WO2018020060A1 - Method for predicting the clinical response to a treatment with anti-inflammatory agents - Google Patents

Abstract

The present invention relates to the use of allelic variants of human TLR10, including the allelic variant of human TLR10, I473T, as biomarkers for predicting gravity or prognosis in inflammatory diseases, including rheumatoid arthritis, and/or for predicting the response to a treatment using anti-inflammatory agents such as anti-TNFα agents.

Description

 METHOD TO PREACH THE CLINICAL RESPONSE TO A TREATMENT WITH

 ANTI-INFLAMMATORY AGENTS

FIELD OF THE INVENTION

The present invention can be included in the field of personalized medicine. More specifically, the present invention relates to the use of allelic variants of human TLR10, including the allelic variant of human TLR10 I473T, as a biomarker to predict severity or prognosis in inflammatory diseases, including rheumatoid arthritis and / or to predict response to a treatment with anti-inflammatory agents, such as anti-TNFα agents.

BACKGROUND OF THE INVENTION

Rheumatoid arthritis (RA) is a systemic autoimmune disease primarily characterized by chronic inflammation of the synovial lining and activation of Th1, which leads to the progressive destruction of the joints. It has been suggested that viruses and bacteria may contribute to initiate or aggravate RA by binding to Toll-like receptors (TLR) [Iwahashi M, et ai., Arihrítis Rheum 2004, 50 (5): 1457-1467 .; Pierer M, et a /., J Immunoi 2004, 172 (2): 1256-1265].

TLRs belong to a family of transmembrane proteins that constitute one of the primary defense mechanisms in infectious and some non-infectious diseases in mammals [Gdor I, et ai., Chem Phya 2011, 13 (9): 3782-3787]. In particular, TLRs are transmembrane type I glycoproteins with extracellular leucine-rich (LRR) repeats and a homology domain with the intracellular Toll / IL-1 receptor (TIR) [Ulevitch RJ, Nat Rev Immunoi 2004, 4 (7 ): 512-520]. Inappropriate activation of TLR-mediated pathways has been implicated in the loss of self-tolerance that leads to autoimmunity and chronic inflammation [Wagner H, Adv Immunoi 2006, 91: 159-173; Deighton K, et ai., Appetite 2014, 81: 52-59] In addition, it has been suggested that TLRs are involved in responses to pathogens in RA [Cromartie WJ, et ai., J Exp Med 1977, 148 (8) : 1585-1602].

TLR signaling involves interaction with adapters that contain TIR domain, including MyD88, TRIF, TRAM, TIRAP and SARM1 [O'Neiil LA. Bowie AG; Nat Rev immunoi 2007, 7 (5): 353-364] that promotes the activation of the nuclear factor enhancer of Kappa light chains of activated B cells (NFKB) among other functions. Activation TLR-dependent NFKB leads to the production of chemokines, cytokines and proinflammatory cell adhesion molecules [O'Neili LA, Bowie AG; Nat Rav Immunol 2007, 7 (5): 353-364 .; Drexler SK, eí al., Int J Biochem Cell Biol 2010, 42 (4): 506-518]. There is increasing evidence that NFKB is an important, if not the main, transcription factor that controls inflammation [Abu-Soud HM, at al., Biochemistry 1998, 37 (11): 3777-3786] and decipher the mechanisms that regulate the activity of NFKB is considered of great importance to understand the response to inflammatory stimuli.

TLR10 remains the only orphan member among human TLRs because their ligands remain unknown and there are discordant data about their function [Oostíng M, eí al., Proc Nati Acad Sci U S A 2014. 111 (42): E4478-4484 .; Hasan U, at al., J Immunol 2005, 174 {5): 2942-2950]. The expression of TLR10 has been described mainly in B cells, dendritic cells, eosinophils, neutrophils and non-immune cells such as trophoblasts [Hasan U eí al., J Immunol 2005, 174 (5): 2942-2950. Homung V et al. ., J Immunol 2002, 168 {9): 4531-4537 .; Nagase H. et al. J Immunol 2003, 171 (8): 3θ77-3982; Mulla MJ, at al., Am J Reprod Immunol 2013, 69 (5): 449-453]. In mammals, TLR10, TLR1, and TLR6 share a common locus on chromosome 4p14 and are structurally similar to each other [Hasan U, et al., J Immunol 2005, 174 (5): 2942-2950]. Despite interacting with My088 [Hasan U, et al., J Immunol 2005, 174 (5): 2942-2950], TLR10 differs from other TLRs due to the lack of a classical posterior signaling route [Guan Y, et al., J Immunol 2010, 184 (9): 5094-5103]. Phylogeny supports the idea that TLR10 arose before the gene duplication that generated TLR1 and TLR6 [Abhishek A, eí al .. J Clin Rheumatol 2010. 16 (1): 15-18 .; Zhou H. at al., J Mol Evol 2007. 65 (2): 119-123]. TLR1 and TLR6 can form a protein complex with TLR2 and TLR10 [Hasan U, ei al., J Immunol 2005. 174 (5): 2942-2950 .; Mulla MJ ef al., Am or Reprod Immunol 2013, 69 (5): 449-453], although the individual contribution of each protein to the function of the complex is largely unknown.

Today, TLR10 is the only member of the TLR family without a well-defined biological function. Several studies describe that TLR 10 acts as a pro-inflammatory receptor by activating NFKB signaling [Hasan U, eí al., J Immunol 2005, 174 (5): 2942-2950; Regan T, eí al., J Immunol 2013, 191 (12): 6084-6092]. Another study showed that TLR10 does not activate typical TLR-induced signaling, including transcriptional activation mediated by NFKB or interferon-beta (IFNp) (Guan Y, eí al., J Immunol 2010, 184 (9): 5094-5103]. Recently. , it has been shown that TLR10 is a modulator with mainly inhibitory effects [Stappers MH, eí al., J Infect Dis 2015], In line with this, block TLR10 with Antagonist antibodies potentiate the production of prainflammatory cytokine [Oosting M, ei., Proc Nati Acad Sci USA 2014, 111 (42}: E4478-4484] .In addition, evidence has been provided that TLR10 induces apoptosis through the activation of caspasa-3 [Kuuiiaia K, et al., Ann Rheum Oís 2006, 65 (9): 1241-1243].

Genetic variants in members of the TLR family have been mainly associated with disease propensity in patients with RA with varying levels of significance and even discordant results [Kuuiiaia K, et al., Ann Rheum Dia 2006, 65 (9): 1241 - 1243 .; Radstake TR. et al., Arthritis Rheum 2004, 50 {3): 999-1001 .; Etem EO, et al., Rheumatoi Int 2011, 31 (10): 1369-1374 .; Coenen MJ, et al., PLoS One 2010, 5 (12}: e14326; Zheng B, et al., Rheumatoi Int 2010. 30 (9): 1249-1252].

A previous study investigated the association between variants of TLR10 and propensity to RA but no statistical significance was found [Etem EO, et al., Rheumatoi Int 2011, 31 (10): 1369-1374]. Although single-nucleotide variants of the TLR10 gene have been associated with other autoimmune diseases [Requena T, ei., Immunogenetics 2013, 65 (5): 345-355], tumors [Kutikhin AG, Hum Immunol 2011, 72 (11 ): 1095-1116], infectious and inflammatory diseases such as extrapulmonary tuberculosis and asthma [Ma X, et al., PLoS One 2007, 2 (12): e1318; Lazarus R, et al., Am J Reapir Crit Face Med 2004. 170 (6): 594-600], the functional activity of this protein and the clinical significance of the gene variants are still controversial [Hasan U, et al., J Immunol 2005, 174 (5): 2942-2950 .; Stappers MH, eí al., J lnfect Dis 2015].

The allelic variant I473T has only been described in a previous study associated with a decreased meningioma bias [Rajaraman P, et al., Epidemial Cancer Biamarkers Prev 2010, 19 (5): 1356-1361].

Despite significant progress in recent years in the discovery of prognostic and / or predictive markers related to RA and inflammatory diseases, there is a continuing need for improved methods to assess the severity and prognosis of the disease, to predict the evolution of the disease and the risk of recurrence or relapse, to allow the classification of the patient population according to the prognosis and select the most appropriate treatment accordingly. It is also desired to identify polymorphic regions within a gene, such as human TLR10, that are associated with the response to one or more drugs used in the treatment of RA and others. Inflammatory diseases, such as disease-modifying antirheumatic drugs (DMARDs) or biological therapies, including TNFα inhibitors.

SUMMARY OF THE INVENTION

NFKB is a transcription factor involved in many chronic inflammatory disorders, including RA. For the first time, the authors of the invention have described that TLR10 can inhibit NFKB signaling in hematopoietic cells. In vitro functional studies showed that TLR10 rounds the activation of the inflammatory NFKB pathway in hematopoietic cells, while the I473T variant did not have this inhibitory capacity. The inventors observed that, after stimulation of the NFKB pathway with TNFα brings ia exposure to infliximab (a TNFα inhibitor) cells expressing the I473T variant showed a further NFKB activity compared to cells carrying TLR10 of wild type .

It is known that TNFα inhibitors help control the evolution of RA. However, not all patients respond adequately to the initial anti-TNFα treatment [Alten R. et al., Int J Rheum Dis 2014, 17 (1): 5-18]. The inventors analyzed the association of the contradictory variant of human TLR10, I473T, which is located in the LRR18 domain, with AR observing that the I473T variant is not associated with propensity to AR. However, it was found that this vain correlates significantly with erosive disease in seropositive patients for antibodies against citrullinated proteins (ACPA) (p = 0.017 in the total cohort and p = 0.0049 in women) and with a lower response to infliximab treatment measured by the DAS28 Index (p = 0.012) and by the criteria of the EULAR (p = Q, 049), as shown in Figure 2B.

The data shows that a genetic variant of TLR10 selects a group of patients with a more severe and resistant disease. Likewise, said genetic variant of TLR10 may be a good candidate marker of response to infliximab or other anti-TNFα treatments in patients with inflammatory disease and in particular with RA.

In a first aspect, the present invention refers to an in vitro method for predicting the clinical response of a subject who has or is suspected of having an inflammatory disease to an anti-TNFα agent, wherein said method comprises: a) determine, in an isolated biological sample of said subject, the presence of one or more TLR10 polymorphisms that give rise to a variant of TLR10 that has lost the ability to inhibit NFKB. In a preferred embodiment, the present invention refers to a mobile method for predicting the dynamic response of a subject who has or is suspected of having an inflammatory disease to an anti-TNFα agent, wherein said method comprises: a) determine, in an isolated biological sample of said subject, the genotype of the single nudeotide polymorphism (SNP) rs11466657.

In another aspect, the invention relates to an in vito method for identifying a subject who has or is suspected of having an inflammatory disease with a higher risk of not responding to treatment with an anti-TNFα agent, wherein said method comprises the stage a) according to the first aspect.

In a related aspect, the invention refers to an in vivo method for selecting a treatment for a subject who has or is suspected of having an inflammatory disease, wherein said method comprises step a) defined above; and also comprises: b) selecting an anti-TNFα agent when the allele (A) is present in at least one of the alleles in the SNP locus rs11466657. preferably when the allele (A) is present in both alleles.

In a further aspect, the invention refers to an in vitro method for determining the severity and / or prognosis of the disease in a subject having an inflammatory disease, said method comprising step a) defined above. It also refers to an in vitro method to obtain useful data to determine the clinical response of a subject, who has or is suspected of having an inflammatory disease, to an anti-TNFα agent, the severity and / or prognosis of said disease. wherein said method comprises step a) defined above. Also, the invention provides a method for treating a subject having an inflammatory disease, wherein said method comprises step a) defined above; and further comprising: b) administering to said subject an anti-TNFα agent when the allele (A) is present in at least one of the alleles in the SNP locus rs11466657, preferably when the allele (A) is present in both alleles.

Furthermore, the present invention refers to an anti-TNFα agent for use in a subject that has or is suspected of having an inflammatory disease, in that said subject has the allele (A) in at least one of the alleles in the SNP locus rs11466657, preferably in which said subject is homozygous for the allele (A).

In a further aspect, the invention provides a kit for determining in a biological sample isolated from a subject and the genotype of one or more TLR10 polymorphisms that give rise to a variant of TLR10 that has lost the ability to inhibit NFKB, where said kit understands:

 - a reagent to determine the genotype of said polymorphisms;

 - optionally, instructions for the use of said reagent in determining the genotype of said polymorphisms in an isolated biological sample.

The invention also relates to the use of a kit, as described in the previous aspect, to predict the clinical response of a subject having an inflammatory disease, to identify a subject having an inflammatory disease at risk of not responding. to a treatment with an anti-TNFα agent, to select a treatment for a subject having an inflammatory disease, or to determine the severity and / or prognosis of an inflammatory disease in a subject, according to the methods of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Variants of the TLR10 gene. (A) Schematic representation of the TLR10 protein that illustrates the position of fmissense "in English" nonsense variants identified by new generation sequencing (NGS). LRR, rich repeats in leucine; TM, transmembrane domain; TIR, Toll-interieucina receptor domain. (B) Genotype distribution of the I473T variant in the independent cohort of 1493 healthy controls (HC) and 1201 patients with rheumatoid arthritis (RA). (C) TLR10 genotype frequencies in HapMap populations (HapMap is a map of haplotypes in the human genome; International HapMap consortium et al. (2010), Integrating common and rare genetic variation in diverse human populations, Natura. 467, 52- 8).

Figure 2. Association of the I473T variant with the severity of the disease in two cohorts of (A) 453 and (B) 1201 patients with RA. The beta value (regression coefficient), used to evaluate the effect of the I473T variant (genetic risk), was estimated for several clinical findings. A positive regression coefficient means that the variant increases the risk. Dashed lines indicate cut-off values. The clinical parameters were categorized as binary variables. Eccp / years, association with erosions in patients who are positive for ACPA, including the evolution of the disease; Eccp / years in women, association with erosions in female patients positive for ACPA including years of disease progression; IFX-EULAR, association with the response to infliximab (EULAR response criteria: moderate / good versus none); IFX-DAS28, association with change in DAS28 in patients treated with infliximab.

Figure 3. Regulation of the transcriptional activity of NFKB by the I473T variant. A) TLR10 3D structure using the Jmol viewer that shows the location of the I473 residue. NAG, 2- (acetylamino) -2-deoxy-beta-D-glucopyranose. (B) and (C) K562 and U937 cells (respectively) were co-transformed with wild-type TLT10 (wild type or wt) or its mutated variant (mut) and an indicator vector containing sequences that respond to NFKB. The cells were stimulated with 10 ng / ml TNFα for 24 h and then the cell extracts were prepared and analyzed for lucifer activity. Likewise, cells (D) K562 and (E) U937 transfected with the TLR10 variants indicated in the presence or absence of infliximab were treated with TNFα and luciferase activity was analyzed. The histog branches show the mean & SD of three independent experiments. * p <0.05.

Figure 4. Regulation of the levels of NFKB target genes by the I473T variant. U937 (A, B, C) and K562 (D, E, F) cells were transfected with TLR10 (wild type or mutated variant) and then treated or not treated with TNFα 20 ng / ml in the presence or absence of infliximab ( IFX) for 24 h. The expression of NFKB target genes, CCL2 (A, D, F) TRAIL (B, E) and TNFα (C) by quantitative RT-PCR. Β-actin was used for normalization. C. cells transfected with empty vector as additional control. Histograms show the mean ± SD of three independent experiments. * p <0.05. DETAILED DESCRIPTION OF THE INVENTION

Definitions

 The term "subject" as used herein refers to a mammalian subject. Preferably, it is selected from a human, companion animal, non-domestic livestock or zoo animal. For example, the subject can be selected from a human, dog, cat, cow, pig, sheep, horse, bear, etc. In a preferred embodiment, said mammalian subject is a human subject.

The term "subject who has a disease" as used herein refers to those subjects who have been diagnosed with that disease. For example, a "confirmed diagnosis of rheumatoid arthritis" or "definitive rheumatoid arthritis" may be based on the confirmed presence of synovitis in at least one joint, absence of an alternative diagnosis that better explains synovitis, and the achievement of a total score of 6 or greater (from a maximum possible of 10) from the individual scores in four domains that include: number and location of affected joints (range 0-5), serological alterations (range 0-3), high acute phase response (range 0-1) and duration of symptoms (two levels, range 0-1). The meaning of these parameters is as defined in the EULAR classification criteria (Ann Rheum Dis 2010; 69: 1580-1588).

The term "subject suspected of having a disease" as used herein refers to a subject presenting one or more signs or symptoms indicative of said disease. A subject that is suspected of having a disease may also have one or more risk factors (i.e., a subject that is suspected of developing or presenting a risk of developing a disease). It also covers an individual who has received a preliminary diagnosis but for whom a confirmation test has not been performed. In addition, it includes those individuals in remission.

The term "examination" as used herein refers to identifying, determining or distinguishing those subjects or individuals that present the characteristics or the defined phenotype A 'test' test may be used when the presence of such characteristics or phenotype is suspected.

The term "treatment" encompasses both a prophylactic and therapeutic treatment. The term therapeutic treatment "or" therapy "as used herein refers to bringing a body from a disease or disease state back to its normal, healthy state. The term" prophylactic treatment "as used in this document it refers to preventing a pathological state The term "response to a treatment" as used herein refers to the degree to which a treatment achieves the desired or expected results, for example the ability of a therapy or a drug to achieve the desired clinical effect.

The term "inflammatory disease" as used herein refers to any disease in which there is an excessive or altered inflammatory response that leads to inflammatory symptoms. Such inflammatory diseases include, but are not limited to, Addison's disease, acne vulgaris, alopecia a reata, amyloidosis. ulcerations, aphthous stomatitis, arteriosclerosis, arthritis, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, sponduroarthropathy, ankylosing spondylitis, bronchial asthma, Behcet's disease, Boeck's disease, Inflammatory bowel disease, Crohn's disease, choroid ulcer celiac disease cryoglobulinemia, macular degeneration, dermatitis, herpetiform dermatitis, dermatomyositis, insulin-dependent diabetes, juvenile diabetes, inflammatory demyelinating disease, Oupuytren's contracture, encephalomyelitis, allergic encephalomyelitis, endophlalmits, allergic enteritis, autoimmune enteropathy syndrome, erythema nodosum , chronic fatigue syndrome, rheumatic fever, cystic fibrosis, gingivitis, glomerulonephritis, Goodpasture syndrome, Graves syndrome, Hashlmoto's disease, chronic hepatitis, histiocytosis, regional ileitis, iritis, systemic lupus erythematosus, cutaneous lupus erythematosus, erythomatous lupus lymphogranuloma, infectious mononucleosis, myasthenia g ra vis, transverse myelitis, primary idiopathic myxedema, nephrosis, obesity, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, panniculitis, vulgar perphigo, periodontitis, polyarteritis nodosa, chronic polyarthritis, polymyositis, acute polyradiculitis, psoriasis, chronic obstructive pulmonary disease, purpura, gangrenous pyoderma, Reiter's syndrome, diabetic retinopathy, rasacea, sarcoidosis, lexical sclerosis, progressive systemic sclerosis, scleritis, scleroderma, multiple sclerosis, disseminated sclerosis uveitls, vitiligo, Whipple's disease, diseases associated with AIDS, severe combined immunodeficiency and Epstein Barr virus: such as Sjogren's syndrome, osteoarticular tuberculosis and parasitic diseases: such as ia leishmaniasis.

The term "inflammatory rheumatic disease" as used herein refers to a disease of the musculoskeletal system and / or connective tissues in which inflammatory mechanisms play an important role. A rheumatic disease can affect joints, bones, cartilage, tendons, ligaments and muscles.

A "sample" in the context of the present invention is a biological sample that contains any type of body cell and may include, as illustrative and non-limiting examples, body fluids and / or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are routinely obtained from a tissue biopsy. A biological sample useful in the present invention includes blood, urine, saliva, synovial fluid, cerebrospinal fluid, bronchial lavage, ascitic fluid, bone marrow aspirate, pleural effusion, as well as any tissue, fluid or any other body constituent that may contain cells. . In a preferred embodiment, the sample to be tested is saliva or blood. In a more preferred embodiment, the sample is a blood sample. The term "allele" has the meaning that is commonly known in the art, that is, an alternative form of a gene (a member of a pair) that is placed in a specific position on a specific chromosome that, when translated, gives as result functional or dysfunctional gene products (including non-existent). The term "polymorphism" or "allelic variant" refers to a common sequence variation of a gene. Allelic variants can be found in exons, introns, untranslated regulatory regions of the gene. or in the sequences that control gene expression. Complete gene sequencing can often identify numerous allelic variants for a particular gene. The significance of allelic variants is often unclear until further study of the genotype and the corresponding phenotype is carried out in a sufficiently large population.

The term "single nucleotide polymorphism" or "SNP" refers to a type of DNA polymorphism that involves the variation of a single base pair. There are millions of SNPs in the human genome. Commonly, these variations are found in gene coding sequences, non-gene coding regions or in intergontic regions between genes. The Presence of a SNP within a gene or in a regulatory region near a gene, can play a more direct role in the disease affecting the function of the gene.

The term "probe" as used herein refers to synthetic or biologically produced nucleic acids, between 10 and 28S base pairs in length that contain specific nucleotide sequences that allow specific and preferred hybridization under predetermined conditions to select target nucleic acid sequences, and optionally contain a moiety to detect or enhance assay performance. Generally a minimum of ten nucleotides is necessary in order to obtain statistically specificity and form stable hybridization products, and a maximum of 285 nucleotides generally represents an upper limit for the length at which the reaction parameters can be easily adjusted to determine mismatched sequences and preferential hybridization. The probes may optionally contain certain constituents that contribute to their proper or optimal operation under certain test conditions. For example, the probes can be modified to improve their resistance to degradation with nuclease (for example, by occupation of ends), to carry detection ligands (for example, fluorescein) or to facilitate their capture on a solid support (for example, "tails" of poly-deoxyadenosine).

The term "primers" as used herein refers to oligonucleotides that can be used for example in an amplification method, such as a polymerase chain reaction ("PCR"), to amplify a nucleotide sequence. Primers are designated based on the polynucieotide sequence of a particular target sequence. The design and validation of primers and probes are well known in the art. For quantitative real-time PCR methods, see for example Rodríguez A ef e /. (Methods Mol Biol., 2015, 1275: 31-56).

The term "specific" as used herein in relation to a nucleotide sequence means that a nucleotide sequence will hybridize with / amplify a predetermined target sequence and will not hybridize with / substantially amplify a non-target sequence under the conditions. For testing, generally rigorous conditions are used. The term "hybridization" as used herein refers to a process by which, under predetermined reaction conditions, two are allowed to come together. partially or completely complementary nucleic acid chains in an antiparallel way to form a double stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules corresponding to which nucleic acid bases can be matched together.

The term "substantive hybridization means that the amount of hybridization observed will be such that someone who observes the results could consider the positive result with respect to hybridization data in positive and negative controls. The data that are considered" background noise "are not substantial hybridization.

The term "stringent hybridization conditions" means approximately 35 ° C to 65 ° C in a NaCl salt solution approximately 0.8 molar. The rigor may also be governed by reaction parameters such as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and the hybridization temperature. Generally as hybridization conditions become stricter, longer probes are preferred if stable hybrids are to be formed. As a rule, the rigor of the conditions under which hybridization is performed will dictate certain characteristics of the preferred probes to be used.

The term "identity" as used herein refers to an exact correspondence of nudeotide with nucleotide or amino acid with amino acid of two polypeptide or potinucleophide sequences or, respectively. Two or more sequences (of polynucleotides or amino acids) can be compared by determining their "percent identity." The "percent identity" of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shortest sequence and multiplied by 100. Suitable programs to calculate identity in percentage or similarity between sequences they are well known in the art, such as the NCBI BLAST program, used for example with the default parameters (http://www.ncbLnlm.gov/cgi-bin/BLAST).

The term "partially identical / complementary" as used herein refers to a sequence that is identical / complementary in at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to a reference sequence. In some embodiments, the partially identical / complementary sequence may be substantially identical / complementary to a reference sequence, which is identical / complementary to at least about 95%, 96%, 97%, 98% or 99% to said reference sequence. In one embodiment, the partially identical / complementary sequence is 100% identical / complementary to a reference sequence.

The term "kit" indicates a set of reagents and adjuvants required for an analysis. Although a kit consists of most of the cases of several units, the various elements of analysis presented in a single unit, which should be considered as kits, may also be available.

Description

A method for predicting the clinical response to an antl-TNFq agent In a first aspect, the present invention refers to an in vttro method for predicting the clinical response of a subject who has or is suspected of having an inflammatory disease to an anti agent. -TNFα, in which said method comprises: a) determining, in an isolated biological sample of said subject, the presence of one or more TLR10 polymorphisms that give rise to a variant of TLR10 that has lost the ability to inhibit NFKB.

The NF-kB (nuclear factor enhancing kappa binding chains of activated B cells) plays a key role in the regulation of the immune response due to infection. Defective regulation of NF-kB is also related to cancer, inflammatory and autoimmune diseases, septic shock, viral infections or inadequate immune development. It is also involved in processes of synaptic plasticity and memory. In a particular embodiment, said variant of TLR10 has at least one polymorphism that results in a contradictory amino acid substitution. Said polymorphisms include but are not limited to those indicated in Table 2. Preferably, said polymorphism is selected from rs11466657 and rs11466650. More preferably, said polymorphism is rs11466657. Thus, in a preferred embodiment, the present invention refers to an in vibro method for predicting the clinical response of a subject who has or is suspected of having an inflammatory disease to an anti-TNFα agent. wherein said method comprises: a) determining, in an isolated biological sample of said subject, the genotype of the single nucleotide polymorphism (SNP) rs11466657.

It also refers to an in vitro method to identify a subject who has or is suspected of having an inflammatory disease with a higher risk of not responding to treatment with an anti-TNFα agent. wherein said method comprises step a) according to the first aspect.

The invention also refers to an in vitro method for obtaining useful data to determine the clinical response of a subject, who has or is suspected of having an inflammatory disease, to an anti-TNFα agent, wherein said method comprises step a) described. previously.

The single nucleotide polymorphism (SNP) rs11466657 has been previously described and its sequence is available in public databases (http://www.ncbi.nlm.nih.gov/project8/SNP/snp_ref.cgi7rss114e6657). As Figure 1A shows, rs11466657 is located in the region that codes for the human TLR10 LRR18 domain.

The rs11466657 is located on chromosome 4, at position 38774173 and corresponds, for example, to position 2056 of the human TRLR10 transcript NM_030956.3 (SEQ ID NO: 1). For this position (locus) the ancestral allele is T and an allelic change from AJT to A £ T has been described which results in a change in the protein sequence (NP_112218.2; SEQ ID NO: 2) of isoleucine (lie) to threonine (Thr) at position 473 (I473T). When said polymorphism is determined in the complementary chain, the allelic change is from A to G (see example 2). In the present invention the allelic variant of SNP rs11466657, which results in the change of amino acid I473T is referred to as allele (G).

According to the data presented in Example 2, patients with the I473T variant show a significantly lower response to anti-TNFα agents, according to the DAS28 Index (Preveo ML, van 1 Hof MA, Kuper HH, van Leeuwen MA, Go from Putte LB, go

PL rail (1ΘΘ5). Modified disease activity scores that include twenty-eight-joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum., 38, 44-48) and the EULAR response criteria (Karonitsch T, Aletaha D, Bcers M, Bombardierl S, Combe B, Dougados M, Emery P, Felson D, Gomez-Kingdom J. Keystone E, Kvien TK, Martin-Mola E, Matucci-Cerinic M, Richards P, van Riel P, Siegel J, Smoten JS, Sokka T, van der Heijde D, van Vollenhoven R, Ward M, Wells G, Zink A, Landewe R. ( 2008) Methods of deriving EULAR / ACR recommendations on reporting disease activity in cfinical triáis of patients with rheumatoid arthritis. Ann Rheum Dís, 67 (10), 1365-73), which also indicates that this variant selects a group of patients with a more serious or severe illness.

Thus, the determination of the presence of the allele (G) in at least one of the alleles in the SNP locus rs11466657, preferably the determination of the allele (G) in both alleles (Le., Genotype GG) is indicative of a greater refusal to respond to the anti-inflammatory agent. In other words it is predlct ^'vo absence of clinical response or resistance to treatment.

The determination of the presence or absence of said SNP can be determined by DNA sequencing, PCR analysis or any other genotyping method known in the state of the art. Examples of such methods include, but are not limited to, chemical assays, such as hybridization of the specific allele, primer extension ('first extension'), oligonucleotide ligation of specific allele, sequencing, enzymatic cleavage, 5 'discrimination (fiap ) endonuclease, and detection methods, such as fluorescence, chemiluminescence, and mass spectrometry, for example, the presence or absence of said polymorphisms can be detected in a DNA sample, preferably after amplification, for example, isolated DNA can be subjected to amplification by polymerase chain reaction (PCR), using oligonucleotide primers specific for polymorphism or allowing amplification of a region containing the polymorphism.For example, the conditions for primer hybridization can be selected to ensure specific amplification, so the appearance of an amplification product n is indicative of the presence of the polymorphism according to the invention. Alternatively, the DNA can be amplified, so that the SNP can be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art. Numerous strategies for genotype analysis have been described (Cooper et al., 1991; Grompe, 1993). For example, the presence or absence of a restriction site in a nucleic acid can be determined. When a polymorphism creates or removes the recognition site of a restriction enzyme, this allows genotyping of the polymorphism simply by direct PCR. Other strategies include, but are not limited to. direct sequencing, restriction fragment length polymorphisms (RFLP); hybridization with allele specific oligonucleotides (ASO), allele specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; Gradient denaturing gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), single chain conformational polymorphism (SSCP) and denaturing high resolution liquid chromatography (Kuklin et al, 1997).

Direct sequencing can be performed by any method known in the state of the art, including, but not limited to, chemical sequencing, using the Maxam-Gilbert method; enzymatic sequencing, using the Sanger method; pyrosequencing, mass spectrometry sequencing; sequencing using arrays; o Quantitative real-time PCR. Typically, the DNA to be sequenced is first subjected to PCR amplification using specific amplification primers. However, several other methods are available, allowing DNA to be studied independently of the PCR reaction, for example, rolling circle amplification (RCA), invader® assay, or oiigonucieotide ligand assay (OLA).

Another preferred quantification procedure is quantitative PCR (qPCR). The qPCR or real-time PCR is well known by a person skilled in the art. Different instruments are commercialized to carry out said reaction, such as ABI Prlsm 7700 SDS, GeneAmp 5700 SDS, ABI Prísm 7900 HT SDS of Applied Biosystems; iCycler iQ from Bio-Rad; Cepheid Smart Cyder; Rotor-Gene from Corbett Research; LightCycler by Roche Molecular Biochemicals and Mx4000 Multiplex by Stratagene. The qPCR procedure allows the exact quantification of the PCR product in real time by measuring the accumulation of the PCR product very early in the exponential phase of the reaction, thus reducing the bias in the quantification linked to the efficiency of the PCR amplification that is produces in endpoint PCR. Real-time PCR is well known in the art and therefore, is not described in detail herein. An overview of the technology and protocols for qPCR are available, for example, from the providers mentioned above, for example, or

http: //twMnw.8igmaaldrích.com/life-scienceAnolecular-b

technical-guide.html. A review of the use of qPCR in the quantification of mRNA is found for example in Wong ML and Medrano JF, Biotechniques 2005, 39 (1): 75-85. Different biochemical detection are available for qPCR. All of them can be used with the qPCR instruments mentioned above. The term "biochemical detection" refers to a procedure for reporting the amplification of the specific PCR product in real-time PCR. These biochemical detection are classified into two main groups. The first group comprises double chain AON interleaving molecules: such as SYBR Green 1 and EvaGreen; and the second group includes oligonucleotides typically labeled with a fluorophore. The latter, in turn, has been divided into three subgroups: (i) primers-probes (Scorpions, Amplifluor®, LUX ™, Cyciicons, Angler®); (ii) hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon ™, MGB-Pleiades, MGB-Edipse, ResonSense®, Yin-Yang or displacing) probes; and (ii) nucleic acid analogs (PNA, LNA®, ZNA ™, unnatural bases: Plexor ™ primer, Tiny-Molecuiar Beacon), see E. Navarro eta I., Clínica Chimica Acta, Volume 439, 15 January 2015 , Pages 231-250.

For example, said probes are oligonucleotides can be double-labeled, such as hydrolysis probes or molecular beacons. The 5 'end of the oiigonucieotide is typically labeled with a fluorescent indicator (reporter) molecule such as FAM. TET or JOE while the 3 'end is marked with a quencher molecule, such as TAM or BHQ1. The probe sequence is specific for a region of interest in the amplified target molecule. In a preferred embodiment, said probe is a hydrolysis probe that is designed so that the length of the sequence places the fluorophore 5 'and the deactivating molecule 3' in close proximity sufficiently narrow to suppress fluorescence. Several indicator and extinguishing molecules for use in qPCR probes are well known in the art. These are available, for example, from https://Www.eurofinsgenomics.eu/en/dna-ma<)iigonudeotídes/optimi

oligos / qpcr-probes.aspx.

Likewise, the present invention relates to oligonucleotide sequences (primers and / or probes) that hybridize specifically with one of the alleles of the polymorphisms. The term "a primer and / or probe" specifically includes "primers and / or probes", encompassing for example a primer, a probe, a primer and a probe, a pair of primers, and a pair of primers and a probe. Preferably, a probe and / or primer is a polynucleotide sequence of between 10 and 30 nucleotides, more preferably between 15 and 26 nucleotides. even more preferably of between 18 and 22 nucleotides, and even more preferably of about 20 nucleotides. In a particular embodiment, said primers and / or probes have been modified for detection or to enhance assay performance.

In a related aspect, the invention refers to an in vivo method for selecting a treatment for a subject who has or is suspected of having an inflammatory disease, wherein said method comprises step a) defined above; and further comprising: b) selecting an anti-TNFα agent when the allele (A) is present in at least one of the alleles in the SNP locus rs11466657, preferably when the allele (A) is present in both alleles.

In a preferred embodiment, the subject is ACPA positive. In a more preferred embodiment, said subject is an ACPA positive woman. An anti-tumor necrosis factor agent (in English "tumor necrosis factor", TNF) decreases, blocks, inhibits, cancels or interferes with TNF activity in vitro and / or in vivo. The term anti-TNFα and TNFα inhibitor are used interchangeably herein. In a preferred embodiment, said anti-TNFα agent is an antibody, preferably a specific anti-TNFα antibody. Methods for obtaining antibodies against a known antigen are part of the state of the art and one skilled in the art will know how to obtain anti-TNFα antibodies.

The term "antibody" is used in the broadest sense and includes fully assembled antibodies, chimeric antibodies, humanized antibodies, monodonal antibodies, polydone antibodies, multispecific antibodies (eg, bispecific antibodies), antigen-binding antibody fragments (eg. , Fab, Fab ', F (ab') ₂ , Fv, scFv, diabodies). single domain antibodies (sdAb) and recombinant peptides comprising the above, as long as they show the desired biological activity. The term "antibody" also refers to a fusion protein that includes a region equivalent to the Fe region of an immunoglobulin. Anti-TNFct agents are well known in the state of the art. By way of illustration, anti-TNFct agents include but are not limited to anti-TNFα agents selected from the group consisting of etanercept, adalimimab, infliximab, golimumab, certolizumab, rítuximab, abatacept, anakinra and tocizumab. In a preferred embodiment, said anti-TNFα agent is infliximab. Singh et al., Arthritis & Rheumathology, 2016, 88 (1), 1-16 provides a manual for the treatment of RA from the American College of Rheumatology. In particular, Table 1 provides a list of treatments for RA that includes anti-TNFα agents. In a particular embodiment, said inflammatory disease is an inflammatory rheumatic disease. Preferably, said inflammatory rheumatic disease is selected from the group consisting of arthritis, psoriasic arthritis, rheumatoid arthritis, systomic lupus erythematosus, Sjogren's syndrome, juvenile rheumatoid arthritis, and spondiioarthropathy. In another particular embodiment, said inflammatory disease is selected from the group consisting of inflammatory bowel disease (eg, Crohn's disease or ulcerative colitis) psoriasis, arthritis, psoriasic arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, juvenile rheumatoid arthritis , spondiioarthropathy, uveftis and Behcet's disease. In a preferred embodiment, said inflammatory disease is rheumatoid arthritis.

A method for the prognosis and / or determination of the severity of a subject suffering from an inflammatory disease In a further aspect, the invention refers to an in vitro method for determining the severity and / or prognosis of the disease in a subject that has an inflammatory disease, said method comprising step a) defined above.

It also refers to an in vitro method to obtain useful data to determine the severity and / or prognosis of the disease in a subject who has or is suspected of having an inflammatory disease, said method comprising step a) defined above.

For each disease or group of inflammatory diseases, criteria have been established by the clinical community to determine the severity or degree of progress and / or activity of the disease that are well known to a person skilled in the art. How I know The DAS28 Index and the ACR / EULAR criteria are commonly described above to be used to determine the degree of severity of RA. For example, patients with severe disease are typically considered to be those with one or more, preferably all, of the following characteristics: positive for rheumatoid factor (FR) and / or antibodies against citrullinated protein antigens (ACPA), presence of erosions and resistance (for example, DAS28≥ 3.2 or intolerance) to at least one disease-modifying antirheumatic drug {DMARDs) DMARDs are well known in the state of the art and include, for example, methotrexate, leflunomide, hydroxychioroquine and sulfasalazine. A person skilled in the art will understand that a greater severity or severity of the disease is associated with a worse prognosis.

According to the data in Example 2, a correlation has been found between the severity of the disease and the presence of the allele (G), in particular the I473T variant is associated with an increase in erosion, especially in ACPA positive patients, since a lower response to anti-TNFα agents (Le., infliximab). Thus, in a particular embodiment of the method of the invention, the presence of the allele (G) in at least one of the alleles in the SNP locus rs11466857, preferably in both alleles, is indicative of an increased severity of the inflammatory disease. . Likewise, the invention provides a method for treating a subject having an inflammatory disease, in which said method comprises step a) defined above; and further comprising: b) administering to said subject an anti-TNFα agent when the allele (A) is present in at least one of the alleles in the SNP locus rs11466657, preferably when the allele (A) is present in both alleles.

In a preferred embodiment, the subject is ACPA positive. In a more preferred embodiment, said subject is an ACPA positive woman.

Furthermore, the present invention refers to an anti-TNFα agent for use in a subject who has or is suspected of having an inflammatory disease, wherein said subject has the allele (A) in at least one of the alleles in the SNP locus rs11466657, preferably in which said subject is homozygous for the allele (A). The methods of the present invention may further comprise the determination of other biomarkers associated with said inflammatory disease, preferably, said blomarkers are selected from the group consisting of citrullinated anti-protein antibodies (ACPA), rheumatoid factor (FR) antibodies, anti antibodies - maninous binding (MBL), globular sedimentation rate (VSG), C-reactive protein (CRP), platelet count, hemoglobin levels and hematocrit. More details on such biomarkers are provided for example in Gupta B et al. J Autoimmun. 2006. 27: 125-133, Afzal Net al. Clin Lab. 2011. 57: 895-899, Takizawa Y et al. Ann Rheum Dis. 2006. 65: 1013-1020, Vossenaar ER et al. Arthritis Res Ther. 2004. 6: R142-R150 and EP0175270 which are incorporated herein by reference.

Rheumatoid factor (RF) is the autoantibody that was first identified in rheumatoid arthritis. It is defined as an antibody against the Fe portion of IgG. RF and IgG bind to form immune complexes that contribute to the disease. On the other hand, antibodies against citrullinated protein antigens (ACPA) are autoantibodies that are directed against peptides and proteins that are citrullinated. ACPAs are present in most patients with rheumatoid arthritis.

In a preferred embodiment of the methods of the invention, citrullinated anti-protein antibodies (ACPA) are also determined.

Optionally, said methods may further comprise the determination of clinical parameters. Examples of signs and / or symptoms whose presence / absence can be determined are: morning stiffness, presence of arthritis in three or more joints. involvement of the hand joints, symmetric arthritis, rheumatoid nodules, and radiographic changes (see Table 1 of Rindfleisch and Muller, and classification criteria of the ACR / EULAR (Ann Rheum Dis 2010; 69: 1580-1588).

The method of the invention may further comprise the storage of the results obtained in a data storage device. In one embodiment, said data storage device is a sheet of paper. In a preferred embodiment, said data storage device is a computer readable medium. As used herein, "a computer-readable medium" may be any apparatus that may include, store, communicate, propagate or transport the results of the determination of the process of the invention. The medium can be an electronic, magnetic, optical, electromagnetic, infrared system (or apparatus or device) or semiconductor or propagation medium.

The methods of the present invention can be implemented by a computer. Therefore, a further aspect of the invention relates to a method implemented by computer, in which the method is any of the methods described herein or any combination of themselves.

Thus, any computer program capable of implementing any of the methods of the present invention or used to implement any of these methods or any combination thereof, is also part of the present invention. Likewise, any device or apparatus comprising or carrying a computer program capable of carrying out, or for the application of any of the methods of the present invention or any combination thereof, is also included as part of the present specification. descriptive

Finally, the methods of the present invention can be applied with individuals of both sexes, that is, men or women, and at any age. The profile determined by the present invention is predictive and prognostic. Kit of the invention and uses thereof »

In a further aspect, the invention provides a kit for determining in a biological sample isolated from a subject the genotype of one or more TLR10 polymorphisms that give rise to a variant of TLR10 that has lost the ability to inhibit NFKB, where said kit understands:

- a reagent to determine the genotype of said polymorphisms;

 - optionally, instructions for the use of said reagent in determining the genotype of said polymorphisms in an isolated biological sample.

In a preferred embodiment, the invention refers to a kit for determining in a biological sample isolated from a subject the genotype of the SNP laug 1466657, comprising: a reagent for determining the genotype of the SNP laug 1466657;

optionally, instructions for the use of said reagent in determining the genotype of SNP rs11466657 in an isolated biological sample. In a particular embodiment, said reagent is a specific primer and / or probe of SEQ ID NO: 1 that amplifies a region comprising the SNP rs11466857. Typically, said reagent includes a partially identical direct primer, preferably substantially identical, to a fragment of SEQ ID NO: 1, and a probe and / or a reverse primer is partially complementary, preferably substantially complementary, to a fragment of SEQ ID NO: one.

Preferably, said primer and / or probe comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and sequences identical to any of them at least 75%.

The oligonuclear sequences with an identity of at least 75% mentioned in the present invention preferably have an identity of at least 80%, at least 85%, at least 90%. at least 95%, more preferably, 96%, 97%. 98%, 99% or 100% with the respective reference sequences. In addition, these sequences with an identity of at least 75% may have the same number of nucleotides, or have more or less nucteotides than the reference sequence. In a particular embodiment, said kit comprises reagents for carrying out a real-time PCR reaction, which normally contains a DNA polymerase, such as TaQ DNA polymerase, a lamella, magnesium, dNTPs, and opdonally other agents (eg, agents stabilizers such as gelatin and bovine serum albumin). In addition, real-time PCR reaction mixtures also contain reagents for real-time detection and quantification of amplification products as described hereinbefore.

Particular embodiments and preferred features of this aspect of the invention have been defined in previous aspects.

The invention also relates to the use of a kit, as described in the previous aspect, to predict the clinical response of a subject who has an inflammatory disease, to identify a subject who has an inflammatory disease with non-response risk. to a treatment with an anti-TNFα agent, to select a treatment for a subject having an inflammatory disease, or to determine the severity and / or the prognosis of an inflammatory disease in a subject, in a method according to the previous aspects of the invention.

It is contemplated that any embodiment discussed in this specification may be implemented with respect to any method, kit and use of the invention described herein. It will be understood that the particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The main features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or may determine, using only routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specification are indicative of the level of knowledge of those skilled in the art to which this invention relates. All publications and patent applications are incorporated herein by reference to the same extent as if it were indicated that each individual patent publication or application is specifically and individually incorporated by reference.

The use of the word "a" or "a" may mean "one," but it also agrees with the meaning of "one or more," "at least one," and "one or more of one." The use of the term "other" may also refer to one or more. The use of the term "or" in the claims is used to mean "and / or" unless explicitly stated that it refers only to alternatives or that the alternatives are mutually exclusive.

As used in this specification and in the {s) claim / claims, the words "comprising" (and any form of understanding, such as "comprise" and "comprise"), "having" (and any form of having, such as "have" and "has"), "which includes" (and any way of including, such as' includes "and" include ") or" which contains "(and any form of containing, such as" contains "and" contain ") are inclusive or open-ended and do not exclude additional elements or steps of method, not cited. The term" includes "also expressly covers and discloses the terms" consists of "and" consists essentially of " As used herein, the expression 'consisting essentially of "limits the scope of a claim to the specified materials or stages and those that do not materially affect the characteristic (s) Basic (s) and navigable (s) of the claimed invention. Tai as used herein, the expression "consisting of" excludes any element, stage or component not specified in the claim except, for example, impurities normally associated with the element or limitation.

The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed elements preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC or ABC, and if the order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC or CAB. Continuing with this example, combinations containing repetitions of one or more elements or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. The person skilled in the art will understand that there are normally no limits on the number of elements or terms in any combination, unless it is evident otherwise from the context.

As used herein, approximation words such as, without limitation, "approximately", "about", "approximately" refer to a condition that when modified in this way is understood to be not necessarily absolute or perfect but could be considered close enough to those of ordinary skill in the art to ensure the designation that the condition is present. The degree to which the description may vary will depend on the extent to which a change can be instituted and on which a person skilled in the art can still recognize that the modified distinctive feature still has the required characteristics and capabilities of the unmodified distinctive feature. In general, but subject to the preceding discussion, a numerical value in this document that is modified by an approximation word such as "approximately" may vary from the value set at £ 1, 2. on 3. on 4, the 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15%. Preferably the term "approximately" means exactly the indicated value (* 0%).

The following examples serve to illustrate the present invention and should not be construed as limiting the scope thereof.

EXAMPLES Example 1.- Material and methods

1.1 Samples

 Two cohorts of patients with RA have been included in this work, a first cohort of 453 unselected patients with follow-up at Marqués de Valdeciila University Hospital (Santander, Spain) and La Paz University Hospital (Madrid, Spain), and a second of 1201 patients recruited by the Consortium of Inflammatory Diseases Mediated by the Immune System (Spain) [Avila-Pedretti G. ef al., PLoS One 2015, 10 (4): e0122088]. The clinical information, including demographic data, disease characteristics and treatment, is summarized in Table 1. All patients were diagnosed according to the classification criteria of the American College of Rheumatology [Arnett FC, et al., Arthrítis Rheum 1988, 31 ( 3): 315-324]. As a control population, 1702 healthy individuals from the same genetic background were also genotyped (ie, a population from southern Europe) [Julia A, et al., Gut 2013, 62 (10): 1440-1445]. All control individuals had been examined to determine the presence of an autoimmune disease or a family history of autoimmune disorders, and were excluded if they were positive.

Table 1. Main characteristics of two cohorts of patients with RA.

1.2 Cell lines KS62 (ATCC, Middlesex, United Kingdom) and U937 (ATCC Middlesex, United Kingdom) cells were maintained in RPMi 1640 medium (Life Technologies. Paisley, United Kingdom) supplemented with 10% fetal bovine serum (FBS) at 10% (Lonze, Verviers, Belgium). The medium was replaced every 2-3 days.

1.3 TLR10 gene sequence analysis

 Exonee coding and flanking regions of the TLR10 gene were sequenced in 66 selected RA patients with severe disease. Patients with serious disease were considered those with positive for rheumatoid factor (FR) and / or antibodies against citrullinated prolel antigens (ACPA), presence of erosions and resistance to at least one disease-modifying anti-rheumatic drug (DMARDs) and 30 healthy controls by next generation sequencing (NGS). The FR was measured by nephelometry (BNII, Siemens Healthcare) and the cut-off value 22 Ul / mi. ACPAs were measured by an ELISA trade test! (CCP2, Eurodiagnostica) and were considered positive above 50 U / ml. Those that presented a lack of response in the DAS28 Index (DAS28≥ 3.2) or intolerance were classified as resistant to DMARDS.

DNA libraries were processed for hybrid enrichment using a SeqCap EZ design (Roche Nimblegen, Basel, Switzerland) containing the TLR10 coding sequences. Next, double barcode libraries were sequenced using a MiSeq NGS platform (lllumina, Madison, Wl). For Sanger sequencing. whole blood DNA was extracted using the QlAamp blood DNA kit (Qiagen, Hamburg, Germany) and amplified with primers for human TLR10 (SEQ ID NO: 3): 5'-CATGGCCAGAAACTGTGGTC-3 'and (SEQ ID NO: 4): 5'-ACCATCCAACCATCATGACC-3 '. Sequence analysis of the amplified fragment was carried out using a genetic analyzer (Applied Biosystems, Foster City, CA).

1.4 Single SNP analysis

The I473T variant of TLR10 (rs11466657; http.7 / www.ncb¡.nlm.nih.gov / projects / SNP / snp_ref.cg¡? Rs = 11466657) was analyzed in an independent cohort of 1201 patients with RA and 1493 healthy controls using the TaqMan® genotyping platform (Assay Id C_25643390_30, Life Technologies, Carísbad, CA). All endpoint fluorescence and PCR reaction readings were performed using an ABI PRISM 7900 HT (Applied Biosystems) sequence detection system. The PCR reaction conditions were as follows: 50 ° C for 2 min and 95 ° C for 10 min, followed by 40 cycles of 92 ° C for 15 s and 60 ° C for 1 min. Genotyping error was estimated by genotyping 20% of the samples in duplicate (error <1%) [Lopez-Lasanta M, Julia A, Maymo J, Fernandez-Gutierrez B, Urema-Garnica i, Blanco FJ, Canete JD, Alperi -Lopez M, Olive A, Coraminas H ef a /: Variation at lnterleukirt-6 Receptor Gene Is Associated to Joint Damage in Rheumatoid Arthritis. Arthritis Res Ther 2015, 17: 242].

1.5 Transfect to perform Indicator gene tests

TLR10 cDNA (NM_030956; http://wvvw.ncbi.nlrn.nih.gOv/nuccore/NM_03Q956.3; SEQ ID NO: 1) was cloned into pCMV6 (Origene, Rockville, MD). The I473T variant was generated by site-directed mutagenesis using the Quick Change mutagenesis kit (Agilent Technologies, Santa Clara, CA) with the following primers: SEQ ID NO: 5 S'-GGCCTTACGAGAACTAAATACTGCATTTAATTTTCTAACTGATC-3 'and SEQ ID NO: 6 S ^, -ATCAGTTAGAAAATTAAATGCAGTATTTAGTTCTCGTAAGGCC-3. The modified graft was sequenced to verify the mutation. K562 and U937 cells were co-transfected with 2 μg of TLR10 constructs of wild-type or mulant type, 1 of plasmid pBVI-Luc indicator, containing six tandem repeats of the NFKB recognition sites within the promoter region linked to the gene of the luciferase [Inohara N, ef al., J Biol Chem 2001, 276 (4): 2551-2554] and 0.2 pRSV-B-gai using Lipofectamine 2000 (Sigma-Aldrich, St Louls, MO).

1.6 Indicator gene assays

 After 24 h of transfection, K562 and U937 cells were incubated with 10 ng / ml and 20 ng / ml of tumor necrosis factor-alpha (TNFα) in the presence or absence of 200 ug / ml of infiiximab and after 24 h The cell extracts were prepared and analyzed for relative luciferase activity by means of a Dual-Light indicator gene test system (Applied Biosystems). The results were normalized to determine the efficiency of transfection with values obtained with pRSV-β-gal.

1.7 Expression analysis of target genes from NFKB

To evaluate the expression of individual genes, a complementary DNA was generated and amplified using primers for human TNFα SEQ ID NO: 7 (5'-CAATGGCGTGGAGCTGAGAG-3 'and SEQ ID NO: 8 S'-GGCTGATGGTGTQGGTGAGG-S '), CCL2 SEQ ID NO: 9 (5'-CTCGCTCAGCCAGATGCAAT-3' and SEQ ID NO: 10

S'-GTCTTCGGAGTTTGGGTTTGC-a '), TRAIL SEQ ID NO: 11 (5 -GAGCTGAAGCAGATGCAGGAC-3' and SEQ ID NO: 12 S-TGACGGAGTTGCCACTTGACT-3 '), and β-actlna SEQ ID NOCC: 13- SCAAC S 'and SEQ ID NO: 14

5'-GATGGAGTTGAAGGTAGTTTCGTG-3). Quantitative real-time PCR was performed in a 7000 sequence detection system (Applied Biosystems). The ratio of the abundance of differentiation markers with respect to that of β-actin transcripts was calculated as 2n, where n is the threshold cycle value of β-actin minus the threshold acid value of the corresponding mRNA and was normalized by Sample value with the lowest expression level of these genes. The specificity of the desired PCR products was determined with melting curve analysis. Real-time PCR was carried out using the Applied Biosystems 7000 Real-Time PCR system (Bio-Rad). Each PCR reaction contains 12.5 μΙ SYBR GREEN PCR Master Mix (Applied Byosystems), 400 nM of each primer, and 1 μΙ diluted cDNA (100 ng) in a total volume of 25 μΙ. The reactions were carried out in a 96-well reaction plate under the following conditions: 2 min at 60 ° C, 10 min at 95 ° C, followed by 40 cycles of 15 s at 95 ° C, 3 s and 1 min at 60 ° C .

The delta Ct method

It was used for the analysis of PCR matrix data. The normalized value for each gene of interest (GOI) was calculated by subtracting the average Ct of the two constitutive expression genes fhousekeeping genes ") from the Ct of each GOI. Then, the double delta Ct

for each GOI it was calculated by subtracting the

mean of each GOI in the control group of the

of each GOI. The change factor ("fold-change") of each GOI compared to the control group was calculated as

1.8 Statistical analysis

All statistical analyzes were performed using the SPSS 20 program (IBM, Armonk, New York) and R statistical software (version 3.2.0). Differences in quantitative variables between groups of patients were compared with the Mann-Whitney U test, and chi-square statistics were used for categorical variables. Statistical significance was performed between groups in in vttro analysis using the bilateral Student t test for independent data. The level of significance was set at p <0.05. Genetic association analysis was performed using linear and logistic regression models. As previously described, covariates in these genetic analyzes included the years of disease progression and baseline disease activity score (DAS28) for the association of the rs11468857 genotype with the level of joint damage [Lopez-Lasanta M, Julia A, Maymo J, Fernandez-Gutiérrez B, Urena-Gamica I, Blanco FJ, Cañete JO, Alperí-Lopez M, Olive A, Coraminas H eí ah Variation at lnterieukin-6 Receptor Gene Is Associated to Joint Damage in Rheumatoid Arthritis. Arthritis Res Ther 2015, 17: 242], and the change in DAS28 at 12 weeks [Acosta-Colman I, Paiau N, Tornero J, Fernandez-Nebro A, Blanco F, González-Alvaro I, Cañete JD, Maymo J, Badina J, Fernandez-Gutiérrez B et at Gwas Replication Study Confirm the Association of Pde3a-Slcold with Anti-Tnf Therapy Responso in Rheumetoid Arthritis. Pharmacogenomics 2013. 14 {7): 727-734], respectively.

Example 2.- The I473T variant is associated with the severity of the disease and response to treatment in patients with RA.

The function of TLR10 is controversial and its association with RA has hardly been studied. In order to assess whether TLR10 variants contribute to modifying disease development in patients with RA, exons coding for the TLR10 gene were sequenced in 66 selected RA patients and 30 healthy controls. After filtering the bases that had at least 30X sequence coverage, sixteen variants were identified (see table 2 below).

Table 2. Sixteen afolic variants of the TLR10 gene found by NGS in control populations and with RA. REF, reference allele; ALT, alternative allele; MAF, minority allele frequency; (+) harmful change; (-) without harmful change; nr, without registration.

Then, a selection was made based on allelic frequencies in both patient and control populations and the expected functional impact on the protein using the SIFT programs (Nq PC and Henikoff S. Nucleic Acids Res. 2003, 31 (13): 3812 - 4) and SNP & 3D (Yue et al., BMC Bioinformatics. 2006, 7: 166). Most of the nonsense changes found were located in the LRR domains (Figure 1A). As a result of the selection procedure, the I473T variant (Table 2) was identified for additional association studies. First, a population of 453 patients was studied and genotype frequencies (86.6% of AA, 11.9% of AG, 1.5% of G6) were found not to be significantly different from those of a population of 209 controls (84.5% of AA, 14.3% of AG, 1.2% of GG). Then several clinical findings were analyzed in the population of patients associated with the severity of the disease, including the presence of typical AR autoantibodies (RF and ACPA). Extra-articular manifestations and the need for specific biological treatments and surgery. All of them indicated a trend correlation between severity and the GG genotype that was not observed with the AA genotype (Figure 2A). To reinforce this observation, a cohort larger than 1201 patients with RA and 1493 healthy controls was studied. As previously noted, the genotype distribution was found to be similar in both patient and control populations (Figure 1B), which is consistent with the genotypes described for the European population in the HapMap database (Nature. 2005 , 437 (7063): 1299-320; Figure 1C). It is interesting, as shown in Figure 2B. that the I473T variant is highly associated with erosive disease in RA positive for ACPA (p = 0.017 in the total cohort and p-0.0049 in female patients) and with a lower response to infliximab treatment measured by changing the DAS28 Index (p = 0.012 in the total cohort) as well as by the EULAR response criteria (p = 0.025 in the total cohort), which indicates that this variant selects a group of patients with a more serious disease.

Example 3.- The I473T variant modifies the NFKB naoulatoria capacity of TLR10

Based on a 3D structure model using the Jmoi viewer (Nepomnyachiy S1, et al., Structure. 2015 May 5; 23 (5): 941-8; Figure 3A), position 473 is within a beta chain in the domain LRR18 and is occupied by an isoleucma, a highly hydrophobic amino acid, hidden inside the protein nucleus. In the TLR10 variant, isoleucine is replaced by threonine, a polar amino acid that can participate in hydrogen bonds and is usually located on the surface of the protein. The LRR domains provide an outstanding framework to achieve various protein interactions. Therefore, this structural change decreases hydrophobic contacts and can alter functionally relevant protein-protein interactions. It is known that members of the TLR family are regulators of NFKB activity, a key route in inflammation. To experimentally demonstrate the functional consequences of the I473T variant on NFKB activation, the codon alteration nucleotide was introduced into a construct containing TLR10 cDNA by site-directed mutagenesis and the ability of this variant to modify transcriptional activity was studied. from NFKB. As shown in Figures 3B and 3C, wild-type TLR10 regulates by decreasing the transcriptional activity of NFKB both in conditions not stimulated and in conditions stimulated by TNFα in the hematopoietic cell lines K562 and U937. However, the substitution of I473T blocks the inhibitory capacity of TLR10. In order to translate the reduced response to Infliximab in patients with the ín vHro model, TNFα cells were stimulated in the presence or absence of infliximab. Consequently, cells expressing the I473T vanant had a higher level of NFKB activity that remained after treatment with infliximab compared to cells expressing the wild-type variant (Figures 3D and 3E). Then, this result was confirmed by analyzing the expression of NFKB target genes. The expression levels of CCL2, TRAIL and TNFα were regulated by decrease in the presence of wild-type TLR10 after stimulation of the NFKB pathway with TNFα. In line with the previous results, the regulation by reduction of NFKB target genes was canceled when the cells were transfected with the construct containing the variant and cultured with TNFα (Figure 4A-E). The expression of CCL2 also confirmed that the variant generates a lower response to infliximab (Figure 4F).

In conclusion, the I473T variant leads to a reduced capacity of TLR10 to inhibit the activation of NFKB in response to inflammatory stimuli. This effect could be explained because the amino acid change decreases the hydrophobicity of an LRR domain, which can alter the interaction with TLR proteins necessary for TLR10 signaling [Govindaraj RG. et a / .. PLoS One 2010, 5 (9): e12713],

Claims

1. In vibro method to predict the clinical response of a subject, who has or is suspected of having an inflammatory disease, to an anti-TNFα agent, in which said method comprises: a) determining in an isolated biological sample of said subject the single nucleotide polymorphism (SNP) genotype rs11466657; where the presence of the allele (G) in at least one of the alleles in the iocus of the SNP rs11466657 indicates an increased risk of not responding to the anti-TNFα agent; and where said inflammatory disease is selected from the group consisting of inflammatory bowel disease, psoriasis, psoriasis arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, juvenile rheumatoid arthritis, spondlloarthropathy, uveitis and Behcet's disease.

2. M vitro method to identify a subject, who has or is suspected of having an inflammatory disease, who presents a higher risk of not responding to treatment with an anti-TNFα agent, in which said method comprises step a) according to claim 1; where the presence of the allele (G) in at least one of the alleles in the Iocus of SNP rs11466657 indicates an increased risk of not responding to the anti-TNFα agent; and where said inflammatory disease is selected from the group consisting of inflammatory bowel disease, psoriasis, psoriasis arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, juvenile rheumatoid arthritis, spondlloarthropathy, uveitis and Behcet's disease.

3. The method according to any of claims 1 or 2, wherein the presence of the allele (G) in both alleles indicates an increased risk of not responding to the anti-TNFα agent.

4. In vitro method for selecting a treatment for a subject, who has or is suspected of having an inflammatory disease, wherein said method comprises step a) according to claim 1; and further comprising: b) selecting an anti-TNFα agent when the allele (A) is present in both alleles in the SNP locus rs11466657; where said inflammatory disease is selected from the group consisting of inflammatory bowel disease, psoriasis, psoriasis arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sjógren's syndrome, juvenile rheumatoid arthritis, spondyloarthropathy, uveitis and Behcefs disease.

5. In vito method for determining the severity and / or prognosis of the disease in a subject having an inflammatory disease, wherein said method comprises step a) according to claim 1; where the presence of the (G) allele in at least one of the alleles in the SNP locus rs11466657 is indicative of severe disease; and where said Inflammatory disease is selected from the group consisting of inflammatory bowel disease, psoriasis, psoriasic arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sjógren's syndrome, juvenile rheumatoid arthritis, spondyloarthropathy, uveitis and Behcefs disease.

6. The method according to claim 5 wherein the presence of the allele (G) in both alleles is indicative of severe disease.

7. In vitro method to obtain useful data to determine the clinical response of a subject, who has or is suspected of having an inflammatory disease, to an anti-TNFα agent, the severity and / or prognosis of said disease where said method comprises step a) according to claim 1; where said inflammatory disease is selected from the group consisting of inflammatory bowel disease, psoriasis, psoriasis arthritis, rheumatoid arthritis, Systemic lupus erythematosus, Sjfigren's syndrome, juvenile rheumatoid arthritis, spondyloarthropathy, uveitis and Behcefs disease.

8. The method according to any one of claims 1 to 7, wherein the subject is positive for anti-citrullinated peptide antibodies (ACPA).

The method according to any of claims 1 to 8, wherein the biological sample is a blood sample.

10. The method according to any one of claims 1 to 9, wherein the SNP genotype rs11466657 is determined by direct sequencing or quantitative PCR.

11. The method according to any of claims 1 to 10, wherein said method further comprises storing the results of the method in a data storage device.

12. Computer implemented method, wherein the method is as defined in any of claims 1 to 11.

13. The method according to any of claims 1 to 12. wherein said anti-TNFα agent is selected from the group consisting of etanercept, adalimimab, infliximab, golimumab, certolizumab, rituximab, abatacept, anakinra and tocizumab. .

14. The method according to any of claims 1 to 12, wherein said anti-TNFα agent is infliximab.

15. Use of infliximab in the preparation of a medicament for the treatment of an inflammatory disease in a subject, who has or is suspected of having said inflammatory disease, in which said subject has the allele (A) in both alleles in the locus of SNP rs11466657, where said inflammatory disease is selected from the group consisting of inflammatory bowel disease, psoriasis, psoriasis arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, juvenile rheumatoid arthritis, spondyloarthropathy, uveftis and Behcet's disease.

16. The method according to any of claims 1 to 14 or the use of infliximab according to claim 15, wherein said inflammatory disease is rheumatoid arthritis.

17. The method according to any of claims 1 to 14 or 16. or the use of infliximab according to any of claims 15 to 16, wherein said subject is a human subject.

18. Kit for determining in a biological sample isolated from a subject the genotype of SNP rs11466657, which comprises:

 - a reagent to determine the genotype of SNP rs11466657;

 - optionally, instructions for the use of said reagent in the determination of the genotype of SNP rs11466657 in an isolated biological sample.

19. The kit according to claim 18, wherein said reagent is a primer and / or specific probe of SEQ ID NO: 1 that amplifies a region comprising SNP rs11466657.

20. The kit according to claim 19 wherein said specific primer and / or probe is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and sequences identical to any of them at least 75%.

21. Use of a kit according to any of claims 18 to 20 to predict the clinical response of a subject who has an inflammatory disease, to identify a subject who has an inflammatory disease with a refusal to respond to treatment with an agent. anti-TNFα. to select a treatment for a subject who has an inflammatory disease, and / or to determine the severity and / or prognosis of an inflammatory disease in a subject.