgG1-C). FIG. 7A illustrates evolution of the clinical severity of the cutaneous injuries, evaluating the appearance and severity of erythema, desquamation of the skin and thickness of the ear treated in comparison with the non-treated contralateral ear. FIG. 7B illustrates histologic severity of the cutaneous injuries. The top photographs show representative examples (×10) of histologic cuts of the ears stained with haematoxylin and eosin. The lower panels show the values of the thickness of the epidermis (left panel) and of the dermis (right panel) in the different animals of each experimental group. The statistical differences are represented as: *p<0.05, *** p<0.001.

[0074] FIGS. 8A-8C illustrate the therapeutic effect of the treatment with B101-37 on the development of DSS colitis. Normal B6 or BAMBI-KO mice, treated or not from the beginning of the experiment with mAb B101-37 (2 mg/mouse/week) received DSS dissolved at a ratio of 3% in the water of the bottle for 5 days. The severity of the colitis was assessed by means of DAI quantification (FIG. 8A) or analyzing the shortening of the colon (FIG. 8B). FIG. 8C illustrates mortality in the different experimental groups. The statistical differences are represented as: *p<0.05, **p<0.01.

EXAMPLES

[0075] The invention is illustrated below by means of tests carried out by the inventors which reveal the effectiveness of the product of the invention.

Material and Methods

Obtention and Characterization of the Murine Anti-BAMBI Monoclonal Antibodies.

[0076] B6.BAMBI-KO mice were immunized with the murine BAMBI (109-133) peptide conjugated to keyhole limpet hemocyanin (KLH) and emulsified in complete Freund's adjuvant (CFA). The mice were immunized on two more occasions (with one month difference between each immunization) with the same peptide emulsified in incomplete Freund's adjuvant (IFA). The murine BAMBI (109-133) peptide is located in the extracellular region of BAMBI and differs from its human homologue by 4 amino acids (positions 8, 9, 10 and 23 of the SEQ ID NO: 1). The presence of murine anti-BAMBI (109-133) circulating antibodies in immunized mice was evaluated 15 days after each immunization by means of ELISA. Mice with higher titers of these antibodies were used for obtaining the murine anti-BAMBI monoclonal antibodies (mAb). To do so, spleen cell suspensions were fused with non-secreting myeloma cell line SP2/O-Ag14 as was previously described (Yokoyama W M. et al. Curr Protoc Immunol. 2013, Unit 2.5). Hybridoma that produce human anti-BAMBI mAb were selected by enzyme-linked immunosorbent assay (ELISA). In the present invention, two of the mAbs obtained have been characterized; clone B101-37 (igG1, k anti-BAMBI) and clone B143-14 (IgM, k anti-BAMBI). The specificity of the mAbs was subsequently evaluated by means of Western Blot in heart cell membrane of hearts from normal B6 and B6.BAMBI-KO mice and of human myocardial samples obtained from surgical biopsies.

[0077] Ribonucleic acid (RNA) of mAbs B101-37 and B143-14 were isolated by means of the commercial kit RNeasy Mini Kit (Qiagen). For defining the encoding sequences for the complementarity-determining regions of (CDRs) in the heavy and light chain of both mAbs, RT-PCRs were carried out based on the purified RNAs, as previously described (Wang Z. et al. J Immunol Methods. 2000, 233:167). For defining the CDR of the heavy chain of B101-37, the following amplicons were used: degenerate amplicon 5′ from the FR1 region of the heavy chain: 5′-CTT CCG GAA TTC SAR GTN MAG CTG SAG SAG TC-3 (SEQ ID NO: 7)′; amplicon 3′ of the constant region of IgG1: 5′-GGA AGA TCT ATA GAC AGA TGG GGG TGT CGT TTT GGC-3′ (SEQ ID NO: 8). For defining the CDR of the heavy chain of B143-14, the previously mentioned degenerate amplicon from the FR1 region of the heavy chain and the amplicon 3′ of the constant region of IgM were used: 5′-GGA AGA TCT GAC ATT TGG GAA GGA CTG ACT CTC-3′ (SEQ ID NO: 9). For defining the CDR of the heavy chain of B101-37 and B143-14, the following amplicons were used: degenerate amplicon from the FR1 region of the light chain k: 5′-GG GAG CTC GAT ATT GTG MTS ACM CAR WCT MCA-3′ (SEQ ID NO: 10); amplicon 3′ of the constant region of the light chain k. 5′-GGT GCA TGC GGA TAC AGT TGG TGC AGC ATC-3′ (SEQ ID NO: 11). The PCR products were subsequently sequenced (STABVida, Caparica, Portugal) and the sequences were analyzed by means of the IgBLAST program.

Study of BAMBI Expression in Murine and Human CD4 T Lymphocytes.

[0078] By means of flow cytometry BAMBI expression in CD4+ T lymphocytes of normal B6 mice after the stimulation thereof using biotinylated mAb B101-37. The isolated CD4+ T lymphocytes of the spleen of normal B6 mice were stimulated in vitro for 48 hours with antibodies anti-CD3 (1 μg/well) and anti-CD28 (0.5 μg/well) bound to the plate in the presence or absence of 2 ng/ml of recombinant murine TGFβ and/or 1 ng/ml of recombinant murine IL-2 (PeproTech, London). As negative controls, spleen cells of B6.BAMBI-KO mice stimulated in the same way and colored with biotinylated B101-37 and normal B6 mice colored with a biotinylated isotype control were used. The colored cells were analyzed in a FACSCanto II cytometer equipped with FACSDiva software (BD Biosciences).

[0079] BAMBI expression in human CD4+ T lymphocytes after the in vitro stimulation thereof was analyzed by means of Western Blot. Said lymphocytes were purified from 50 ml of buffy coats coming from healthy donors of the Blood and Tissues Bank of Cantabria (Marqués de Valdecilla University Hospital, Santander). The mononuclear cells obtained after the Ficoll gradient were subjected to a positive selection after marking with specific mAb of CD4 conjugated to magnetic microparticles (MACS) using a magnetic separator (AutoMACS, Miltenyi Biotec). The CD4+ T lymphocytes were subsequently stimulated in vitro for 48 hours with antibodies anti-CD3 (1 μg/well) and anti-CD28 (0.5 μg/well) bound to the culture plate. The cell membrane lysates of the activated lymphocytes were obtained as described above.

In Vitro Differentiation Cultures of Murine and Human CD4+ T Lymphocytes into Treg and TH17 Cells.

[0080] The inhibiting capability of anti-BAMBI mAbs directed against the BAMBI (109-133) peptide were explored in vitro in murine and human CD4+ T lymphocyte cultures differentiated into Treg and TH17 cells. In these experiments, mAb B143-14 was used. In the experiments with murine lymphocytes, naïve CD4+ cells (CD4+CD25−CD62L+CD44−) from spleens of normal B6 mice were purified by means of cell sorting (FACSAria, BD Biosciences). 5×10.sup.5 naïve CD4+ cells were stimulated for 5 days with anti-CD3 and anti-CD28 antibodies bound to the plastic of the culture plate, in Treg polarizing conditions (2 ng/ml of murine TGFβ) or TH17 (1 ng/ml of murine TGFβ and 10 ng/ml of murine IL-6) in the presence of 20 μg/ml of B143-14 or 20 μg/ml of murine IgM (Sigma, St Louis, Mo.) as an isotype control. The percentages of lymphocytes TCD4+FoxP3+ (Treg) and CD4+IL-17+ (TH17) at the end of the culture were analyzed by means of flow cytometry, as was previously described (Iglesias M. et al. Arthritis Rheum 2013, 65:343).

[0081] Human naïve CD4+ T lymphocytes (in Treg differentiation) or CD45RO+ memory T lymphocytes (in TH17 differentiation), purified by magnetic separation, were activated in vitro for 10 days with anti-CD3 and anti CD28 antibodies (aBs) conjugated to beads in Treg differentiation conditions (5 ng/ml of TGFβ) or TH17 (20 ng/ml of IL-1β, 30 ng/ml of IL-6, 30 ng/ml of IL-23, 3 ng/ml of TGFβ1, 1 μg/ml of anti-IFNγ and 2.5 μg/ml of anti-IL-4) in the presence of 20 μg/ml of B143-14 or 20 μg/ml of murine IgM. The percentages of lymphocytes TCD4+FoxP3+ (Treg) and CD4+IL-17+ (TH17) at the end of the culture were analyzed by means of flow cytometry.

Experimental Model of Arthritis after Immunization with Bovine Type II Collagen Emulsified in CFA (CIA).

[0082] Groups of 10 B10RIII (MHC H-2r) BAMBI-deficient or not mice were immunized before their 12.sup.th week of age by intradermal route at the root of the tail with 150 μg of bovine type II collagen (MD Biosciences, Zurich) emulsified (vol. 1/1) in CFA containing a concentration of Mycobacterium tuberculosis of 4 mg/ml (MD Biosciences) as was previously described (Iglesias M. et al. Arthritis Rheum 2013, 65:343). To study the effect of BAMBI inhibition in the development of CIA, normal B10RIII mice were intraperitoneally (IP) treated during the first 4 weeks after immunization with 2 or 0.3 mg/week of B101-37 or with 2 mg/week of anti-TNP IgG1 used as an isotype control (IgG1-C). The evolution of arthritis was monitored on a weekly basis from the 3rd week (the date on which arthritis began) to the eighth week after immunization. The severity of the arthritis was evaluated in each of the four extremities by means of the method described by Wooley et al (Wooley P H. et al. J Immunol 1985, 135:2443). Likewise, on the eighth week of immunization the articular injuries on the front and back legs were evaluated by means of radiology, as we previously described (Iglesias M. et al. Arthritis Rheum 2013, 65:343).

Experimental Model of Dextran Sulfate Sodium Colitis (DSS-Colitis).

[0083] For the induction of colitis, DSS (MPbio.com), DSS was dissolved in water at 3%. This solution (with daily changes of water) was administered in a bottle to the mice of the different experimental groups for a variable time period [until the mice of the control group reached an activity ratio of the disease (DAI) of between 1.5-2 (approximately 4-5 days)]. The water consumed by each experimental group was measured daily and the DAI was evaluated calculating the clinical score of the following parameters: A) weight loss: 0=no weight loss, 1=weight loss of 1.5%, 2=:weight loss of 5-10%, 3=weight loss of 10-20%, and 4=weight loss of more than 20%; B) fecal consistency (the same value is given to all animals that are in the same box). 0=normal fecal consistency, 2=soft feces and 4=diarrhea; C) rectal bleeding (the same value is given to all animals that are in the same box): 0=no blood, 2=minor bleeding and 4=intense bleeding. The final DAI value was calculated by adding the values of the different parameters and dividing by 3.

Mannan Induced Psoriatic Arthritis.

[0084] For the induction of acute symptoms of psoriatic arthritis, groups of B10RIII BAMBI-deficient mice, or mice not BAMBI-deficient received an IP injection of 10 mg of mannan obtained from S. cerevisiae yeast (Sigma-Aldrich) dissolved in 200 μl of PBS (Khmaladze I. et al. Proc Natl Acad Sci USA 2014, 111:E3669). Additionally, a chronic form of the process was induced in non-transgenic B10RIII mice by means of repeated injections of the same mannan dose once a week. To study the effect of the treatment with B101-37 on the prevention of psoriatic arthritis, mice that received a single mannan injection were treated from the moment of the administration of mannan (induction of the disease) with 2 mg/week of B101-37 or with 2 mg/week of an anti-TN P IgG1 used as IgG1-C. To evaluate the therapeutic effect of the treatment with B101-37, the non-transgenic B10RIII mice with the chronic form of the disease were treated with 2 mg/week of B101-37 (divided into 3 equal doses every two days) from the moment of the first mannan injection with (preventative treatment) or from the moment the first clinical signs of the process were observed (3 days after the first manna injection; therapeutic treatment) until the end of the experiment. Once again, as controls, non-transgenic B10RIII mice treated with an anti-TNP IgG1 from the moment of the appearance of the first signs of the disease were analyzed. The psoriatic injuries were evaluated at the level of the auricles, quantifying the thickness of the same with the help of a digital gauge. The severity of the arthritis was evaluated in each of the four legs, on a scale of 0-10 (total range of 0-40) in the following way: severe inflammation of the carpus/tarsus=5 points, adding an additional point for each inflamed toe. If the inflammation is minor=3 points per carpus/tarsus+0.5 points for each inflamed toe.

Experimental Model of Imiquimod Induced Cutaneous Psoriasis.

[0085] For the induction of cutaneous psoriasis, 12.5 mg of imiquimod (Aldara®) was topically applied for 6 days on the right ears of B10RIII mice, BAMBI deficient or not. The different experimental groups were treated at the moment of the first application of imiquimod with a single dose of 2 mg of B101-37 or of irrelevant murine IgG1. Erythema, desquamation and the increase in the thickness of the ear were evaluated daily. Erythema and desquamation were evaluated using a clinical score of 0 to 4 in the following way: none=0, minor=1, moderate=2, severe=3, highly severe=4. The increase in the thickness of the treated ear was calculated with the help of a digital gauge in comparison with the thickness of the non-treated left ear in the following way: no increase=0, increase of 0.1-10%, increase of 10.1-20%=2, increase of 20.1-30%=3, increase of 30.1-40%=4, increase of 40.1-50%=5. The animals were sacrifices 24 hours after the last application of imiquimod and skin samples were collected for the histological study. The thickness of the epidermis and the dermis in the histological samples from the ears treated with imiquimod were quantified in each animal using the ImageJ program, in relation to the respective thicknesses of the histological samples from the contralateral ears that were not treated.

Example 1

Molecular Characterization of Anti-BAMBI mAb

[0086] The specificity of the anti-BAMBI B101-37 and B143-14 mAbs was initially determined by means of ELISA (during the hybridoma screening process) and subsequently by means of Western Blot. Both mAbs recognize a band of approximately 27-29 kDa and another of approximately 54 kDa, compatible with BAMBI monomers and dimers resistant to sodium dodecyl sulfate (SDS) and reducing conditions, as was previously described (Xavier S et al. 2010 PLoS One 5:e12995) in lysates of B6 mice but not in those of B6.BAMBI-KO mice (FIG. 1A).

[0087] Although the murine BAMBI (109-133) peptide used for the development of anti-BAMBI mAb differs from its human homologue in 4 amino acids studied by Western Blot in myocardial plasma membrane lysates indicate that B101-37 also recognizes human BAMBI (FIG. 1B).

[0088] We subsequently characterize the CDRs of the heavy and light chains of both anti-BAMBI mAbs by means of deoxyribonucleic acid (DNA) sequencing (SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO; 6). The comparison of the DNA sequences with the IgBLAST database indicates that the CDR of the heavy and light chains of mAb B101-37 are the result of the reordering of the V9-3/D1-3/J1 and V15-103/J5 segments, respectively, while the CDRs of the heavy and light chains of mAb B143-14 are the result of the reordering of the V1-55/D4-1/J2 and V5-43/J1 segments, respectively (FIG. 1C).

Example 2

BAMBI Expression in CD4+ T Lymphocytes

[0089] CD4+ T lymphocytes play a fundamental role in the development of inflammatory and autoimmune pathologies. For this reason, the regulation of BAMBI expression in this lymphocytic population in mice is studied by means of flow cytometry and subsequently in humans by means of Western Blot, in both cases using mAb B101-37. In murine naïve CD4+ T lymphocytes BAMBI expression was not detected, but it was induced 48 hours after the in vitro stimulation thereof with anti-CD3 and anti-CD28 antibodies (FIG. 2A). This expression increased after the addition of TGFβ, but not of IL-2, to the activated CD4+ T cells (FIG. 2A). As specificity controls of mAb B101-37, markers in flow cytometry were not detected in activated CD4+ T lymphocytes of BAMBI-KO mice colored B101-37 or in normal B6 mice colored with an isotype control IgG1, respectively (FIG. 2A, bottom panels).

[0090] Just like in the mouse, BAMBI expression was very low in human naïve CD4+ lymphocytes, being induced after the stimulation thereof with anti-CD3 and anti-CD28 antibodies (FIG. 2B).

Example 3

BAMBI Inhibition with Murine Anti-BAMBI (109-133) mAb B143-14 Alters In Vitro Differentiation of the CD4+ T Lymphocytes of Mice and Humans into Subtypes Treg and TH17

[0091] In previous studies we showed that the absence of BAMBI in CD4+ T lymphocytes of BAMBI-KO mice strengthened and inhibited the in vitro differentiation thereof into Treg and TH17 populations, respectively (Postigo J et al. Doctoral Thesis defended on the 19 Apr., 2013, University of Cantabria). To evaluate the inhibiting effect of the anti-BAMBI mAbs directed against the murine BAMBI (109-133) epitope, we analyzed the capability of B143-14 to alter the functional differentiation of the CD4+ T lymphocytes of normal mice in the same sense as that observed in BAMBI-KO mice. To do so, naïve CD4+ T lymphocytes of normal B6 and BAMBI-KO mice were activated in vitro in polarization conditions to Treg or TH17 cells, in the presence of B143-14 mAb or a murine IgM used as an isotype control. Just as observed with the lymphocytes of BAMBI-KO mice, the BAMBI inhibition after the addition to the B143-14 culture, but not of the IgM control, increased and reduced the Treg and TH17 in vitro differentiation, respectively, of CD4+ T lymphocytes of normal B6 mice (FIG. 3A).

[0092] Lastly, we observed that in the presence of mAb B143-14, but not of the IgM control, the in vitro differentiation of human naïve CD4+ T lymphocytes (a Treg) or memory T lymphocytes (a Th17) activated in polarizing conditions to Treg (FIG. 3B) or TH17 (FIG. 3C) is altered in the same way as in the mouse: an increase in Treg and reduction in TH17.

Example 4

The Therapeutic Effect of mAb B101-37 in the Development of CIA, Mannan Induced Psoriatic Arthritis, Imiquimod Induced Psoriasis and DSS Colitis

[0093] The previous results indicate that: 1) we have mAbs able to recognize BAMBI in mice and humans, 2) the expression of BAMBI is induced in the CD4+ T lymphocytes after the activation thereof in mice and humans; and 3) in both species the in vitro inhibition of BAMBI with a murine anti-BAMBI (109-133) mAb alters the functional differentiation of the CD4+ T lymphocytes in the same was as that described in B6.BAMBI-KO mice (increase in Treg cells and reduction of TH17 cells). These findings raise the possibility that the in vivo inhibition of BAMBI has a therapeutic effect on inflammatory/autoimmune pathologies.

[0094] In the present invention we have characterized the therapeutic potential of B101-37 in the development of CIA (the experimental model for rheumatoid arthritis most used by the scientific community), mannan induced psoriatic arthritis, imiquimod induced psoriasis (the experimental model of cutaneous psoriasis most used by the scientific community) and DSS colitis.

[0095] Both from a clinical and radiological point of view, the treatment of the normal B10RIII mice with 2 mg/week of B101-37 during the first 4 weeks after immunization with bovine type II collagen inhibited the development of CIA in these animals, unlike that which was observed in mice treated with 0.3 mg/week of B101-37 or with 2 mg/week of IgG1-C (FIGS. 4A and 4B). CIA inhibition after treatment with the high dose of B101-37 was similar to that observed in B10RIII.BAMBI-KO immunized mice (FIGS. 4C and 4D).

[0096] Just like the CIA model, treatment with 2 mg/week of B101-37 from the moment of mannan administration to the normal B10RIII mice reduced the severity of the articular and cutaneous legions in a highly significant way in this experimental model of acute psoriatic arthritis induced after a single mannan injection, similar to that which is observed in B10RIII.BAMBI-KO (FIGS. 5A and 5B).

[0097] The previous results show a preventative effect of treatment with B101-37 on the development of acute cutaneous and articular injuries after the administration of a single injection of mannan. The therapeutic potential of B101-37 in the chronic variety of this experimental model was subsequently analyzed. FIG. 6 shows that the weekly mannan injection into B10RIII mice causes the appearance of articular and cutaneous injuries that are maintained over time. Preventative treatment with 2 mg/week of B101-37 37 (begun at the moment of the mannan injection and maintained until the end of the experiment) significantly reduced the severity of said injuries throughout the study (FIG. 6). Likewise, a significant reduction, similar to that observed in preventative treatment, of the cutaneous and articular manifestations was observed after the beginning of the treatment with B101-37 once the first signs of the disease were already apparent (three days after the first mannan injection (FIG. 6).

[0098] The therapeutic potential of B101-37 in psoriasis was confirmed in the cutaneous psoriasis experimental model most widely used in the scientific community, imiquimod induced psoriasis. The topical application of imiquimod for 6 consecutive days to non-transgenic B10RIII mice induces cutaneous legions with histological characteristics of psoriasis (FIGS. 7A and 7B). Unlike the previous model, the administration of imiquimod does not cause the development of arthritis. The treatment of non-transgenic B10RIII mice with B101-37, from the moment of the first application of imiquimod and with the same dose used in the preceding models, significantly reduced the severity of the disease from a clinical (FIG. 7A) and histological (FIG. 7B) point of view, as observed in B10RIII-BAMBI.KO mice.

[0099] Lastly, we evaluated the therapeutic effect of B101-37 in the experimental model of DSS colitis. Just like in B6.BAMBI-KO mice, normal B6 mice treated with 2 mg/week of B101-37 from the moment of the administration of DSS developed a significantly less sever colitis than the non-treated controls (FIG. 8A). However, the protective effect of the treatment with B101-37 in B6 mice was not as important as that observed in BAMBI-KO animals, especially with regard to the degree of shortening of the colon (FIG. 8A, right panel). It is worth pointing out that both in the B6 animals treated with B1010-37 and in BAMBI-KO mice, the mortality associated with the induction of DSS colitis was entirely prevented (FIG. 8B).

[0100] Therefore, the present invention demonstrates the use of monoclonal antibodies against BAMBI for the treatment and prevention of autoimmune diseases.