WO2021011799A1 - Inflammatory bowel diseases therapy involving disrupting ccr9:drd5 heteromer assembly - Google Patents

Abstract

A method for disrupting the formation of CCR9:DRD5 heteromer assembly in a subject involving administering to the subject a pharmaceutical composition having at least on disrupter agent that inhibits or blocks the interaction between CCR9 and DRD5. Also, peptides and peptide analogs for use as the disrupter agent and pharmaceutical compositions containing the peptides and peptide analogs.

Description

INFLAMMATORY BOWEL DISEASES THERAPY INVOLVING DISRUPTING

CCR9;DRD5 HETEROMER ASSEMBLY

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority under 35 USC 1 19(e) to U.S. Patent Application No. 62/874610 filed on July 16, 2019, the disclosure of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention provides a method and compositions for inhibiting intestinal inflammation, particularly Inflammatory Bowel Diseases (IBD) The methods and compositions comprised for this invention, are for disrupting the formation of CCR9:DRD5 heteromer assembly, where this disruption inhibit the homing of CD4+ T-Cells with gut-tropism. These methods and compositions uses at least one disrupter agent that inhibit or block the formation of CCR9:DRD5 heteromer assembly, where said agent impair the interaction between CCRS:DRD5, particularly where said agent impair the interaction between transmembrane segments from CCR9 with those from DRD5. Further, the present invention will be an effective treatment for patient with IBD, as for example, Crohn Disease and Ulcerative colitis

BACKGROUND OF THE INVENTION

Gut mucosa Immunity involves a tight equilibrium between inflammatory responses against ora!iy administered dangerous foreign antigens and the generation of tolerance to food-derived and commensal microbiota-derived antigens (Cassani et a!., 201 1 ). Effector CD4⁺ T-cells, including T-helper 1 (Thl ) and Th17 cells (which display an inflammatory potential) and regulatory CD4^÷ T- ce!!s (Treg, which display a suppressive potential) constitute central players In maintaining this equilibrium between tolerance and inflammation (Graniund et a!., 2013; Olsen et at., 2011 ). Importantly, the evidence has shown that the recruitment of inflammatory Th1 and Th17 lymphocytes as well as of Treg cells into gut mucosa is mediated by two key molecules: the C-C chemokine receptor 9 (CCR9) and the a4b7 integrin (Cassani et ai._; 201 1 ; Elgueta et a!. , 2008). The loss of oral tolerance may result in inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC) The overall IBDs prevalence is in the range of 500-900 cases per 100,000 individuals, which has been increasing during the last decade, mainly in westernized countries (Molodecky et a!., 2012). Evidence from inflammatory colitis mouse models and from samples obtained from IBD patients has indicated that gut inflammation in IBDs is driven mainly by the inflammatory effector CD4⁺ T-cell subsets Th1 and Thu (Olsen et ai., 201 1 ) whereas Treg remains dysfunctional in these disorders.

Cells residing in the gut may encounter dopamine, which can be produced from different sources, including the intrinsic enteric nervous system, the intestinal epithelial layer (Pacheco et ai., 2014), some components of the gut microbiota (Clark and Mach, 2016), and certain immune cells, including dendritic cells and Treg (Cosentino et ai. , 2007; Prado et a!., 2012) Interestingly, inflamed gut mucosa from CD and UC patients involves a marked decrease of dopamine levels (Magro et ai., 2002), which may affect the function of immune cells expressing dopamine receptors (DRs), Including T-cells. Importantly reduced levels of intestinal dopamine have been also observed in inflamed gut mucosa using animal models of inflammatory colitis (Magro et al., 2004; Pacheco et al., 2014).

Dopamine exerts its effects by stimulating five different DRs described so far, termed DRD1- DRD5, all of them G-protein coupled receptors. All these receptors have been found in CD4⁺ T- ceils from human and mouse origin (Pacheco et a ., 2009). It is important to consider that each DR displays different affinities for dopamine: DRD3 > DRD5 > DRD4 > DRD2 > DRD1 (Ki(nM) = 27, 228, 450, 1705, 2340, respectively), thereby their functional relevance depends on dopamine levels (Pacheco et al , 2014). interestingly, high dopamine concentrations, such as those concentrations found in dopaminergic tissues of healthy individuals, have been shown to promote an anti-inflammatory effect at the level of the immune system, thus promoting homeostasis. This immunosuppressive role of high-dopamine levels seems to be mediated by the stimulation of low- affinity DRs in immune cells, including DRD1 and DRD2 (Shao et ai., 2013; Van et ai., 2015). Indeed, DRD1 -signalling in macrophages/microglia and astrocytes has been shown to attenuate the activation of the NLRP3 infiammasome (Van et al., 2015). in addition, DRD2 stimulation has been associated with the production of the anti-inflammatory cytokine !L-10 by CD4¹ T-cells (Besser et al., 2005) and with the inhibition of both increased gut motility and ulcer development (Miyazawa et al. , 2003). Moreover, a genetic polymorphism of DRD2 gene, which results in decreased receptor expression, has been reported as a risk factor to develop refractory CD (Magro et al., 2006). Conversely, low-dopamine levels, which selectively stimulate high-affinity DRs, seem to represent a“danger signal” in dopaminergic tissues In this regard, our recent study showed that Drd3-6e ficient naive CD4⁺ T-ce!ls display impaired Th1 differentiation and reduced expansion of Th17 cells and consequently an attenuated manifestation of inflammatory colitis (Contreras et al., 2016). Taking info account the reduction in intestinal dopamine levels («1000 nM In healthy individuals; «100 nM in CD and UC patients (Asano et al., 2012; Magro et al., 2002) and the fact that DRD3 may be selectively stimulated at low dopamine concentrations, these results suggest that low dopamine levels present in the inflamed gut mucosa favour the inflammatory potential of CD4⁺ T-cei!s, thus promoting chronic inflammation indeed, our previous results have shown that D/x/3-deficiency results in attenuated inflammatory colitis in mouse (Contreras et al., 2016). Although DRD5 constitutes another high-affinity DR expressed in CD4⁺ T-ce!!s (Franz et al., 2015; Osorio-Barrios et al., 2018), the involvement of this receptor in the development of gut inflammation remains unexplored.

Current treatments for inflammatory bowel diseases (consisting mainly of Crohn’s disease and Ulcerative Colitis), are general anti-inflammatory drugs, which inhibit the immune response in general, including azathioprine (Azasan, Imuran), mercaptopurine (Purinetbol, Purixan), cyclosporine (Gengraf, Neoral, Sandimmune) and methotrexate (Trexal!).

There are also biological therapies, most of them focused on neutralizing the action of TNF-a, such as infliximab (Remicade), adalimumab (Humira) and go!imumab (Simponi). Other biological therapies currently used inhibit the action of the cx4 integrin subunit (natalizumab, Tysabri), a4b7ntegrin (vedo!izumab, Entyvio), IL-12 and IL-23 preventing binding to its IL-12RB1 receptor

(ustekinumab; Stelara).

In general, the limitations of the drugs currently used for the treatment of intestinal inflammatory diseases is the non-specificity, which inhibits the pathological inflammatory response, but also inhibits the beneficial immune responses that defend us against infectious pathogens and against the development of tumors.

The biological therapies mentioned above also involve the problem of non-specificity in different degrees: in this regard, TNF-a, IL-12 and IL-23 are inflammatory cytokines involved in a general way in the pathogenic inflammatory immune responses, but also in immune responses beneficial for the organism. With respect to integrin-blocking antibodies, these have a greater degree of specificity since they affect the entry of inflammatory cells into specific tissues. With respect to this, the blockade of <x4 by natalizumab inhibits the infiltration of lymphocytes both in the centra! nervous system (blocking a4b1 ) and in the intestinal mucosa (blocking a4b7). In the case of vedolizumab, blockade of a4b7 allows a specific inhibition of the recruitment of inflammatory ceils in the intestinal mucosa. However this drug, besides inhibiting the entry of inflammatory T cells into the intestinal mucosa, also inhibits the infiltration of regulatory T ceils (Tregs) in this tissue, which carry out an immunosuppressive function and therefore beneficial in the context of intestinal inflammatory diseases. On the other hand, blockade of a4b7 vedolizumab prevents the entry of T cells into both the colonic mucosa and the mucosa of the small intestine, the site of elimination of inflammatory cells.

In this context, to solve the technical problem consisting in the absence of IBD therapies with high degree of specificity, the present invention discloses methods and compositions useful for an effective IBD therapy, based on the interruption of the functioning of the CCR9: DRD5 heteromer (by means of disassembling or dual antagonism), that display the advantages of, in addition to specifically affecting the infiltration of inflammatory CD4+ T-cells (and not Tregs) in the colonic mucosa, it promotes a redirection of these cells towards the mucosa of the small intestine, where they are eliminated by the intestinal lumen. Thus, the present invention provides new solutions for the design of improved therapeutic and preventive strategies against IBD, with greater level of specificity.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides therapeutic methods, pharmaceutical compositions, disrupter agents (e.g peptides and peptides analog), polynucleotides (e.g. encoding peptides and peptides analog), and kits, useful for the treatment of intestinal inflammation, particularly useful for the treatment of Inflammatory Bowel Diseases (IBD), and more particularly useful for the treatment of Crohn Disease and Ulcerative colitis.

In a particular embodiment, the methods and compositions useful for the treatment of IBD comprised by this invention, are for disrupting or inhibiting the homing of CD4+ T-Ceils with gut- tropism.

In another particular embodiment, the methods and compositions useful for the treatment of IBD comprised by this invention, are for disrupting the formation of CCR9:DRD5 heteromer assembly.

In a particular embodiment, the methods and compositions useful for the treatment of IBD comprised by this invention, are for disrupting or inhibiting the horning of CD4+ T-Ceiis with gut- tropism in a subject comprising administering to the subject a pharmaceutical composition comprising and inhibitor or disruptor agent able to disassemble the CCR9:DRD5 heteromer. In another particular embodiment, the methods and compositions useful for the treatment of IBD comprised by this invention, are for disrupting the formation of CCR9:DRD5 heteromer assembly, in a subject comprising administering to the subject a pharmaceutical composition comprising and inhibitor or disrupter agent able to disassemble the CCR9:DRD5 heteromer.

in a particular embodiment, the methods and compositions useful for the treatment of IBD comprised by this invention, are for disrupting or inhibiting the homing of CD4+ T-Ceiis with gut- tropisrn in a subject comprising administering to the subject a pharmaceutical composition comprising at least one peptide analog to TM5 and/or TMS transmembrane segments from CCR9, and/or at least one peptide analog to TM5 and/or TM6 transmembrane segments from DRD5. in another particular embodiment, the methods and compositions useful for the treatment of IBD comprised by this invention, are for disrupting the formation of CCR9:DRD5 heteromer assembly in a subject comprising administering to the subject a pharmaceutical composition comprising at least one peptide analog to TMS and/or TM6 transmembrane segments from CCR9, and/or at least one peptide analog to TM5 and/or ΪM6 transmembrane segments from DRD5.

in another embodiment, the present invention comprises methods for disrupting the formation of CCR9:DRD5 heteromer assembly in a subject in need comprising administering to the subject a pharmaceutical composition comprising one or more disrupter agent that inhibit or block the interaction between CCR9 and DRD5

In a particular embodiment, the present invention comprises methods for disrupting the formation of CGR9:DRD5 heteromer assembly in a subject in need comprising administering to the subject a pharmaceutical composition comprising one or more disrupter agent that inhibit or block the Interaction between CCR9 and DRD5, wherein the disrupter agent Inhibit or block the interaction between, the transmembrane segment 5 (TMS) and/or transmembrane segment 6 (TM6) of CCR9, and the TM5 and/or TM6 of DRD5.

In a particular embodiment, the present invention comprises methods for disrupting the formation of CCR9:DRD5 heteromer assembly in a subject in need comprising administering to the subject a pharmaceutical composition comprising one or more disrupter agent that Inhibit or block the interaction between GCR9 and DRD5, wherein the disrupter agent inhibit or block the interaction between, the TMS and/or TM6 of CCR9, and the TMS and/or TM6 of DRD5, and where the disrupter agent Is a peptide. In a more particular embodiment, these methods wherein the disrupter agent is a peptide, the peptide is a peptide analog to TM5 or TM8 from CCR9 or DRD5. As all the methods of this invention, these methods are for use in the treatment of inflammatory bowel disease, particularly in the treatment of Crohn Disease and Ulcerative Colitis.

In another embodiment, the present invention comprises methods for inhibiting the homing of CD4+ T-celis with gut-tropism in a subject in need comprising administering to the subject a pharmaceutical composition comprising one or more disrupter agent that inhibit or block the formation of CCR9:DRD5 heteromer assembly.

in a particular embodiment, the present invention comprises methods for inhibiting the homing of CD4+ T-ce!is with gut-tropism in a subject in need comprising administering to the subject a pharmaceutical composition comprising one or more disrupter agent that inhibit or block the formation of CCR9:DRD5 heteromer assembly, wherein the disrupter agent inhibits or block the interaction between CCR9 and DRD5.

In a particular embodiment, the present invention comprises methods for inhibiting the homing of CD4+ T-celis with gut-tropism in a subject in need comprising administering to the subject a pharmaceutical composition comprising one or more disrupter agent that Inhibit or block the formation of GGR9:DRD5 heteromer assembly, wherein the disrupter agent inhibits or block the interaction between CCR9 and DRD5, and where the disrupter agent is a peptide. In a more particular embodiment, these methods wherein the disrupter agent is a peptide, the peptide is a peptide analog to TM5 or TM6 from CCR9 or DRD5. As all the methods of this invention, these methods are use in the treatment of inflammatory bowel disease, particularly in the treatment of Crohn Disease and Ulcerative Colitis.

In another embodiment, the present invention comprises methods for treating inflammatory bowel disease in a subject in need comprising administering to the subject a pharmaceutical composition comprising one or more disrupter agent that inhibit or block the interaction between CCR9 and DRD5, and therefore disrupting the formation of CCR9:DRD5 heteromer assembly.

in a particular embodiment, the present invention comprises methods for treating inflammatory bowel disease in a subject in need comprising administering to the subject a pharmaceutical composition comprising one or more disrupter agent that inhibit or block the interaction between CCR9 and DRD5, and therefore disrupting the formation of CCR9:DRD5 heteromer assembly, wherein the disrupter agent inhibits or block the interaction between CCR9 and DRD5, and where the disrupter agent inhibit or block the Interaction between, the TM5 and/or TM6 of CCR9, and the TM5 and/or TM6 of DRD5. in a particular embodiment, the present invention comprises methods for treating inflammatory bowel disease in a subject in need comprising administering to the subject a pharmaceutical composition comprising one or more disrupter agent that inhibit or block the interaction between GCR9 and DRD5, and therefore disrupting the formation of CCR9:DRD5 heteromer assembly, wherein the disrupter agent inhibits or block the interaction between CCR9 and DRD5, and where the disrupter agent specifically inhibit or block the interaction between, the TM5 and/or TM6 of CCR9, and the TM5 and/or TM6 of DRD5, and where the disrupter agent is a peptide. In a more particular embodiment, these methods wherein the disrupter agent Is a peptide, the peptide Is a peptide analog to TM5 or T 6 from CCR9 or DRD5. As all the methods of this invention, these methods are for use in the treatment of inflammatory bowel disease, particularly in the treatment of Crohn Disease and Ulcerative Colitis.

In another embodiment, the present invention comprises methods, wherein the one or more disrupter agents are peptides analogue to transmembrane segments comprises an amino acid sequence selected from the group consisting of TM1 C (SEQ ID No.1 ), TM2C (SEQ ID No.2), TM3C (SEQ ID No.3), TM4C (SEQ ID No.4), TIV15C (SEQ ID No.5), TM6C (SEQ ID No.6), TM7C (SEQ ID No.7), TM1 D (SEQ ID No.8), TM2D (SEQ ID No.9), TM3D (SEQ ID No.10), TM4D (SEQ ID No.11 ), TM5D (SEQ ID No.12), TM6D (SEQ ID No.13) and TM7D (SEQ ID No.14)

In another embodiment, the present invention comprises methods, wherein the one or more disrupter agents are peptides analogue to transmembrane segments comprises an amino acid sequence selected from the group consisting of TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM5D (SEQ ID No 12) and TM6D (SEQ ID No.13).

In another embodiment, the present invention comprises methods, wherein the peptides analogues comprises an amino acid sequence that has at least 80% sequence identity to amino acid sequence selected from the group consisting of TMI C (SEQ ID No.1 ), TM2C (SEQ ID No.2), TM3C (SEQ ID No.3), TM4C (SEQ ID No.4), TM5C (SEQ ID No.5), T 6C (SEQ ID No.6), TM7C (SEQ ID No.7), TM1 D (SEQ ID No.8), TM2D (SEQ ID No.9), TM3D (SEQ ID No.10), TM4D (SEQ ID No.11 ), TM5D (SEQ ID No.12), TM6D (SEQ ID No.13) and TM7D (SEQ ID No.14). These methods comprise a percent sequence identity that is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%

In another embodiment, the present invention comprises methods, wherein the peptides analogues comprise an amino acid sequence that has at least 80% sequence identity to amino acid sequence selected from the group consisting of TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM5D (SEQ ID No.12) and TM6D (SEQ ID No.13). These methods comprise a percent sequence identity that is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%.

in another embodiment, the present invention comprises pharmaceutical compositions comprising one or more disrupter agent that inhibit or block the interaction between CCR9 and DRD5, and therefore disrupting the formation of CCR9.DRD5 heteromer assembly, wherein the disrupter agent inhibits or block the interaction between CCR9 and DRD5, and where the disrupter agent inhibit or block the interaction between, the TM5 and/or TM6 of CCR9, and the TM5 and/or TM6 of DRD5.

In another embodiment, the present invention comprises pharmaceutical compositions comprising one or more disrupter agent that inhibit or block the interaction between CCR9 and DRD5, and therefore disrupting the formation of CCR9:DRD5 heteromer assembly, wherein the disrupter agent inhibits or block the interaction between CCR9 and DRD5, and where the disrupter agent inhibit or block the interaction between, the TM5 and/or TM6 of CCR9, and the TM5 and/or TM6 of DRD5, and where the disrupter agent is a peptide in a more particular embodiment, these pharmaceutical compositions wherein the disrupter agent is a peptide, the peptide is a peptide analog to TM5 or TM6 from CCR9 or DRD5. As all the pharmaceutical compositions of this invention, these pharmaceutical compositions are for use in the treatment of inflammatory bowel disease, particularly in the treatment of Crohn Disease and Ulcerative Colitis

in another embodiment, the present invention comprises pharmaceutical compositions, wherein the one or more disruptor agents are peptides analogue to transmembrane segments comprises an amino acid sequence selected from the group consisting of TM1 C (SEQ ID No.l ), TM2C (SEQ

In another embodiment, the present invention comprises pharmaceutical compositions, wherein the one or more disruptor agents are peptides analogue to transmembrane segments comprises an amino acid sequence selected from the group consisting of TM5C (SEQ ID No.5), TIV16C (SEQ ID No.6), TM5D (SEQ ID No.12) and TM6D (SEQ ID No.13)

In another embodiment, the present invention comprises pharmaceutical compositions, wherein the peptides analogues comprises an amino acid sequence that has at least 80% sequence identity to amino acid sequence selected from the group consisting of TM1 C (SEQ ID No.1 ), TM2C (SEQ ID No.2), TM3C (SEQ ID No.3), TM4C (SEQ ID No.4), TM5C (SEQ ID No.5), TM6C

(SEQ ID No 14). These methods comprise a percent sequence identity that is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%

In another embodiment, the present Invention comprises pharmaceutical compositions, wherein the peptides analogues comprise an amino add sequence that has at least 80% sequence identity to amino acid sequence selected from the group consisting of TM5C (SEQ ID No.5), TM6C (SEQ ID No 6), TIV15D (SEQ ID No 12) and TW16D (SEQ ID No 13) These methods comprise a percent sequence identity that is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%.

In another embodiment, the present invention encompasses kits comprising any of the pharmaceutical compositions comprised by the present invention, and instructions for administering the pharmaceutical composition to disrupt the formation of CCR9:DRD5 heteromer assembly.

In another embodiment, the present invention encompasses kits comprising pharmaceutical compositions that comprises one or more disrupter agent that inhibit or block the interaction between CCR9 and DRD5, and therefore disrupting the formation of CCR9:DRD5 heteromer assembly, wherein the disrupter agent inhibits or block the interaction between CCR9 and DRD5, and where the disrupter agent Inhibit or block the interaction between, the TM5 and/or TM6 of CCR9, and the TM5 and/or TM6 of DRD5

In another embodiment, the present invention encompasses kits comprising pharmaceutical compositions that comprises one or more disrupter agent that inhibit or block the interaction between CCR9 and DRD5, and therefore disrupting the formation of CGR9:DRD5 heteromer assembly, wherein the disrupter agent inhibits or block the interaction between CCR9 and DRD5, and where the disrupter agent inhibit or block the interaction between, the TM5 and/or TM6 of CCR9, and the TM5 and/or TM6 of DRD5, and where the disrupter agent is a peptide. In a more particular embodiment, these pharmaceutical compositions wherein the disrupter agent is a peptide, the peptide is a peptide analog to TM5 or TM6 from CCR9 or DRD5. As all the pharmaceutical compositions of this invention, these pharmaceutical compositions are for use in the treatment of inflammatory bowel disease, particularly in the treatment of Crohn Disease and Ulcerative Colitis. in another embodiment, the present invention encompasses kits comprising pharmaceutical compositions that comprises one or more disrupter agent, wherein the disrupter agents are peptides analogue to transmembrane segments which comprises an amino acid sequence selected from the group consisting of TM1 C (SEQ ID No.1 ), TM2C (SEQ ID No.2), TM3C (SEQ

In another embodiment, the present invention encompasses kits comprising pharmaceutical compositions that comprises one or more disrupter agent, wherein the disrupter agents are peptides analogue to transmembrane segments which comprises an amino acid sequence selected from the group consisting of TM5C (SEQ ID No.5), TM6C (SEQ ID No.8), T 5D (SEQ ID No 12) and TM6D (SEQ ID No 13).

In another embodiment, the present invention encompasses kits comprising pharmaceutical compositions that comprises one or more disrupter agent, wherein the disrupter agents are peptides analogue to transmembrane segments which comprises an amino acid sequence that has at least 80% sequence Identity to amino acid sequence selected from the group consisting of

No.13) and TM7D (SEQ ID No.14) These kits comprise peptides with a percent sequence identity that is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%.

In another embodiment, the present invention encompasses kits comprising pharmaceutical compositions that comprises one or more disrupter agent, wherein the disrupter agents are peptides analogue to transmembrane segments which comprises an amino acid sequence that has at least 80% sequence identity to amino acid sequence selected from the group consisting of TM5C (SEQ ID No.5), TM6C (SEQ ID No.8), TM5D (SEQ ID No.12) and TM6D (SEQ ID No.13). These kits comprise a percent sequence identity that is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%.

In another embodiment, the present invention encompasses kits comprising instructions for administering a pharmaceutical composition to disrupt the formation of CCR9.DRD5 heteromer assembly, The present invention encompasses kits comprising instructions for administering a pharmaceutical composition to inhibit or block the interaction between CCR9 and DRD5

The present invention encompasses kits comprising instructions for administering a pharmaceutical composition to inhibit the homing of CD4+ T-ce!ls with gut-tropism.

The present invention encompasses kits comprising instructions for administering a pharmaceutical composition to treat an inflammatory bowel disease.

The present invention encompasses kits comprising instructions for administering a pharmaceutical composition to treat Crohn disease.

The present invention encompasses kits comprising instructions for administering a pharmaceutical composition to treat ulcerative colitis.

In another embodiment, the present invention comprises the use of disrupter agents that inhibit or block the formation of CCR9:DRD5 heteromer assembly, for preparation of a medicament for treatment of inflammatory bowel diseases, preferably for the treatment of Crohn Disease and Ulcerative Colitis, wherein the disruptor agents are peptides or peptides analog to TM5 or TM6 from CCR9 or DRD5.

In another embodiment, the present invention comprises the use of peptides or peptides analog for preparation of a medicament for treatment of inflammatory bowel diseases, preferably for the treatment of Crohn Disease and Ulcerative Colitis, wherein the peptides comprises: An amino acid sequence selected from the group consisting of TMI C (SEQ ID No.1), TM2C (SEQ ID No.2),

TM7C (SEQ

amino acid sequence that has at least 80% sequence Identity to amino acid sequence selected from the group consisting of TM1 C (SEQ ID No.1 ), TM2C (SEQ ID No.2), TM3C (SEQ ID No.3), TM4C (SEQ ID No.4), TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM7C (SEQ ID No.7), TM1 D (SEQ ID No.8), TM2D (SEQ ID No.9), TM3D (SEQ ID No.10), TM4D (SEQ ID No 11 ), TM5D (SEQ ID No.12), TM6D (SEQ ID No.13) and TM7D (SEQ ID No.14); or an amino acid that has percent sequence identity at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%, to amino acid sequence selected from the group consisting of TM1 C (SEQ ID No.1 ), TM2C (SEQ ID No.2), TM3C (SEQ ID No.3), TM4C (SEQ ID No.4), TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM7C (SEQ ID No 7), TM1 D (SEQ ID No.8), TM2D (SEQ !D No.9), TM3D (SEQ ID No.10), TM4D (SEQ ID No.1 1 ), TM5D (SEQ ID No.12), TM6D (SEQ ID No.13) and TM7D (SEQ ID No.14).

In another embodiment, the present invention comprises the use of pharmaceutical compositions for preparation of a medicament for treatment of inflammatory bowel diseases, preferably for the treatment of Crohn Disease and Ulcerative Colitis, wherein the pharmaceutical compositions comprises peptides or peptides analog encoded by sequences that comprises: An amino acid sequence selected from the group consisting of TM1C (SEQ ID No.1 ), T 2C (SEQ ID No.2), TM3C (SEQ ID No.3), TM4C (SEQ ID No.4), TM5C (SEQ ID No 5), TM6C (SEQ ID No 6), TM7C (SEQ ID No.7), TM1 D (SEQ ID No.8), TM2D (SEQ ID No.9), TM3D (SEQ ID No.10), TM4D (SEQ ID No.1 1 ), TM5D (SEQ ID No.12), TM6D (SEQ ID No.13) and TM7D (SEQ ID No.14); or an amino acid sequence that has at least 80% sequence identity to amino acid sequence selected from the group consisting of TM1 C (SEQ ID No.1 ), TM2C (SEQ ID No.2), TM3C (SEQ ID No.3), TM4C (SEQ ID No.4), TM5C (SEQ ID No.S), TIVI6C (SEQ ID No.6), TM7C (SEQ ID No.7), TM1 D (SEQ ID No S), TM2D (SEQ ID No.9), TM3D (SEQ ID No.10), TM4D (SEQ ID No.11 ), TM5D (SEQ ID No.12), TM6D (SEQ ID No.13) and TM7D (SEQ ID No.14); or an amino acid that has percent sequence identity at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%, to amino acid sequence selected from the group consisting

(SEQ ID No.4), TM5

ID No.8), TM2D (SE

No.12), TM6D (SEQ ID No.13) and TM7D (SEQ ID No.14). In a particular embodiment, the inhibitor or disruptor agent able to disassemble the CCR9:DRDS heteromer comprised by the pharmaceutical compositions useful for the treatment of !BD, is a chemical molecule or a biological molecule or any other molecule, able to disrupt the CCR9:DRD5 heteromer assembly, and/or produce a dual antagonism for CCR9 and DRD5.

In a particular embodiment, the inhibitor or disruptor agent able to inhibit or blocking the homing of CD4+ T-Cells with gut-tropism comprised by the pharmaceutical compositions useful for the treatment of IBD, is a chemical molecule or a biological molecule or any other molecule, able to disrupt the CCR9:DRD5 heteromer assembly, and/or produce a dual antagonism for CCR9 and DRD5.

In a more particular embodiment, the above mentioned Inhibitor or disruptor agent able to disassemble the CCR9:DRD5 heteromer and inhibit or blocking the homing of CD4+ T-Celis with gut-tropism, is a chemical molecule or a biological molecule, as for example, peptides, antibodies, nanobodies, aptamers, small molecules, and any other chemical or biological molecule with de property of disrupt the formation of the GCR9:DRD5 heteromer assembly.

in a still more particular embodiment, the above mentioned inhibitor or disruptor agent able to disassemble the CCR9:DRD5 heteromer and inhibit or blocking the homing of CD4+ T-Cells with gut-tropism, is a peptide. A preferably embodiment of this invention, the peptide is select from the group of peptides comprised in the Table 1 , which could be normal peptides or modified peptides, where in this last case, peptides could be modified at terminus and/or Internal regions, with linkers, spacers, PEG modifications, special amino acids, modified peptide bonds, sequences that give proper delivering of TM peptides with the correct orientation in the plasma membrane (e.g. TAT), inter alia.

Table 1 . Peptides analogue to transmembrane segments designed to breakdown the formation of the CCR9:DRD5 heteromers

^*Transmembrane regions were predicted by 3D modeling of DRD5 (access code Q8BLD9.1 ) or CGR9 (access code Q9WUT7.1 ) following the criteria deduced from crystals of G-protein coupled receptors (William et al., 1992). To give a proper delivering of transmembrane peptides with the correct orientation in the plasma membrane, the TAT peptide (marked in red) was added in direct orientation (YGRKKRRQRRR) in the C-terminal of odd transmembrane segments and in the inverse orientation (RRRGRRKKRGY) in the N-terminal of even transmembrane segments. The TAT peptide is a cell-penetrating peptide derived from the transactivator of transcription protein of the human immunodeficiency virus. In addition, to avoid the formation of disulfide bridges, cysteines were replaced with serines (marked in green}. In a more particular embodiment, the above mentioned inhibitor or disrupter agent able to disassemble the CCR9:DRD5 heteromer and inhibit or blocking the homing of CD4+ T-Celis with gut-tropism, is a peptide or peptide analog comprising an amino add sequence selected from the group consisting of TM1 C (SEQ ID No.1 ), TM2C (SEQ ID No.2), TM3C (SEQ ID No.3), TM4C (SEQ ID No.4), TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM7C (SEQ ID No/7), T 1 D (SEQ ID No.8), TM2D (SEQ ID No.9), TM3D (SEQ ID No.10), TM4D (SEQ ID No.1 1 ), TM5D (SEQ ID No.12), TM6D (SEQ ID No.13) and TM7D (SEQ ID No.14).

In a still more particular embodiment, the above mentioned inhibitor or disruptor agent able to disassemble the CCR9:DRD5 heteromer and inhibit or blocking the homing of CD4+ T-Ceils with gut-tropism, is a peptide or peptide analog comprising an amino acid sequence is selected from the group consisting

TM6D (SEQ ID No.1

in a still more particular embodiment, the above mentioned Inhibitor or disruptor agent able to disassemble the CCR9:DRD5 heteromer and inhibit or blocking the homing of CD4+ T-Ceiis with gut-tropism, is a peptide or peptide analog comprising an amino add sequence that has at least 80% sequence identity to amino acid sequence selected from the group consisting of TM1 C (SEQ

and TM7D (SEQ ID No.14).

In a still more particular embodiment, the above mentioned inhibitor or disruptor agent able to disassemble the CCR9:DRD5 heteromer and inhibit or blocking the homing of CD4+ T-Cel!s with gut-tropism, is a peptide or peptide analog comprising an amino acid sequence that has at least 80% sequence identity to amino acid sequence selected from the group consisting of TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM5D (SEQ ID No 12) and TM6D (SEQ ID No.13).

In another embodiment, the present invention comprises methods as any of the methods before mentioned, wherein the peptides analogues comprise an amino acid sequence that has at least 80% sequence identity to amino add sequence selected from the group consisting of TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM5D (SEQ ID No.12) and TM6D (SEQ ID No.13). These peptides comprise a percent sequence identity that is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%. In a still more particular embodiment, the above mentioned inhibitor or disrupter agent able to disassemble the CCR9:DRD5 heteromer and inhibit or blocking the homing of CD4+ T-Cells with gut-tropism, are peptides or peptides analogues that comprise an amino acid sequence that has at least 80% sequence identity to amino acid sequence selected from the group consisting of TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM5D (SEG ID No.12) and TM6D (SEQ ID No.13) These peptides comprise a percent sequence Identity that is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%.

In a still more particular embodiment, the above mentioned inhibitor or disrupter agent able to disassemble the CCR9:DRD5 heteromer and inhibit or blocking the homing of CD4÷ T-Celis with gut-tropism, are peptides or peptides analogues, wherein the amino acid sequence of the peptide or peptide analog is between 9 and 50 mer. The large of the amino add sequences of these peptides or peptides analog is at least between 9 and 50 mer, preferably at least between 12 and 40 mer, more preferably at least between 15 and 30 mer, even more preferably at least between 18 and 25 mer, and most preferably at least between 19 and 24 mer.

In another embodiment of this invention, the above mentioned inhibitor or disrupter agent able to disassemble the CCR9:DRD5 heteromer and inhibit or blocking the homing of CD4+ T-Ceils with gut-tropism, comprises a polynucleotide encoding, upon expression, any of the peptide or peptide analog that comprises: An amino acid sequence selected from the group consisting of TM1 C (SEQ ID No.1 ), TM2C (SEG ID No.2), TM3C (SEQ ID No.3), TM4C {SEG ID No.4), TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM7C (SEQ ID No.7), TM1 D (SEG ID No.8), TM2D (SEQ ID No 9) TM3D (SEQ ID No.10), TM4D (SEQ ID No.11 ), TM5D (SEG ID No.12), TM6D (SEG ID No.13) and TIVS7D (SEQ ID No.14); or an amino add sequence that has at least 80% sequence identity to amino add sequence selected from the group consisting of TM1 C (SEQ ID No.1 ),

(SEG ID No.14); or an amino acid that has percent sequence identity at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%, to amino acid sequence selected from the group consisting of TM1 C (SEQ ID No.1 ), TM2C (SEG ID No.2), TM3C (SEQ ID No 3), TM4C (SEG ID No.4), TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM7C (SEQ ID No.7), TM1 D (SEQ ID No.8), TM2D (SEG ID No.9), T 3D (SEQ ID No.10), TM4D (SEQ ID No.1 1 ), TM5D (SEQ ID No.12), TM6D (SEQ ID No.13) and TM7D (SEQ ID No.14)

In other embodiment of the invention, the methods and compositions that disrupt the CCR9:DRD5 heteromer useful for the treatment of the SBD, could be combined with any of the standard therapies actually available for the treatment of IBD.

In another embodiment, the present invention comprises methods as any of the methods before mentioned, wherein the method further comprises administering one or more additional therapies, preferably wherein the one or more additional therapies comprise anti-inflammatory bowel disease therapy, and more preferably wherein the one or more additional therapies comprise those therapies use in the treatment of Crohn disease or ulcerative colitis.

In a particular embodiment, the present invention comprises combined therapeutic methods, where any of the methods before mentioned comprised by the present invention, are combined with one or more additional anti-inflammatory bowel disease therapies, wherein the additional anti-inflammatory bowel disease therapies comprises therapeutics selected from the group consisting of neutralizers of anti-TNFalpha (infliximab (Remicade), adaiimumab (Humira), goiimumab (Simponi), Cerfo!izumab), a!pha4 integrin subunit inhibitors (nataiizumab (Tysabri)), alpba4beta7 integrin inhibitors (vedolizumab (Entyvio)), IL~12Rbeta1 blocker that avoid its union to ligands IL-12 and !L-23 (ustekinumab (Steiara)), anti-MAd-CAM-1 monoclonal antibody (PF- 00547659), anfi-IL-23 monoclonal antibody (MEDI2070), sphingosine-1 -phosphate (S1P) receptor agonist (Ozanimod), antisense oligodeoxynucleotide complementary to mRNA of Smad7 (Mcngersen), aminosalicylates (5-aminosa!icy!ic acid (5-ASA); 5-ASA derivatives (Sulfasalazine, Asacol HD, Deizicoi, Pentasa, Coiazal, Dipentum, Lialda, Apriso, Mesalazine), corticosteroids (hydrocortisone, methylpredniso!one, prednisone, Budesonide, Budesonide MMX, hydrocortisone), antibiotics (ciprofloxacin, metronidazole, rifaximin, omidazole, tinidazole, anti- tuberculosis therapy, macroiides, fluoroquinolones, 5-niiroimidazoles, antimycobacterials), and immunomodulaiors (thiopurines (Purinethol, Purixan), 6-mercaptopurine, azathioprine (Azasan, Imuran), methotrexate (Trexa!l), cyclosporine (Gengraf, Neoral, Sandimmune).

Also this invention comprises methods for inhibiting intestinal inflammation in a subject comprising administering to the subject a pharmaceutical composition comprising a peptide analog to TM5 or TM6 transmembrane segments from CCR9 and DRD5, and/or any other inhibitor or disruptor agent able to disassemble the CCR9:DRD5 heteromer and inhibit or blocking the homing of CD4+ T-Ceils with gut-tropism. Other embodiment of the invention are peptides analog to any of the seven transmembrane segments of the CCR9 or peptides analog to any of the seven transmembrane segments of the DRD5, for example, those peptides analog codified by the amino acidic sequences pointed out in the Table 1

Other preferred embodiment are pharmaceutical composition for inhibiting intestinal inflammation in a subject, which comprise any of the peptide analog mentioned in this application, and/or any other inhibitor or disrupter agent able to disassemble the CCR9:DRD5 heteromer and inhibit or blocking the homing of CD4+ T-Ce!ls with gut-tropism, and a pharmaceutically acceptable carrier, diluent or excipient.

These methods and compositions uses at least one disrupter agent that inhibit or block the formation of CCR9:DRD5 heteromer assembly, where said agent inhibit or disrupt the interaction between CCR9:DRD5, particularly where said agent impair the interaction between transmembrane segments from CCR9 with those from DRD5. Further, the present invention will be an effective treatment for patient with IBD, as for example, Crohn Disease and Ulcerative colitis.

DEFINITIONS

IBP: is an umbrella term used to describe disorders that involve chronic inflammation of the digestive tract. Types of IBD include: Ulcerative colitis, which is a condition causes long-lasting inflammation and sores (ulcers) in the innermost lining of your large intestine (colon) and rectum; and Crohn's disease, which is a type of IBD that is characterized by inflammation of the lining of your digestive tract, which often spreads deep into affected tissues. Both ulcerative colitis and Crohn's disease usually involve severe diarrhea, abdominal pain, fatigue and weight loss. IBD can be debilitating and sometimes leads to life-threatening complications.

Peptide analog: As used herein describes a peptide comprising one or more amino acid modifications, such as but not limited to substitution and/or one or more deletion(s) and/or one or more addition(s) of any one of the amino acid residues with any natural or unnatural amino acid, synthetic amino acids or peptidomimetics and/or the attachment of a substituent to any one of the natural or unnatural amino acids, synthetic amino acids or peptidomimetics at any available position. The addition or deletion of amino acid residues can take place at the N-termina! of the peptide and/or at the C-terminal of the peptide. BRIEF DESCRIPTION OF FIGURES

Figure 1. DRD5 is upregulated upon CD4^* T-cefl activation. CD4⁺ T-cells purified from wild- type mice were left untreated or activated with anti-CD3e plus anti~CD28 mAbs for 24 h. (A) The expression of DRD5 was evaluated by Western blots b-actin was used as a control. (B) Cells were immunostained with anti-DRD5 antibody (open histograms) or with irrelevant isotype matched control {filled histograms) and analysed by flow cytometry. (A-B) Representative results from one out of three independent experiments are shown.

Figure 2. Deficiency of DRDS-signaOIng in CD4^÷ T-celis avoids the development of inflammatory colitis and results in the absence of CD4^÷ T-ceiis in the colonic lamina propria. Naive CD4^÷ T-celis (CD3⁺ CD4⁺ CD45RB' : Tn) were isolated from the spleen of DRD5 sufficient ( Drd5^+/+ , black bars/symbols) or DRD5 deficient ( Drd5^~'' white bars/symbols) mice and then i.p. injected (5x10⁵ per mouse) into RagT^ mice and the extent of body weight loss and CD4^* T-cell infiltration in different tissues were determined. (A) A scheme illustrating the experimental design is shown. (B) Body weight loss was monitored once a week throughout 10 weeks and represented as % respective to the initial body weight. Values represent mean ± SEM. (C) Ten weeks after T-cell transfer, mice were sacrificed and the frequency of CD4⁺ T-cell was evaluated in secondary lymphoid organs (spleen and mesenteric lymph nodes) and In the coionic lamina propria (cLP) by flow cytometry Values represent mean with SEM. Data from 4-9 mice per group is shown. *, p<G.05.

Figure 3. DRDS-deficiency in CD4⁺ T-celis affects the gut-tropism without effects in T-cell differentiation. Naive CD4* T-celis (CDS^* CD4⁺ CD45RB^high; Tn) were isolated from the spleen of DRD5 sufficient ( Cd45.1 ^+A Drd5⁺' black bars/symbols) or DRD5 deficient (Cd45.? Drd5 ^'. white bars/syrnbois) mice and then i p. injected (5x10⁵ per mouse) into RagT^f~ mice. Another group of mice received a 1 :1 mix of Drd5^*/÷ and Drd5^A Tn (5x1 Q⁵ total cells per mouse, grey bars/symbols). Twelve weeks later, the extent of body weight loss and CD4⁺ T-cell phenotype and infiltration in different tissues were determined. (A) A scheme illustrating the experimental design is shown. (B) Body weight loss was monitored once a week throughout 12 weeks and represented as % respective to the initial body weight. Values represent mean ± SEM from 4-6 mice per group. ^*, p<0.05 (black versus white); #, p<0.05, ##, p<0,01 (grey versus white). (C-G) Mononuclear ceils were Isolated from the cLP of mice that received the 1 : 1 mix of Drd5^*/+ and Drd5 Tn (C-D) Ceils were restimulated with PMA and ionomycin in the presence of brefeidin A for 4h and inflammatory functional phenotypes of CD4⁺ T-cel!s were assessed by intracellular immunostaining of IFN-y and IL-17 followed by flow cytometry analysis In the TCRp⁴ CD4⁺ population. (C) Representative contour plots of IFN-g versus IL-17 from the CD45. T ( Drd5^+/ , left panel) and CD45.2⁺ (Drd5^~/; right panel) CD4⁺ T-cells. Numbers indicate the percentage of ceils in the corresponding quadrant.

(D) Quantification of the frequency of single producers IFN-y⁺ or IL-17⁴ or double producers IFN- f IL-17^* wild-type CD45.1⁴ { Drd5^+/+ ) or DRD5-deficient CD45.2⁺ (Drd5n CD4 T-cells. Values represent mean ± SEM from 15 mice per group. (E-F) The relative abundance of DRD5 sufficient (CD45.T^f) or DRD5 deficient (CD45.2⁴) CD4⁺ T-cells was analysed in the 1 :1 mixture before the transfer in recipient mice (input) of infiltrated in different tissues 12 weeks after the ceil transfer

(E) Representative contour plots analysing the relative abundance of CD45.1 ⁺ (Drd5^+/+) versus CD45.2^* ( Drd5 ) CD4⁴ T-celis in the input or infiltrating the spleen, MLN or cLP are shown. Numbers indicate the percentage of cells in the corresponding region. (F) Quantification of the relative abundance of CD45.1⁴ (Drd5^+A) versus CD45 2⁴ ( Drd5^{~ ~} ) CD4⁺ T-celis relative to the input composition Data is the % of GD45.1 ⁺ or CD45.2⁺ from CD4⁴ T-cells in a given tissue divided by the % of CD45. T⁴ or CD45.2 from CD4⁴ T-cells in the input. Values represent mean ± SEM from 13-18 mice per group. **, p<0.01. (G) CCR9 and a4B7 expression was analysed in CD45.T^* ( Drd5^+/+ ) and CD45.2⁴ (Drcf5^/) CD4^f T-celis isolated from different tissues. Values represent the percentage of CCR9⁴ a4[37\ CCR9⁴ a4p7⁴ or CCR9 a4p7⁴ ceils in the TCRp⁴ CD4⁴ population. Data from 7-13 mice is shown ^*, p<0.05.

Figure 4 DRDS deficiency in CD4^÷ T-cells results in increased expression of molecules associated to gut-tropism upon inflammatory colitis. Naive CD4⁺ T-ce!!s (CD3⁴ CD4⁴ CD45RB^high; Tn) were isolated from the spleen of DRDS sufficient {Cd45.1*^/* Drd5^+/+, black bars/symbols) or DRDS deficient ( Cd45.2^+/* Drd5^/ white bars/symbols) mice, mixed at 1 : 1 ratio and then i.p. injected (5x10⁵ total cells per mouse) into RagT^A mice Twelve weeks later, the surface expression of CCR9 and a4b7 was determined in the TCRp⁺ CD4⁺ population infiltrating different tissues. (A) Gating strategy showing how CD45 1 ⁺ and CD45.2⁺ were selected from alive (Zombie Aqua-; ZmAqp TCRp⁺ CD4⁺ T-cells according to the characteristic FSC-A and SSC-A gate for lymphocytes. (B) Representative contour-plots of CCR9 versus «4b7 expression in the Drd5^+/+ (CD45.1⁴) and Drd5^f~ (CD45.2⁴) populations infiltrating the spleen, MLN and cLP are shown (left panel). Representative histograms of a4b7 expression in the Drd5^+/+ (CD45.1⁴; grey histograms) and DrdS^ (CD45.2⁺; white histograms) populations Infiltrating the spleen, MLN and cLP are shown (right panel). (C) Density of CCR9 and a4b7 expression was quantified in CD45.1 ⁺ ( Drd5^{+/ +}) and CD45.2⁴ (DrdS^) CD4⁺ T-celis isolated from the different tissues as the mean fluorescence intensity (MFI). Values represent mean with SEM. Data from 7-13 mice is shown.

**, p<0.01 .

Figure 5. DRDS-signallsng Is required for gut homing of CD4^: T-cells In inflammatory conditions. Naive CD4’ T-celis (CD3⁺ CD4⁺ CD45RB^;"'^9h) were isolated from the spleen of DRD5 sufficient {Cd45.1*^f* Drd5^+/ black bars) or DRD5 deficient (Cd45.2^*/* Drd5^~ , white bars) mice and then activated with anti-CD3/anti-CD28 mAbs coated dynabeads in the presence of IL-2 and RA for 5 d to induce gut tropism. Afterward, GD45.1⁺ ( Drd5^w+ ) and CD45.2⁺ (Drd5^~/~) cells were mixed in a 1 :1 ratio and i.v. injected (20^® total cells per mouse) into Rag†’⁴ recipient mice. Mice were sacrificed 72h later and the relative composition (CD45. T versus CD45.2 ) and expression of gut-homing molecules on CD4⁺ T-cei!s isolated from different tissues was analysed (A) A scheme illustrating the experimental design is shown. (B) Representative contour plots analysing the relative abundance of CD45.1⁺ ( Drd5^+/+ } versus CD45.2⁺ (DrdS^) CD4⁺ T-celis in the input or infiltrating the spleen, MLN or cLP are shown (C) Quantification of the relative abundance of CD45.1⁺ (Drd5^+/+) versus CD45.2⁺ {Drd5^~A) CD4⁺ T-ceils relative to the Input composition. Data is the % of CD45.1⁺ orCD45.2⁺ from CD4⁺ T-ceils in a given tissue divided by the % of CD45.1 ⁺ or CD45.2⁺ from CD4⁺ T-ceSIs in the input. Values represent mean ± SEM from 7 mice per group. ^*, p<0.05; ***_, p<0 001. (D-F) CCR9 and a4b7 expression was analysed in CD45.1⁺ ( Drd5^+/* ) and CD45.2⁺ ( Drd5 ) CD4⁺ T-celis isolated from different tissues. (D) Values represent the percentage of CCR9⁺ a4b7^~, CCR9⁺o4p7⁺ or CCR9 ct4p7⁺ cells in the TCRj3⁺ CD3^* CD4⁺ population from the input. Data from 4 mice per group is shown (E) Quantification of the percentage (left panel) and the mean fluorescence intensity (MFI, right panel) of CCR9 expressed in CD4⁺ T-ceiis isolated from the spleen, MLN and cLP. Values represent mean ± SEM from 3-8 mice per group. ^**, p<0.01. (F) Quantification of the percentage (left panel) and the mean fluorescence intensity (MFI, right panel) of a4b7 expressed in CD4⁺ T-cells isolated from the spleen, MLN and cLP. Values represent mean ± SEM from 3-8 mice per group.

Figure 6. DRD5 deficiency In CD4⁺ T-ceils bearing gut tropism results in increased expression of CCR9 in those cells infiltrating gut-associated tissues. Naive CD4⁺ T-ce!!s (CDS’ CD4⁺ CD45RB^hi9h) were isolated from the spleen of DRD5 sufficient ( Cd45 1^+/+ Drd5^+/+. grey histograms) or DRD5 deficient ( Cd45. ^A Drd5^A , white histograms) mice and then activated with anti-CD3/anti-CD28 mAbs coated dynabeads in the presence of IL-2 and RA for 5 d to induce gut tropism. Afterward, CD45.1 ⁺ ( Drd5^+/+ ) and CD45.2⁺ ( Drd5^A ) cells were mixed in a 1 : 1 ratio and i.v. injected (2G⁶ total ceils per mouse) into Rag ^A recipient mice. Mice were sacrificed 72h later and the expression of CCR9 and a4b7 was determined in the CD45.1 ⁺ and CD45.2^* subsets in the alive TCRp⁺ CD4⁴ population infiltrating different tissues. Representative histograms for a4b7 (left panels) and CCR9 (right panels) expression are shown.

Figure 7. DRDS-signa!isng does not affect the expression of several surface molecules involved in CD4⁺ T-ce!I migration under steady-state conditions. CD4⁺ T-cel!s (TCRp⁺ CD4Q were isolated from the spleen of DRD5 sufficient (Drd5^÷/+, black bars) or DRD5 deficient [Drd5 . white bars) mice and the expression of several molecules involved in lymphocyte migration was analysed by flow cytometry. Quantification of the percentage of CD4⁺ T-ceils positive for a number of surface molecules is shown. Values represent mean with SEM. Data from seven mice per group is shown. No significant differences were found.

Figure 8. GGR9 and DRD5 form heteromers in CD4⁺ T-ceils. (A) A CD4⁺ T-cei! line, Jurkat cells, were transfected with constant amount of cDNA codifying for Reni!la !uciferase (RLuc)- fusioned to DRD5 (as donor) and increasing amounts of cDNA codifying for the Yellow Fluorescent Protein (YFP) fusioned to CCR9 (as acceptor) or increasing amounts of cDNA codifying for Ghrelin receptor l a fusioned to YFP (GHSR-1 a-YFP; as a negative control). The relative amount of BRET was quantified as the ratio between YFP fluorescence and RLuc activity (x1000) and expressed as mils BRET units (mBU). Values are the mean ± SEM from six independent experiments. A saturation curve indicates dose proximity (10-100 A) of both receptors and therefore suggesting physical interaction. (B-C) Naive CD4⁺ T-cells (GD3⁺ GD4⁺ CD45RB^hi9h; Tn) were isolated from the spleen of DRD5 sufficient (Drd5^+/+) or DRD5 deficient {Drd5^/) mice and then i.p. injected (5x10^s per mouse) into RagT mice. Another group of mice received a 1 :1 mix of DrdS^ and DrdS’' Tn (5x10^s total cells per mouse). Twelve weeks later, mice were sacrificed and proximity-!igation-assay (PLA) analysis was performed in histological sections of the colon (n = 8 mice per group; 8 sections were analysed per mice) (B) representative images for each group are shown. Nuclei were stained with Hoechst (blue) and CCR9-DRD5 molecular interaction in these samples was detected using the Duolink II in situ PLA detection kit (red) (C) Quantification of the number of positive spots (red spots, Indicating GGR9-DRD5 proximity) per cell in the population of PLA⁺ cells (r) is represented as mean ± SEM (left panel). The percentage of PLA⁺ cells among cells with leukocyte size was also quantified and represented as mean ± SEM (right panel). ^*p<0.05, **p<0.01 , ^***p<0.001 compared to control (WT) by oneway ANOVA followed by Dunnet's posthoc test. Figure 9.€CR9:DRD5 heteromers assembly in HEK293 cells. A human embrionic kidney cell line, HEK293 cells, were transfected with constant amount of cDNA codifying for Renilia !uciferase (RLuc)-fusioned to DRD5 (as donor) and increasing amounts of cDNA codifying for the Yellow Fluorescent Protein (YFP) fusioned to CCR9 (as acceptor) or increasing amounts of cDNA codifying for Ghre!in receptor 1 a fusioned to YFP (GHSR-l a-YFP; as a negative control). The reiative amount of BRET was quantified as the ratio between YFP fluorescence and RLuc activity (x1000) and expressed as mili BRET units (mBU). Values are the mean ± SEM from six independent experiments. A saturation curve indicates close proximity (10-100 A) of both receptors and therefore suggesting physical interaction.

Figure 10. Transmembrane segments 5 and 6 from DRD5 and from CCR9 constitute the interface of interaction required for the assembly of functional CCR9:DRD5 heteromers. A set of peptides analogue to transmembrane segments of CCR9 and DRD5 (TM-peptides; see Table 1) were synthesized to assess the disruption of the CCR9:DRD5 heteromer assembly by competition with the interphase of interaction. (A) A scheme illustrating the experimental strategy used to disrupt the CCR9:DRD5 heteromer assembly is shown. (B) Jurkat ceils were transfected with N-termina! fragment of Venus protein fused to DRD5 (DRD5-nVenus) and with C-terminal fragment of Venus protein fused to CCR9 (CCR9-cVenus) and Bimoiecular Fluorescence Complementation (BiFC) assay was performed. 48 h later, cells were left without treatment (Control, black bar) or incubated with different TM-peptides (0,4 mM) from CCR9 (blue bars) or from DRD5 (red bars) for 4h and Venus-associated fluorescence was determined. Data corresponds to the fluorescence units from 12 independent determinations. Values are mean ± SEM. ^*, p<0.05; ^****, p<0 0Q01 versus the control. (C-D) Jurkat cells were transfected with CCR9 and DRD5 and incubated for 48h. Afterward, cells were pre-incubated in serum-free medium for 2h, seeded in white ProxiPiate 384-well microplates (3x10⁴ ceils/well) and then treated with indicated antagonists for 20 mins followed by incubation with indicated agonists for 7 mins ERK1/2 phosphorylation was then determined by alpha-screen bead-based technology. (C) Ceils were treated with CCR9-agonisi (CCL25 at 300 ng/ !), DRD5-agonist (SKF81297; SKF at 1 mM) or both together (left panel). Ceils were treated with CCR9-agonist or DRDS-agonist, each alone or in the presence of a DRD5-antagonist (SCH23390; SCH at I mM). Data Is represented as % of phosphorylation of total ERK1/2. Values are mean ± SEM from six independent determinations. *, p<0 05; **, p<0.01 ; **^*, p<0.001 ; ^****, pO.0001. (D) Cells were pre-incubated with TM-peptides (0,4 mM) from CCR9 (blue tones bars) or from DRD5 (red-orange tones bars) for 4h and then treated with CCR9-agonist (CCL.25 at 300 ng/ml), DRD5-agonisi (SKF81297; SKF at 1 mM) or both

L L together. Data is represented as % of phosphorylation of total ERK1/2 Values are mean ± SEM from six independent determinations. ^*, p<0.05; ^**, p<0 G1 , ^***, p<0.001 , ^****, p<0.0001. (E-F) Jurkat cells were transfected with CCR9 and DRD5 and incubated for 48h. Afterward, ceils were pre-incubated in serum-free medium for 4h, seeded in white ProxiPlate 384-we!l microplates (10³ cells/well) and then treated with indicated antagonists for 15 mins followed by incubation with indicated agonists for 15 mins. cAMP production was quantified by a TR-FRET methodology. (E) Cells were treated with the same conditions described in (C) Data is represented as % of cAMP accumulation. Values are mean ± SEM from 4-8 independent determinations. ^*, p<G.Q5; **, p<0.Q1 ; ^****, p<Q.0Q01. (F) Ceils were treated with the same conditions described in (D). Data is represented as % of cAMP accumulation. Values are mean ± SEM from six independent determinations *, p<0.05; **, p<0.01 ; ***, p<Q.G01 ; ^****, p<0.Q001.

Figure 11. Dose-response curves of ERK1/2 phosphorylation and cAMP production upon GGR9 and DRD5 stimulation. Jurkat ceils were transfected with CCR9 and DRD5 and incubated for 48h. (A-B) Afterward, ceils were pre-incubated In serum-free medium for 2h, seeded in white ProxiPlate 384-well microplates (3x10⁴ celis/weil) and then treated with increasing concentrations of CCR9~agonist (CCL25 (A)) or DRD5-agonist (SKF81297 (B)) for 7 mins. ERK1/2 phosphorylation was then determined by alpha-screen bead-based technology. Data is represented as % of phosphorylation of total ERK1/2. Values are mean ± SEM from six independent determinations. ^*, p<0.05; ^**, p<0 01 ; ^****, pO.OOOl (C-D) Ceils 'were pre-incubated in serum-free medium for 4h, seeded in white ProxiPlate 384-well microplates (10³ celis/weil) and then treated with increasing concentrations of CCR9-agonist (CCL25 (C)) or DRD5-agonist (SKF81297 (D)) for 15 mins. cAMP production was quantified by a TR-FRET methodology. Data is represented as % of cAMP accumulation. Values are mean ± SEM from 4-8 independent determinations. ^*, p<0.Q5; ^**, p<G.Q1 ; ^***, p<Q.0Q1 ; ^****, p<Q.00Q1.

Figure 12. The disruption of the CCR9:DRD5 heteromer formation impairs CD4^÷ T-ce!l migration into the gut mucosa. Naive CD4⁺ T-ce!ls (CD3⁺ CD4⁺ CD45RB^hi9 ) were isolated from the spleen of Cd45.1^*/+ (black symbols) or 0 45.2^*'^'* (red symbols) wild-type mice ( Drd5^*/+ ) and then activated with anti-CD3/anti-CD28 mAbs coated dynabeads in the presence of IL-2 and RA for 5 d to Induce gut fropism. Afterward, Cd45. 1^*/* were incubated with a TM-peptide irrelevant for CCR9:DRD5 interaction (TM1 C or TM7C, 4mM), whilst Cd45.2^*/+ were incubated with a TM- peptide that disrupt CCR9:DRD5 interaction (TM5C or TM6C, 4mM) for 4h. (A-B) Then cells were mixed In a 1 :1 ratio and i.v injected (20⁶ total cells per mouse) into Rag1 ^~ recipient mice. Mice were sacrificed 72b later and the relative composition (CD45.1 ⁺ versus CD45.2⁴) and expression of gut-homing molecules on CD4⁺ T-ceils isolated from different tissues was analysed. (A) A representative contour plot analysing the relative abundance of CD45.1 ⁺ (treated with TM1 C) versus CD45.2⁺ (treated with TM5C) CD4⁺ T-ceils in the input is shown (left panel). Representative contour plots analysing the expression of CCR9 and a4b7 in the CD45. T (middle panel) or in the CD45.2⁺ (right panel) CD4⁺ T-ceils in the input are shown. (B) Quantification of the relative abundance of CD45.T^f (treated with TM1 C) versus CD45.2⁺ (treated with TM5C) CD4⁴ T-cells isolated from the indicated tissue relative to the input composition. Data is the % of CD45.1⁺ or CD45.2⁴ from CD4⁺ T-cells in a given tissue divided by the % of CD45.1⁴ or CD45.2⁺ from CD4⁺ T-cells in the input. Values represent mean + SEM from 3-4 mice per group. ^*, p<G.G5; n.s., no significant differences. (C) Cells were mixed in a 1 :1 ratio and i v. injected (6x10^® total cells per mouse) into wild-type cd45. 1^+/Vcd45 2^+/ recipient mice that previously received DSS for 48h. Mice were further treated with 1.75% DSS for 72h after T-cell transfer and then were sacrificed and the relative composition (CD45.1 ⁺ versus CD45.27) of CD4⁺ T-cells isolated from different tissues was analysed. In these analyses, single positive (CD45.1 ⁺ CD45.2^~ or CD45. T CD45.2⁺) transferred CD4⁺ T-cells were distinguished from double positive endogenous CD4^* T- ce!is (CD45.1 ⁺ CD45.2T) by flow cytometry. Data is the % of single positive CD45.1 or CD45.2⁺ from CD4⁺ T-cells in a given tissue divided by the % of single positive CD45.1⁺ or CD45.2⁺ from CD4⁺ T-cells in the input. Values represent mean ± SEM from six mice per group. ^*, p<0.05; ^**, p O.Ql (D) Cell migration to CCL25 was determined in transwe!! assays. Values represent mean ± SEM from 3 Independent determinations *, p<0.05.

Figure 13. TM-peptides do not affect CD4^* T-cells viability. Naive CD4⁺ T-celis (CD3 CD4⁺ CD45RB ^igh) were isolated from the spleen of wild-type mice ( Drd5^+/+ ) and then activated with anti- CD3/anti-CD2.8 rnAbs coated dynabeads in the presence of lL-2 and RA for 5 d to induce gut tropism. Afterward, ceils were left without further treatments (black bar) or incubated with peptide analogues to CCR9 (blue bars) or DRD5 (red bars) transmembrane segments (4mM). Four hours later, the extent of cell death (apoptosis/necrosls) was determined by staining with Annexin V and 7-AAD followed by flow cytometry analysis. The viability was quantified as the percentage of ceils with negative staining for Annexin V and 7-AAD. Values are mean ± SEM from three independent determinations. No significant differences were found.

Figure 14. Analysis of GCR9 expression on CD4^* T-cell upon treatment with T!¥l~peptides followed by in vivo migration assay. Naive CD4⁺ T-ceils (CD3⁴ CD4⁺ CD45RB^h'^9h) were isolated from the spleen of wild-type mice (Drd5^+/+) and then activated with anti-CD3/anti-CD28 mAbs coated dynabeads in the presence of IL-2 and RA for 5 d to induce gut tropism. Afterward, cells were Incubated with peptide analogues to CCR9 (blue bars) or DRD5 (red bars) transmembrane segments (4mM) for 4h and CCR9 expression was evaluated by flow cytometry. CCR9 expression was quantified as the percentage of GCR9⁺ ceils. Values are mean ± SEM from 3-5 independent determinations. No significant differences were found.

Figure 15. CCR9:DRD5 heteromer signalling increases the migratory speed of CD4÷ T-celis in microchanne!s. Naive CD4⁺ T-celis (CDS* CD4^÷ CD45RB^high} were isolated from the spleen of wild-type mice ( Drd5^*/+ ) and then activated with anti-CD3/anti-CD28 mAbs coated dynabeads in the presence of iL-2 and RA for 5 d to induce gut tropism. Afterward, cells were individually tracked in 3pm-width confined microchannels under different conditions and the migratory speed was determined. (A) Migratory speed in response to increasing concentrations of GCL25 was determined; n = 52 - 163 cells per condition. (B) Migratory speed was determined in the absence or in the presence of CGL25 alone or CCL25 and increasing concentrations of dopamine; n = 97 - 224 ceils per condition. (C) Migratory speed was compared when only CCR9 is stimulated or when CCR9 and DRD5 are stimulated together, in the absence or in the presence of a TM-peptide irrelevant for CCR9:DRD5 heteromer assembly (TM1 D) or of a TM-peptide that disrupt CCR9:DRD5 heteromer assembly (TM6D); n = 74 - 134 cells per condition. (A-C) Data corresponds to the median migratory speed of lymphocytes in grn/min. In the box plots, the bars include 90% of the data points, the horizontal line in the box indicates the median and the box contains 75% of the data points. Data from two independent experiments is shown. ^*, p<0.05; ^**, p<0.01 ; ^***, pO.OQl

DESCRIPTION OF THE INVENTION

The present invention, which provides methods and compositions useful for disrupting the formation of CCR9:DRD5 heteromer assembly, thus inhibiting the homing of CD4÷ T-Cells with gut-tropism, and therefore transforming this invention in an effective and specific treatment for patient with IBD, was developed using the following materials and methods:

Mice

Wild-type C57BL/6 (Drd5^+/+; Cd45.T ) and RagT mice were obtained from The Jackson Laboratory. C57BL/6 Drd5^f mice were kindly donated by Dr. David Sibley (Hoilon et al., 2002). B6.SJL -Ptprc⁸ (Cd45.1^+,+) mice were kindly provided by Dr. Maria Rosa Bono. Drd5^~'^'~ Cd45. 1^+/+ and, Cd45.1 ^~ Cd45.2 ^l mice were generated by crossing parental mouse strains. We confirmed the genotype of these new strains by PCR of genomic DNA. Mice from 6 to 10 wk were used in ail experiments. Ali procedures performed in animate were approved by and complied with regulations of the Institutional Animal Care and Use Committee at Fundacion Ciencia & Vida (FCV).

Reagents

Monoclonal antibodies ( Abs) for flow cytometry: and anii-iFN-v (done XMG1 2) conjugated to PE-Cy7, anti-cc4p7 (clone DATK32) conjugated to PE and an†i~CCR9 (done GW.1.2) conjugated to APC or to APC-Cy7 were obtained from eBioscience (San Diego, CA, USA). Anti-CD4 (clone GK1.5) conjugated to APC and APC-Cy7; anti-CD25 (clone PC61 ) conjugated to Fluorescein isothiocyanate (FITC); antl-CD44 (clone 1M7) conjugated to PE; anii-CD62L (done MEL14) conjugated to APC-Cy7; anti-IL-17A (clone TC1 1-181710.1 ) conjugated to APC; anti-CD45.2 (clone 104) conjugated to PE-Cy7; anti-CD45.1 (done A20) conjugated to Brilliant Violet (Bv)421 were purchased from Biolegend (San Diego, CA, USA) mAbs for Cel! Culture: the followings mAbs low in endotoxins and azide free (LEAF) were purchased from Bioiegend: anti-CD28 (done 37.51 ) and anti-CDS (clone 145-2C1 1 ). Carrier-Free IL-2 and CCL25 were purchased from Biolegend. Zombie Aqua (ZAq) Fixable Viability dye detectable by flow cytometry was purchased from Bioiegend. Phorbol 12-myristate 13-acetate (PMA), ionomycin and retinoic add (RA) were purchased from Sigma-Aldrich (San Luis, MO, USA) Brefeidin A and Fetal Bovine Serum (FBS) were obtained from Life Technologies (Carlsbad, CA, USA). The peptide analogues to transmembrane segments derived from CCR8 and DRD5 (Table 1 ) were synthesized by GenScrlpt (Piscataway, NJ, USA). Anti-CD3/anti-CD28 conjugated dynabeads were purchased from Thermo Scientific. Bovine Serum Albumin (BSA) was purchased to Rockland (Limerick, PA, USA). DSS was obtained from MR Biomedicais. Ail tissue culture reagents were bought from Life Technologies.

T-cefl transfer induced chronic inflammatory colitis

Chronic inflammatory colitis was induced as described before (Contreras et a!., 2016). Briefly, Total CD4⁺ T~ee!!s were obtained by negative selection of sp!enocytes (Miitenyi). Naive ( CD3⁺ CD4⁺ CD45RB^hish) T-celi isolation was achieved by cell sorting using a FACS Aria II (BD), obtaining purities over 98%. Recipient Rag1^~,L mice received naive CD4⁺ T-cells i.p. (5x10^s cells per mouse) and the body weight of each animal was recorded weekly. After 10 or 12 wk, mice were sacrificed to obtain spleen, mesenteric lymph nodes, colon and, in some experiments, small intestine. The expression of phenotypic markers and the frequency of transferred T-ceils were assessed by flow cytometry. Dextran Sodium Sulphate induced acute inflammatory colitis

Wild-type cd45.1^+/7cd45.2⁺'^~ mice were treated with 1 75% Dextran Sodium Sulphate (DSS) in the drinking water. DSS was given for a total period of 5 d. Forty-eight hours after the beginning of DSS treatment, mice received an i.v. injection of CD4⁺ T-celis (6x1 Q⁶ total ceils per mouse) bearing single positive congenic markers (CD45.1⁺ CD45.2 or CD45.T CD45 2⁺). Seventy-two hours later, transferred T-cel!s were tracked by analyzing specific alleles of congenic markers on T-celis purified from different tissues of Interest by flow cytometry.

Flow cytometry analysis of CD4^÷ T-cell phenotypes

To determine expression of surface molecules, including «4b7, CCR9, CD3, CD4, CD25, CD44, CD45RB, CD62L, and TCRp, CD4⁺ T-ceils were immunostained with fluorochrome-conjugated monoclonal antibodies (mAbs) for 30 min. In the case of DRD5, a polyclonal Ab (pAb) against intracellular epitope was used. Accordingly, ceils were first fixed with 1% paraformaldehyde in phosphate-buffered saline (PBS, NazHPCTs 8.1 mM, KH2PO4 1 .47 mM, NaC! 64 2 mM, KCI 2.68 mM, pH 7.4) for 15 min at room temperature, and then, treated with permeabilizing buffer (0 5% Saponin, 3% BSA in PBS). Permeabi!ized cells were incubated with unconjugated rabbit anti- DRD5 pAb during 45 min, followed by FITC-conjugated anti-rabbit IgG Abs (Santa Cruz Biotechnology, Santa Cruz, USA). Non-specific rabbit Ig followed by the secondary FITC- conjugated anti-rabbit IgG Ab were included as controls. Fluorescence associated to anti-DRD5 and isotype control staining was analysed by flow cytometry in the CD4⁺ population. For intracellular cytokine staining, CD4⁺ T-celis were stimulated for 4 h with phorbol-12-myristate-13- acetate (PMA, 50 ng mL ¹, Sigma-Aidrich, St. Louis, MO, USA) and ionomycin (1 pg mL^-1, Sigrna- A!drich, St. Louis, MO, USA) in the presence of brefeldin A (5 pg mL ¹, Sigma-Aidrich, St. Louis, MO, USA). Cell surface staining was carried out in PBS with 2% FBS. For intracellular staining, cells were first stained with Zombie Aqua (ZAq) Fixable Viability kit (Bioiegend), followed by staining for cell-surface markers and then resuspended in fixation/permeabiiization solution (3% BSA and 0.5% saponin in PBS) Data were collected with a Canto II (BD) and results were analysed with FAGSDiva (BD) and FlowJo software (Tree Star, Ashlan, OR, USA).

Imprinting gut tropism in CD4⁺ T-celis ex vivo

Naive (CD3⁺ CD4⁺ CD44^’ CD62L⁺) T-ceiis were isolated from the spleen of Drd5^+/* or Drd5 ^÷ mice by cell sorting using a FACS Aria II (BD), obtaining purities over 98%. Gut tropism was imprinted by activation of T-cel!s in the presence of retinoic acid (RA) and !L-2 as described before (Kurmaeva et ai , 2013) Briefly, naive T-celis were resuspended (10⁶ cells/ml) in RPMI1640 medium containing 10% FBS, 2 mM L-glutamine, 1 % Penicillin/Streptomycin, MEM Non-Essential Amino Acids IX and Sodium Pyruvate 1X (all purchased from Gibco), gentamicin 50 pg/mi (Thermofisher Scientific) and b-mercaptoethanol 1 pg/mi (Thermofisher Scientific). Cells were activated with anti-CD3/CD28 coated dynabeads (at a beadsxeils ratio of 1 : 1 ) in the presence of 100 nM all-trans retinoic acid (RA; Sigma Aldrich) and 1000 U/mL recombinant mouse IL-2 (Preprotech) for 5 days Viability and gut tropism were routinely confirmed after 5 d of culture by staining with Zombie Aqua (ZAq) Fixable Viability kit (Biolegend) and CCR9 and a4b7 immunostaining followed by flow cytometry analysis. in vivo T-ce!l migration assay

Naive CD4⁺ T-ceiis were isolated from the spleen of cd45.1 ^+/+ or cd45.2^+/+ congenic mice and incubated in conditions to induce gut-tropism (see above). CD4+ T-celis bearing gut tropism were not further treated or incubated with different TM-peptides (4 mM) for 4h and then congenic CD4⁺ T-cells were mixed in a CD45.1⁺:CD45.2⁺ ratio 1 : 1 and i.v injected (2.0^® total cells per mouse) into RagT' recipient mice. Mice were sacrificed 72h later and the relative composition (CD45 1 ⁺ versus CD45.2⁺) and expression of gut-ho ing molecules on CD4⁺ T-cells isolated from different tissues was analysed, including spleen, mesenteric lymph nodes (MLN), coionic lamina propria (cLP), small intestine lamina propria (SI) and cecum lamina propria (Cecum) Quantification of the relative abundance of CD45.1 ⁺ versus CD45.2⁺ CD4^÷ T-celis was normalized with the input composition.

In vitro transwe!f migration assay

Naive CD4⁺ T-cells were isolated from the spleen of C57BL/6 mice and incubated in conditions to induce gut-tropism (see above). Beads were removed according tc manufacturers instructions (Dynabeads, Thermofisher Scientific) and live cells were counted with Trypan Blue. 3x10⁵ live cells were resuspended in 100 pi of PBS seeded on the top chamber of a 5 pm pore transwell (Corning). The bottom chamber of the transwell was previously incubated with TO pg/mL of fibronectin (Sigma Aldrich) diluted in PBS for 2 h, and 600 mΐ of either RPMI 1640 + 5% bovine serum albumin (BSA) with 300 ng/mL of recombinant mouse CCL25 (Bioiegend) or equal volume of PBS. Cells migration was allowed for 3 h at 37°C and 5% C0₂ and then ceils were recovered from the top and bottom chambers, stained with 1 pL of antibodies for CD4, CCR9, a4i37 and also stained with ZAq for 25 minutes and resuspended in 150 pL of PBS. To quantify the absolute number of cells, 50 pL of 123 count eBeads (Thermofisher Scientific) was added to each sample prior to analysis by flow cytometry and ceil concentration was calculated using the following formula:

Cell Count x eBead Volume-

Cell Concentration (Cells /mV) ~ -— - - x eBead Concentration eBead Count x Cell Volume

Ceil concentration was normalized to the frequency of CGR9+ seeded cells.

T-cel! migration in microchannels

Naive CD4^÷ T-ce!!s were isolated from the spleen of vvi!d-type C57BL/6 mice and incubated in conditions to induce gut-tropism (see above). CCRST cells were purified from CD4⁺ T-cei!s bearing gut tropism using a cell-sorter. Lymphocytes were incubated with or without transmembrane analogue peptides (4 uM) for 4h and then the median speed of ceils was determined in microchannels as described before (Vargas et al., 2014). Briefly, 5 pi of ceils suspension (10⁷ cells/ml) was loaded into a fibronectin (10pg/ml)-coa†ed chip of poiy-dimethylsi!oxane containing several 3 pm-diameter micro-channels. After 30 min of incubation at 37°C, 90% humidity and 5% CO2, 2 mi of complete culture medium containing IL-2 (100 U/m!), GCL25 (0 - 20Q ng/mL) and dopamine (0 - 1 mM) or SKF81297 (0 1 mM) were added. Afterwards, cells phase contrast images were recorded during 10 h with 8 min time-lapses using an automated microscope (Nikon ECLIPSE TE100G-E, and Olympus X71 , with a Marzhauser motorized stage and an HG2 Roper camera) equipped with an environmental chamber to control temperature (37°C), humidity (90%) and CO2 (Life Imaging Services). The analysis of migration parameters was performed using an Imaged Fiji-based script.

Resonance Energy Transfer experiments

For Bioluminescence Resonance Energy Transfer (BRET) experiments, JURKAT ceils transiently co-transfected with a constant amount of cDNA encoding for the protein fused to RLuc and with increasingly amounts of cDNA corresponding to the protein fused to YFP (see figure legends) were used after 48 h transfection. To quantify BRET measurements, 5 m coeienterazine H (Molecular Probes, Eugene, OR) was added to the equivalent of 20 pg of cell suspension. After 1 minute, the readings were collected using a Mithras LB 940 that allows the integration of the signals detected in the short-wavelength filter at 485 nm and the long-wavelength filter at 530 nm. To quantify protein-RLuc expression, luminescence readings were also performed after 10 minutes of adding 5 mM coeienterazine H To quantify protein-YFP expression, fluorescence of ceils (20 pg protein) was also read. The net BRET is defined as [(long-wavelength emission)/(short-wavelength emission)]-Cf where Cf corresponds to [(longwaveiength emission)/(short-wavelength emission)] for the donor construct expressed alone in the same experiment. Data were fitted to a non-linear regression equation, assuming a single phase saturation curve with GraphPad Prism software (San Diego, CA, USA). BRET is expressed as mill BRET units, mBU (net BRET x 1000).

Bimolecular fluorescence complementation assay (BiFC)

Jurkat cells were transiently transfected with equal amounts of the cDNA for fusion proteins of the hemi-truncated Venus (1.5 pg of each cDNA). 48h after transfection cells were treated for 4 h at 37° with TAT-peptides (0.4 mM) before plating 20 pg of protein in 96-well black microplates (Porvair, King's iynn, UK). To quantify reconstituted YFP Venus expression, fluorescence was read in a Fluoro Star Optima Fluorimeter (BMG Labtechnologies, Offenburg, Germany) equipped with a high-energy xenon flash lamp, using a 10 nm bandwidth excitation filter at 400 nm reading. Protein fluorescence expression was determined as fluorescence of the sample minus the fluorescence of cells not expressing the fusion proteins (basal).

In situ proximity ligation assay

Colonic sections of mice undergoing inflammatory colitis were used to analyse the CCR9:DRD5 heteromer in situ by proximity ligation assay (PLA). Tissue sections were fixed in 4% paraformaldehyde for 15 min, washed with PBS containing 20 mM glycine to quench the aldehyde groups and permeabiiized with the same buffer containing 0.05% Triton X-100 for 15 min. Primary antibodies recognizing CCR9 (rabbit anti-mouse CCR9; 1 : 100 dilution; purchased from Abeam) and DRD5 (rabbit anil-DRD5; 1 : 100 dilution: purchased from Calbiochem) were linked directly to PLA probes detecting rabbit antibodies (Duoiink II PLA probe anti-Rabbit plus and Duolink !l PLA probe anti-Rabbit minus). After 1 h incubation at 37° with blocking solution, tissue sections were incubated with the primary antibodies linked to PLA probes and further processed as described before (Sierra et at., 2015). Nuclei were stained with Hoechst (1 :200 dilution; purchased from SigmaAldrich). Coverslips were mounted using mowiol solution. Samples were observed in a Leica SP2 confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with an apochromatic 63X oil-immersion objective (N.A. 1.4), and 405 n and 561 nm laser lines For each field of view a stack of two channels (one per staining) and 3 to 4 Z stacks with a step size of 1 pm were acquired. Quantification of ceils containing one or more red spots versus total cells (blue nucleus) and, in cells containing spots, the ratio r (number of red spots/ cell), 'were determined by Duolink Image tool software. Evaluation of ERK1/2 phosphorylation

Jurkat cells expressing GCR9 and dopamine D5 receptors were incubated in serum-free medium for 2 h ERK 1/2 phosphorylation was determined using AlphaScreen^®SureFire^® kit (Perkin Elmer) following the instructions of the supplier and using an EnSpire^® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). Cells (30 000 ceils / well for transfected Jurkat cells) were seeded in white ProxiPlate 384-well microplates, pre-treated at 25"C for 20 min with vehicle or antagonists in serum-starved DMEM medium supplemented or nor with 1 mM ionomycin and stimulated for an additional 7 min with the Indicated agonists. Phosphorylation was determined by alpha-screen bead-based technology using the Amplified Luminiscent Proximity Homogeneous Assay kit (PerkinElmer, Waltham, MA, USA) and the Enspire Multimode Plate Reader (PerkinElmer).

Determination of cAMP production

Jurkat cells expressing CCR9 and dopamine D5 receptors were incubated in serum-free medium for 4 h. Cells were plated in 384-well white microplates (1000 ce!is/we!!) and incubated for 15 min with the specific agonists followed, when indicated, by 15 min stimulation with 0 5 uM forsko!!n As a general rule derived from Gi-coupling of CCR9 and Gs-coupling of D5 receptors, the treatment with CCR9 receptor agonists was performed in cells pre-treated with forskolin, whereas cells were not pre-treated with the reagent when stimulated with SKF or with SKF plus CCR9 agonist. cAMP production was quantified by a TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) methodology using the LANCE Ultra cAMP kit (PerkinElmer) and the Pherastar Flagship Microplate Reader (BMG Labtech, Ortenberg, Germany).

Western blots

For DRD5 detection, CD4⁺ T-celis were purified from sp!enoeytes and lymph nodes by negative selection using magnetic micro-beads-based kit from Mi!tenyi Biotec (Bergisch Gladbach, Germany). CD4^* T-cel!s (4x10⁶ cells ml/¹) were immediately lysed or activated during 24 h with plate-bound anti-CD3s mAh plus antl-CD28 mAb (see below). After 24 h of T-cell activation ceils were lysed with lysis buffer, protein extract was quantified, resolved by SDS-PAGE and transferred to PVDF membranes. DRD5 was detected using a mouse anti-DRD5 mAb (T.1000, Santa Cruz Biotechnologies, Santa Cruz, USA) followed by HRP-conjugated goat anti-mouse IgG Ab (1 :5000; Rockland, Gilbertsviile, PA, USA). Membranes were stripped and reprobed with mouse anti-S-actln mAb (1 : 10000, Sigma-Aldrich, St. Louis, MO, USA) followed by HRP- conjugated goat anti-mouse !gG Ab (1 :5000; Rockland, Gilbertsvilie, PA, USA) and detected as described above.

Viability assay

Flow cytometry analysis routinely included the staining with Zombie Aqua (ZAq) Fixable Viability kit (Biolegend), which was carried out before the immunostaining for cell-surface markers. Flow cytometry analysis of surface molecules or intracellular cytokines was performed in the viable population (ZAq ). In some experiments, to gain a more detailed view of dying cells, the extent of apoptosis/necrosis was determined with a commercial kit (Pacific Blue TM Annexin V Apoptopsis Detection Kit with 7-AAD: 640926, Biolegend).

Statistical analysis

All values were expressed as mean ± SEM Differences in means between two groups were analysed by 2-tailed Student's ί-test or, when data was not normally distributed, with a non- parametric Mann-Whitney d-test. Comparison between multiple groups was analyzed using 1 - or 2-way ANOVA with time, treatment or genotype as the independent factor. When AN OVA showed significant differences, pair-wise comparison between means was tested by Tukey post-hoc analysis. When data was not normally distributed, ANOVA on ranks was used (Kruskal-Waliist test followed by pair-wise comparison using Dunn test). P value £ 0.05 was considered significant. Analyses were performed with GraphPad Prism 6 software.

To develop the present invention, has been addressed the question of what is the relevance of DRD5-signaiiing in CD4+ T -cells in gut inflammation using an animal mode! that involves the transference of naive CD4⁺ T-cells (in the absence of Treg) into lymphopenic recipient mice (lymphocyte-deficient mice), which develop a chronic inflammatory response against microbiota- derived antigens.

Our results showed a highly relevant role of DRD5--s!gnal!ing in gut inflammation, as mice bearing DRD5-deficient CD4⁺ T-ceils were completely refractory to the manifestation of the disease. Further analysis revealed that, despite DRD5-deficiency results in exacerbated expression of gut homing molecules CCR9 or a4b7, CD4⁺ T-cel! infiltration in the gut mucosa and gut-associated lymphoid tissues was impaired. Mechanistic experiments indicated that DRD5 is assembled with CCR9 to generate a heteromeric receptor in inflammatory CD4⁺ T-ce!ls, which lead the migration of these cells into the inflamed gut mucosa.

This and other experiments made to get the present invention are shown in the following examples:

Example 1, Deficiency of DRDS-signailing in€D4‘ T-cef!s avoids the development of inflammatory colitis and resu ts in the absence of CD4⁺ T-celis in the gut mucosa

To evaluate the role of DRD5-signa!ling in CD4⁺ T-ceiis in the development of gut inflammation, we use the mouse mode! of chronic inflammatory colitis induced by T-ceii transfer, which involves the administration of naive CD4 T-ce!is (defined as GD3⁺ CD4⁺ CD45RB^n¾h, a T-cel! subset devoid of Treg) into lymphopenic Rag1 (recombination activating gene 1 knockout mice, which are devoid of T and B cells) recipient mice (Ostanin et a!., 2009). In these conditions, a fraction of naive CD4⁺ T-celis become activated in the gut-associated secondary lymphoid organs (including Mesenteric Lymph Nodes, MLN) by a polyclonal recognition of microbiota-derived antigens in the absence of Treg. In these conditions, activated CD4⁺ T-ce!ls differentiate in Th1 and Th17 cells. Subsequently, these Teffs migrate into the colonic lamina propria (cLP), where they release inflammatory mediators, including IFNy, IL-17 and others, which recruit and stimulate cytotoxic activity of neutrophils and macrophages, inducing chronic inflammation in gut mucosa (Ostanin et a!., 2009).

Since DRD5-signa!iing has been shown to be relevant in CD4⁺ T-ce!ls (Franz et ai., 2015; Osorio- Barrios et a! , 2018) and its expression increases after T-cell activation (Fig. 1), we first compared how was the severity of inflammatory colitis manifestation of Rag1 mice receiving D/x/5-sufficient or DrdS-de ficient naive GD4⁺ T-cells (Fig, 2A). The results show that mice bearing DrdS-defieient GD4⁺ T-celis did not present colitis manifestation (Fig. 2B) and, indeed they increased their body weight along the time-course of the experiment, such as the expected body weight change for healthy mice according their age. To determine whether the absence of disease manifestation in mice bearing Drc/5-deficient CD4⁺ T-ce!is was due to an altered acquisition of T-cell phenotypes or to a different number of inflammatory T-celis In the inflamed tissue, we next attempted to determine the extent of T-ceil infiltration and the functional phenotypes of T-cells in the gut mucosa. For this purpose, ten-weeks after disease induction, CD4⁺ T-celis infiltrating cLP, MLN and the spleen were isolated and analysed by flow cytometry. Strikingly, the results show a complete absence of CD4^÷ T-ceiis in the cLP of mice bearing Drd5- deficient CD4⁺ T-cells (Fig. 2G) Conversely, an Increased frequency of CD4+ T-cells was observed in the spleen of mice bearing Drd5~defieieni GD4⁺ T-celis in comparison with those mice bearing Drd5- sufficient CD4⁺ T-cei!s. On the other hand, no differences were detected in the extent of CD4 T-ceils infiltrating the MLN when mice bearing DrdS-de ficient or DrdS-sufficient CD4⁺ T-ee!is were compared (Fig. 2G). Thus, these results indicate that Dfu5~deficiency in CD4⁺ T-ceils induces a complete resistance to inflammatory colitis, which was associated to the absence of CD4⁺ -celis in the colonic mucosa.

Example 2. DRDS-signaiiing in CD4^* T-cefls affects the gut-tropism without effects in T-cell differentiation

To analyze the role of DRD5-signal!ing in CD4^* T-celis in the acquisition of functional phenotypes in the context of inflammatory colitis, we next carried out a set of experiments in which the behaviour of Drd5- deficient and DrdS-sufficieni CD4⁺ T-celis was compared inside the same recipients. For this purpose, inflammatory colitis was induced transferring congenic Drd5- sufficient ( Cd45. ^/+) and Drd5- deficient ( Cd45.2^+/+ ) naive CD4⁺ T-ceils into Ragi^A recipients and after 12 weeks, T-ceil phenotype was analysed in different tissues (Fig. 3A). Rag† receiving only DrdS-sufficieni CD4⁺ T-celis or receiving only Drd5-de ficient CD4⁺ T-ceils were used as controls. The results show that mice receiving a 1 :1 mix of DrdS-deflclent and DrdS-sufficient CD4⁺ T-ceils display a disease manifestation similar to those mice bearing only Dr S-sufficient CD4⁺ T-ceils (Fig. 3B), suggesting that Drd5- sufficient CD4⁺ T-celis are able to rescue the pathogenic phenotype of mice, even when the half of CD4⁺ T-celis are Drd5- deficient interestingly, the inflammatory profile of CD4^f T-ceils infiltrating the cLP in these mice was similar in Drc/5-s efficient and Drd5- deficient T-ce!ls (Fig. 3C-D), indicating that DRD5-signailing was not relevant for the acquisition of Th1 and Th17 phenotypes upon gut inflammation. Nevertheless, when the extent of T-cell infiltration was quantified in different tissues, the results show a significant and selective impairment of DrdS-deficient CD4⁺ T-ce!!s to be recruited into the gut mucosa (Fig. 3E-F). Conversely, the recirculation of CD4⁺ T-ce!ls though the spleen and MLN was not affected by Dr d-defieiency (Fig. 3E-F). Intriguingly, when gut-fropism associated molecules were quantified In CD4⁺ T-celis infiltrating different tissues, we observed an increased density of a4b7 expression in Dre/S-deficient lymphocytes in the cLP and MIN, but without effect in those cells recirculating though the spleen (Fig. 4). On the other hand, Drd5- deficiency reduced the frequency of CCR9⁺ a4b7 , whereas increased the percentage of double positive CCR9⁺ a4b7⁺ CD4⁺ T-celis in the MLN (Fig. 3G). DRD5-signalling in CD4⁺ T-cells had no effect in the frequency of CCR9⁺ a4b7 , CCR9 a4b7⁺ or CCR9⁺ a4b7⁺ in the spleen or cLP upon chronic gut inflammation (Fig. 3G). Thus, these results indicate that, despite Drc 5-deficient CD4⁺ T-cells display higher surface density of the gut horning molecule a4b7, these cells are recruited in a lesser extent into the gut mucosa.

Since the results above exposed indicate a role of DRDS-signa!ling in the extent of CD4’ T-celi Infiltration into the gut mucosa upon inflammation and suggest a regulatory role in the expression profile of gut homing molecules, we next attempted to determine whether DRD5-signalling was involved in the migration of inflammatory lymphocytes into the gut mucosa. For this purpose, we set up an in vivo migration assay in which the infiltration of congenic Drd5- sufficient (Cd45. 1^+/+) and Drd5- deficient ( Cd45.2^+/+ ) CD4⁺ T-cells displaying gut tropism into different tissues was compared Inside the same recipients after a short period of time (Fig. 5A). The results show that the arrival of DrdS-defscient CD4 T-celis to the cLP and MLN was significantly impaired in comparison with Drc/5-sufiicient lymphocytes (Fig. 5B-C). Conversely, the recirculation through the spleen was similar for D/T/5-sufiicieni and Dra^'S-deficient GD4⁺ T-cells, thus suggesting that DRD5-signailing has no a significant effect in cell death and/or proliferation of GD4⁺ T-cells during this period of time. To gain a deeper insight of the role of DRDS-signalling in CD4⁺ T-ce!l migration, we also determine the expression profile of gut-ho ing molecules on CD4⁺ T-cells upon arrival to the different tissues analysed. Surprisingly, despite the migration of Drd5- deficient CD4⁺ T-cells to the cLP and MLN was impaired, CCR9 expression was increased in frequency (cLP) and in density (MLN) (Figs. 5D-E and 6). Conversely, CCR9 expression was not affected by DrdS- deficiency in those CD4⁺ T-cells recirculating through the spleen (Figs. SD-E and 6). «4b7 expression profile was similar in Drd5-sufficient and Drc/5-deficient CD4⁺ T-cells In all the tissues analysed in this in vivo migration assay (Figs. 5F and 6). Since the results above indicate a relevant role of DRD5-signa!iing in the recruitment of CD4‘ T-cells displaying gut tropism into the gut mucosa upon inflammation, we attempted to determine whether DRDS-signalling was also relevant for lymphocyte migration under steady-state. For this purpose we determine the surface expression of several molecules involved in homing of CD4⁺ T-cells isolated from the spleen of Drd5-sufficient and Drdo-deficienf mice in steady-state and we found no significant differences between, genotypes (Fig. 7). Thus, together these results indicate that DRD5~signa!iing is required to the arrival of CD4⁺ T-cells into the gut mucosa and, intrlguingiy, Drc/S-deficiency results in increased expression of CCR9 after a short period of time and enhanced density of a4b7 upon chronic inflammation.

Example 3. OCRS and DRD5 form heteromers in CD4⁺ T-cells

During the past decade an increasing number of studies, performed mainly in the nervous system, have reported heteromerisation between different couples of G protein-coupled receptors (GPCRs) to form oligomers (Gomes et a!., 2016). Evidence has shown that GPGRs heieromers involve complex interactions at the level of signalling and ligand affinity which can induce a final physiological outcome quantitatively and qualitatively different of those exerted by the corresponding protomers separately Since we observed an attenuated CD4+ T-cei! migration to the gut mucosa even when CCR9 expression was higher in Drd5-deficient lymphocytes, we suspected that CCR9 could be forming heieromers with DRD5. Accordingly, we next performed in vitro experiments to address this possibility. For this purpose, a CD4 T-ceii !ine, Jurkat cells, was transfected with constant amounts of cDNA encoding Renilia !uciferase (RLuc}-fusioned to DRD5 and increasing amounts of cDNA codifying for the Yellow Fluorescent Protein (YFP) fusioned to CCR9 and Bioluminescence Resonance Energy Transfer (BRET) experiments were performed. These assays were based in that, when both molecules RLuc-DRD5 and YFP-CCR9 are separated for 10- 100 A, the luminescent energy produced by the reaction catalysed by RLuc is able to excite YFP The results show a saturation curve for RLuc-DRD5 and YFP-CCR9, indicating close proximity between these receptors (Fig, 8A). As a control, the BRET of constant RL.UC-DRD5 expression with increasing expression of the ghrelin receptor 1 a fusioned to YFP (YFP-GHSR1 a) was evaluated and just barely signal was detected (Fig. 8A). Similar results were obtained when the same experiments were carried out in HEK293 cells (Fig, 9), thus indicating that, indeed, CCR9 might form heieromers with DRD5 in transfected cell lines in vitro.

Afterwards, to assess whether CCR9.DRD5 heieromers are actually found in vivo, we determined the association of CCR9 and DRD5 in the colonic mucosa of mice upon inflammatory colitis by in situ proximity ligation assay (PLA), which is able to detect when two target proteins are in dose proximity (< 17 riM) (Callen et a!., 2012). To this end, DrdS-sufficient and Dra'5-deficient naive CD4⁺ T-ceils were transferred alone or together (1 :1 mix) into RagY^;~ recipient mice and 12-wk iater mice were sacrificed and PLA was assessed in colonic slices. The results show abundant mark for CCR9:DRD5 association (PLA^* mark, red dots) in colonic samples of mice recipients of Dro'5-sufficieni CD4* T-ce!!s and in a lesser extent in those colonic samples obtained from recipients of 1 : 1 mixture of Drc/5-sufficient and DrdS-deficient naive CD4⁺ T-celis (Fig, 8B). Conversely, the mark for CCR9:DRD5 association was virtually absent in colonic samples obtained from recipients of DrcfS-deficient naive CD4⁺ T-ce!ls (Fig. 8B). Quantitative analysis in recipients of Drcf5-sufficient CD4^* T-ceils shows that aproximately 75% of lymphoid cells infiltrating the coionic mucosa are PLA⁺ (Fig. 8C). In contrast, recipients of Drd5~ deficient naive GD4^* T-cells displayed barely detectable PLA lymphoid ceils infiltrating the colonic mucosa in the same direction, the density of the mark for CCR9:DRD5 association was significantly higher in recipients of Drd5~ sufficient CD4⁺ T-cells in comparison with that of recipients of DraS-deficient CD 4⁺ T-ce!ls (Fig. 8C). Recipients of 1 :1 mixture of DrbS-sufficient and Drd5-deficient CD4⁺ T- ceiis presented an intermediate frequency of PLA cells and density of PLA mark (Fig. 8C). Thus, together these results indicate that CCR9:DRD5 heteromers can be formed in CD4⁺ T-cells, and suggest that these heteromers are found in CD4⁺ T-cells infiltrating the gut mucosa upon inflammation.

To further analyse the molecular interaction between CCR9 and DRD5 required for the CCR9:DRD5 heteromer assembly, we next attempted to decipher the transmembrane (TM) segments from CCR9 and DRD5 involved in the interphase of interaction. For this purpose, we generate a set of peptides analogue to the seven TM segments from CCR9 and to the seven TM segments from DRD5 (herein collectively called TM-peptides; Table 1). Since this kind of peptide has been shown to interfere with BRET assays, we used a bimoiecuiar fluorescence complementation (BiFC) assay to evaluate the disassembly of the CCR9:DRD5 heteromer by competition with TM-peptides (Fig. 10A). In these experiments, Jurkat cells were transfected with the N-terminai fragment of Venus protein fused to DRD5 (DRDS-nVenus) and with C-ierminal fragment of Venus protein fused to CCR9 (CCR9-cVenus) and the fluorescence associated to Venus was evaluated in the presence of the different TM-peptides. The results show that only TM5 and TM6 from CCR9 and TM5 and TM6 from DP.D5 were able to strongly attenuate the assembly of Venus (Fig. 10B), thus indicating that the TM5 and TM8 from each protomers are involved In the interphase of interaction required for CCR9:DRD5 heteromer formation.

To analyse the functional relevance of the CCR9:DRD5 heteromer, we next studied the crosstalk of CCR9 and DRD5 signalling at the level of ERK1 /2 phosphorylation and c.AMP production, which have previously described to be modulated by the stimulation of these receptors (Franz et a!., 2015; Sharma et aL, 2010). To this end, we transfected Jurkat cells with CCR9 and DRD5 and dose-response curves of ERK1/2-phosphorylation and cAMP acumulation were generated with increasing concentrations of the CCR9-agonist CCL.25 and the DRD5-agonist SKF81297 and optimal dose of ligand were obtained for further experiments (Fig. 11 ). Afterward, the crosstalk of CCR9-stimu!ation and DRD5-stimulation was analysed at the level of ERK1/2- phosphoryiation and cAMP acumulation. We observed that despite CCR9-stimu!ation and DRD5- stimulation each alone induce increased ER.K1 /2-phosphorylation, both together had no significant effect in this signalling pathway (Fig, 10C, left panel). Furthermore, a DRD5-antagonist (SCH23390) was able to attenuate the increased ERK1 /2-phosphorylation induced by DRD5- stimulation and by CCR9~s†imuiation, thus Indicating cross-antagonism (Fig. 10C, right panel). Importantly, when Jurkat cells were pre-incubated with TM-peptides that disrupt CCR9:DRD5 heteromer assembly (TM5C, TM6C, TM5D and TM5D). but not when pre-incubated with TM- peptides irrelevant for the heteromer assembly (TM1 C, TM1 D) the cross-talk at the level of ERK1 /2-phosphorylation was abrogated (Fig. 10D). On the other hand, whereas CCR9- stimulation induces an inhibition of cAMP production and the DRD5-stimula†ion promotes increased cAMP accumulation, when both receptors are stimulated together, cAMP levels are reduced (Fig. 10E, left panel). Moreover, a cross-antagonism was observed for CCR9 and DRD5 triggered signalling at the level of cAMP as well (Fig. 10E, right panel). Similar to the effect at the level of ERK1/2-phosphoryiation, when Jurkat cells were pre-incubated with TM-peptides that disassembled the CCR9:DRD5-heieromer, the cross-talk of CCR9 and DRD5 at the level of cAMP production was lost (Fig. 10F). Taken together these results indicate that the CCR9:DRD5 heteromer is functionally relevant, as the cross-talk of CCR9 and DRD5 at the level of intracellular signalling is different for the assembled heteromer in comparison with their separated protomers Of note, these findings indicate that the CGR9:DRD5 heteromer works as an independent receptor, with different function of CCR9 and DRD5.

Example 4. The disruption of the CCR9:DKD5 heteromer formation impairs CD4^* T-cefl migration into the gut mucosa

To address the functional relevance of the CCR9:DRD5 heteromer at the level of CD4⁺ T-celi migration, we next carried out in vivo and in vitro experiments in which the heteromer assembly was impaired by TM-peptides and the recruitment of T-ceiis bearing gut-tropism was analysed in different settings. To this end, we first treated congenic CD4⁺ T-ce!!s bearing gut-tropism with a TM-peptide that disassembled the CCR9:DRD5 heteromer formation (TM5C) or with an irrelevant TM-peptide (TM1 C) and then were transferred together into Rag1^_/ mice and the arrival to different tissues was evaluated 72b later (Fig. 12A) The results show that the disruption of the CCR9:DRD5 heteromer resulted in a higher proportion of ceils recirculating through the spleen, a reduced number of ceils reaching the MLN and without significant effects in the extent of cells arriving to the small intestine (SI) lamina propria (Fig. 12B). Unfortunately, ceils were not detectable in the cLP in these experiments (data not shown). Despite the results suggest that interference with the CCR9:DRD5 heteromer formation would promote recirculation of T-ce!!s through the spleen and impairing the arrival of T-cells into the gut mucosa, this data was not condussive. For this reasons we next used another in vivo migration assay in the context of gut inflammation. For this purpose, acute inflammatory colitis was induced in wild-type congenic mice by treatment with dextran sodium sulphate (DSS) 48 h after initiated the induction of the disease, congenic CD4⁺ T-cells bearing gut-tropism and pre-treated with a TM-peptides were transferred into DSS-rnice and 72n later the arrival of transferred ceils was analysed in different tissues. The results indicate that disruption of the CCR9:DRD5 heteromer impaired selectively the arrival of CD4+ T-cells into the colonic and cecum mucosa (Fig. 12C). Of note, any of the TM-peptides affected significantly the viability of CD4⁺ T-ce!!s (Fig. 13) The expression of CCR9 was also evaluated and was not significantly affected by the treatment with the TM-peptides used for migration assays (Fig. 14). Finally, we attempted to evaluate the functional impact of the CCR9:DRD5 heteromer using in vitro migration assays. For this purpose we assessed the migration of CD4⁺ T-ce!is bearing gut-tropism into CCL25 containing chambers when pretreated with TM-peptides in transweiis assays. The results show that cells pre-treated with an irrelevant TM-peptide (TM7C) present a significant higher migration to CCL25 in comparison with the basal migration into a chamber containing only vehicle. However, when the assembly of the CCR9:DRD5 heteromer was impaired by TM5C or TM6C, the migration to CCL25 containing chambers was abrogated (Fig, 12D). Furthermore, we analysed the migratory speed of CD4⁺ T- ceiis bearing gut-tropism in confined microchannels. In this regard we first demonstrated a dose- dependent increase of the migratory speed of these ceils when CCL25 concentration was enhanced (Fig. 15A) We also observed a potentiation In the migratory speed of CD4⁺ T-ce!!s when both, DRD5 and CCR9 were stimulated together (Fig. 1 SB). Importantly, this potentiation of migratory speed mediated by DRD5-stimuiation and CCR9-stimulation was abrogated when CCR9:DRDRS heteromer formation was disrupted by TM6D, but not when treated with an irrelevant TM-peptide (TM1 D) (Fig, 15C). Taken together these results indicate that the CCR9:DRD5 heteromer constitutes a key molecular sensor driving the migration of CD4⁺ T-celis into the gut mucosa.

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Claims

1. A method for disrupting the formation of€€R9:DR 5 heteromer assembly in a subject in need comprising administering to the subject a pharmaceutical composition comprising one or more disrupter agent that inhibit or block the interaction between CCR9 and DRDS.

2. The method of claim 1, wherein the disrupter agent inhibit or block the interaction between, the transmembrane segment 5 and/or transmembrane segment 6 of CCR9, and the transmembrane segment 5 and/or transmembrane segment 6 of DRDS.

3. The method of claim 2, wherein the disrupter agent is a peptide

4. The method of claim 3 wherein the disrupter agent is a peptide analog to transmembrane segment 5 or transmembrane segment 6 from Q3R9 or DRDS.

5. A method for inhibiting the homing of CD4+ T-cells with gut-tropism in a subject in need comprising administering to the subject a pharmaceutical composition comprising one or more disrupter agent that inhibit or block the formation of CCR9:DRD5 heteromer assembly

6. The method of claim 5, wherein the disrupter agent inhibits or block the Interaction between CCR9 and PROS.

7. The method of claim 6 wherein the disrupter agent inhibits or block the interaction between, the transmembrane segment 5 and/or transmembrane segment 6 of CCR9, and the transmembrane segment 5 and/or transmembrane segment 6 of DRDS.

8. The method of claim 7, wherein the disrupter agent is a peptide.

9. The method of claim 8, wherein the disrupter agent is a peptide analog to transmembrane segment 5 or transmembrane segment 6 from CCR9 or DRDS.

10. A method for treating inflammatory bowel disease in a subject in need comprising administering to the subject a pharmaceutical composition comprising one or more disrupter agent that inhibit or block the interaction between CCR9 an DRDS, an therefore disrupting the formation of CCR9:DRD5 heteromer assembly.

11. The method of claim 10, wherein the disrupter agent inhibits or block the interaction between, the transmembrane segment 5 and/or transmembrane segment 6 of CCR9, and the transmembrane segment 5 and/or transmembrane segment 6 of DRD5.

12. The method of claim 11 , wherein the disrupter agent is a peptide.

13. The method of clai 12, wherein the disrupter agent is a peptide analog to transmembrane segment 5 or transmembrane segment 6 from CCR9 or DRD5.

14. The method of claim 10, wherein said inflammatory bowel disease is Crohn Disease.

15. The method of claim 10, wherein said inflammatory bowel disease is Ulcerative Colitis.

16. The method of any precedent claims 1 to 15. wherein the one or more disrupter agents are peptides analogu to transmembrane segments comprises an amino acid sequence selected from the group consisting of TM 1C (SEQ 11) No 1 ), TM2C (SEQ ID No.2), TM3C (SEQ ID No.3), TM4C (SEQ ID No.4), TM5C (SEQ ID No.5). TMPC (SEQ ID No.6), TM7C (SEQ ID No.7), TM1 D (SEQ ID No.8), TM2D (SEQ ID No.9), TM3D (SEQ ID No.10), TM4D (SEQ ID No.11), TM5D (SEQ ID No.12), TM6D (SEQ ID No.13) and TM7D (SEQ ID No.14).

17. The method of claim 16, wherein the peptide analogues comprises an amino acid sequence that has at least 80% sequence identity_' to amino acid sequence selected from the group consisting of TM 1C (SEQ ID No.1), TM2C (SEQ ID No.2), TM3C (SEQ ID No.3), TM4C (SEQ ID No.4), TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM7C (SEQ ID No 7), TMID (SEQ ID No.8), TM2D (SEQ ID No.9), T. 3D (SEQ ID No J O), TM4D (SEQ ID No.11), TM5D (SEQ ID No.12), TM6D (SEQ ID No.13) and TM7D (SEQ ID No.14).

18. The method of ciaim 17, wherein amino acid sequence is selected from the group consisting of TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM5D (SEQ ID No.12) and TM6D (SEQ ID No.13).

19. The method of any one of claims 1 to IS, wherein the method further comprises administering one or more additional therapies.

20. The method of claim 19, where m the one or more additional therapies comprise ami- inflammatory bowel disease therapy.

21. The method of claim 20, wherein the additional anti-inilammatory bowel disease therap comprises therapeutics selected from the group consisting of neutralizers of anti-TNFalpha (infliximab (Remicade), adafimumab (Hiimira), golimumab (Sin poni), Certolizumab), alpha4 i tegrin subunit inhibitors (natalizumab (Tysabri)), alpha4beta7 integrin inhibitors (vedo!i/umab { Enty vio 11.-42 Rbeta ! blocker that avoid its union to ligands 11. 12 and 11 -23 (ustekimrmab (Stelara)), anti-MAd-CAM-l monoclonal antibody (PF-00S47659), anti-IL-23 monoclonal antibody ( MED! 2070), sphingosine-1 -phosphate (SI P) receptor agonist (Ozanimod), antisense oligodeoxynucleotide complementary to mKMA of Smad7 (Mongersen), aminosalicylates (5-ammosaiieyiic acid (5-ASA); 5-ASA derivatives (Sulfasalazine, Asacol HD, Deizicol, Pentasa, Colazal, Dipentum, Lialda, Apriso, Mesalazine), corticosteroids (hydrocortisone, methylprednisoione, prednisone, Bndesonide, Budesonide MMX, hydrocortisone), antibiotics (ciprofloxacin, metronidazole, rifaximin, omidazole, tinidazoie, anti-tuberculosis therapy macrolides, fluoroquinolones, 5-nitioimidazoles, antimycobacterials), and imimmomodulators (tfriopurines (Pnrinethol, Purixan), 6-mercaptopurine, azathioprine (Azasan, Imuran), methotrexate (Trexall , cyclosporine (Gengxaf, Neoral, Sandimmune).

22. A peptide or peptide analog comprising an a ino acid sequence selected from the group consisting of TM1C (SEQ ID Ho.I), TM2C (SEQ ID No.2), TM3C (SEQ ID No.3), TM4C (SEQ ID o.4), TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM7C (SEQ ID No.7), TM1D (SEQ ID No.8), TM2D (SEQ ID No.9), TM3D (SEQ ID No.10), TM4D (SEQ ID No l 1), TM5D (SEQ ID No 12), TM6D (SEQ ID No 13) and TM7D (SEQ ID No 14)

23. A peptide or peptide analog comprising an amino acid sequence that has at least 80% sequence identity to amino acid sequence selected from the group consisting of TM1C (SEQ ID No. l), TM2C (SEQ ID No.2), TM3C (SEQ ID No.3), TM4C (SEQ ID No.4), TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM7C (SEQ ID No.7), TMID (SEQ ID No.8), TM2D (SEQ ID No.9), TM3D (SEQ ID No.10), TM4D (SEQ ID No. l I), TM5D (SEQ ID No 12), TM6D (SEQ ID No.13) and TM7D (SEQ ID No.14).

24. The peptide of claim 22, wherein the amino acid sequence is selected from the group consisting of TM5C (SEQ ID No.5), TM6C (SEQ ID No.6), TM5D (SEQ ID No.12) and TM6D (SEQ ID No.13).

25. The peptide of claim 22, wherein the amino acid sequence of the peptide or peptide analog is between 9 and 50 mer.

26. The peptide of claim 22, wherein the amino acid sequence of the peptide or peptide analog is between 18 and 25 mer

27. A polynucleotide encoding, upon expression, the peptide or peptide analog according to claim 22.

28. A polynucleotide encoding, upon expression, the peptide or peptide analog according to claim 23.

29. A pharmaceutical composition comprising at least one peptide or peptide analog of claims 22, 23, 24, 25 or 26, and a pharmaceutically acceptable carrier, diluent or excipient.

30. The pharmaceutical composition of claim 29, for use in the treatment of inflammatory bowel diseases.

31. The pharmaceutical composition of clai 29. for use in the treatment of Crohn disease.

32. The pharmaceutical composition of clai 29, for use n the treatment of ulcerative colitis.

33. A kit comprising the pharmaceutical composition of claim 29, and instructions for administering the pharmaceutical composition to disrupt the formation of CCR9:DRD5 heteromer ssembl .

34. A kit comprising the pharmaceutical composition of claim 29, and instructions for administering the pharmaceutical composition to inhibit or block the interaction between CCR9 and DRD5.

35. A kit comprising the pharmaceutical composition of claim 29, and instructions for administering the pharmaceutical composition to inhibit the homing of CD4+ T-ce!ls with gut-tropism.

36. A kh comprising the pharmaceutical composition of claim 29, and instructions for administering the pharmaceutical composition to treat an inflammatory' bowel disease.

37. A kit comprising the pharmaceutical composition of claim 36. and instructions for administering the pharmaceutical composition to treat Crohn disease or ulcerative colitis.

38. Use of the peptide of claim 22 for preparation of a medicament for treatment of inflammatory bowel diseases.

39. Use of the peptide of claim 22 for preparation of a medicament for treatment of Crohn disease.

4C1 Use of the peptide of claim 22 for preparation of a medicament for treatment of ulcerative disease.

41. Use of the pharmaceutical composition of claim 29 for preparation of a medicament for treatment of inflammatory bowel diseases.

42. Use of the pharmaceutical composition of claim 29 for preparation of a medicament for treatment of Crohn disease.

43. Use of the pharmaceutical composition of claim 29 for preparation of a medicament for treatment of ulcerative disease.