ES2656587B1 - Method to predict the clinical response to a treatment with anti-inflammatory agents - Google Patents

Abstract

Method for predicting the clinical response to a treatment with anti-inflammatory agents. # The present invention relates to the use of allelic variants of human TLR10, including the allelic variant of human TLR10 I473T, as a biomarker for predicting the severity or prognosis in inflammatory diseases. , including rheumatoid arthritis and / or to predict the response to a treatment with anti-inflammatory agents, such as anti-TNF agents {al}.

Description

METHOD FOR PREACHING 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 TLR1 or 1473T, as a biomarker to predict severity or prognosis in inflammatory diseases, including rheumatoid arthritis and / or to predict the response to a treatment with anti-inflammatory agents, such as anti-TNFa agents.

BACKGROUND OF THE INVENTION

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

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 1, et al., Cham Phys 2011, 13 (9): 3782-3787J. In particular, TLRs are

Type 1 transmembrane glycoproteins with extracellular leucine-rich (LRR) repeats and a homology domain with the intracellular Tol1 / IL-1 receptor (IRR) [Ulevitch RJ, Nat Rev Immunol 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 Immuno / 2006, 91: 159-173; Deighton

K, et al. Appetite 2014, 81: 52-591. In addition, it has been suggested that the TLRs are involved in responses to pathogens in RA [Cromartie WJ, et al., J Exp Med 1977, 146 (6): 1585-1602].

TLR signaling involves interaction with adapters that contain TIR domain,

including MyD88, TRIF, TRAM, TIRAP AND SARM1 [O'Neill LA, Bowie AG; Nat Rev Immunol

2007, 7 (5): 353-364) that promotes the activation of the nuclear factor enhancer of light chains kappa 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'Neill LA, Bowie AG; Nal Rev Immunol 2007.7 (5): 353-364 .; Drexler SK, al. , Inl J Biochem Ce "Biol2010, 42 (4): 506-518]. Each

There is more evidence that NFKB is an important transcription factor, if not the

main, which controls inflammation [Abu-Soud HM, al., Biochemislry 1998, 37 (11): 3777

3786] and deciphering 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 [005tin9 M,

al., Proc Nall Acad Sci U S A 2014, 111 (42): E4478-4484 .; Hasan U, al., J Immunol 2005, 174 (5): 2942-2950]. TLR10 expression has been described primarily in cells

B, dendritic cells, eosinophils, neutrophils and non-immune cells such as

troloblaslos [Hasan U el al., J Immunol 2005, 174 (5): 2942-2950 .; Hornung V al., J Immunol. 2002, 168 (9): 4531-4537 .; Nagase H, al. , J Immunol 2003, 171 (8): 3977-3982 .; Mulla MJ, al., Am J Reprod / mmuno / 2013, 69 (5): 449-453]. In mammoths, TLR10, TLR1,

and TLR6 share a common locus on chromosome 4p14 and are structurally similar to each other [Hasan U, al., J Immunol 2005, 174 (5): 2942-2950]. Despite interacting with

MyD88 [Hasan U, al., J Immunol2005, 174 (5): 2942-2950], TLR10 differs from TLR olros by

the lack of a classic subsequent signaling route [Guan Y, 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, al., J Cfin Rheumalo / 2010, 16 (1): 15-18 .; Zhou H, al., J Mol Evol. 2007, 65 (2): 119-123]. TLR1 and TLR6 can form a protein complex with TLR2 and TLR10 [Hasan U, al., J Immuno / 2005, 174 (5): 2942-2950 .: Mulla MJ el al., Am J Reprod Immunol2013, 69 (5) : 449-453], although the individual contribution of each

Protect 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 TLR10 acts as a pro-inflammatory receptor by activating NFKB signaling [Hasan U, al., J Immunol 2005, 174 (5): 2942-2950;

Regan T, al., J Immunol 2013, 191 (12): 6084-6092]. Another study showed that TLR10 did not

activates typical TLR-induced signaling, including mediated transcriptional activation

by NFKB or inler / erón-bela (IFNP) [Guan Y, al., J Immunol 2010, 184 (9): 5094-5103].

Recently, TLR10 has been shown to be a modulator with mainly inhibitory effects [Stappers MH, al., J Infect Dis 2015). In line with this, block TLR10 with

antagonist antibodies potentiate the production of proinflammatory citodna [Oosting M. el

to "Proc Natl Acad Sci U S A 2014, 111 (42): E4478-4484J. In addition, they have been provided

evidence that TLR10 induces apoptosis through the activation of caspase-3 [Kuuliala K.

al., Ann Rheum Dis 2006, 65 (9): 1241-1243J.

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 [Kuuliala K, al., Ann Rheum Dis 2006, 65 (9): 1241-1243 .; Radstake TR, al., Arthrilis Rheum 2004, 50 (3): 999-1001.; Etem EO, al., Rheumalollnl 2011, 31 (10): 1369-1374 .; Coenen MJ, al., PLoS One 2010, 5 (12): e14326; Zheng B, al., Rheumalollnl 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., Rheumatollnt 201 1.31 (10): 13691374]. Although single-nucleotide variants of the TLR10 gene have been associated with other autoimmune diseases (Requena T, al., Immunogenetics 2013, 65 (5): 345-355],

tumors [Kutikhin AG, Hum Immuno / 2011, 72 (11): 1095-111 6J, infectious diseases and

inflammatory diseases such as extrapulmonary tuberculosis and asthma [Ma X, al., PLoS One 2007,

2 (12): e1318; Lazarus R, al., Am J Respir Cril Care Med 2004, 170 (6): 594-600J, the

Functional activity of this protein and the clinical significance of the gene variants are

still controversial [Hasan U, al., J Immunol2005, 174 (5): 2942-2950 .; Stappers MH, al., J Infeel Dis 2015J.

The allelic variant 1473T has only been described in a previous study associated with a decreased risk of meningioma (Rajaraman P, et al., Cancer Epidemial Biomarkers Prev 2010,

19 (5): 1356-1361J.

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 TNFo 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 reduced the activation of the inflammatory NFKB pathway in hematopoietic cells, while the 1473T variant did not have this inhibitory capacity. The inventors observed that, after the NFKB route with TNFa was removed after the

Exposure to infliximab (a TNFa inhibitor) cells expressing the 1473T variant showed higher NFKB activity compared to cells carrying wild-type TLR10.

It is known that TNFa inhibitors. help control the evolution of RA Without

However, not all patients respond adequately to anti-TNFa treatment. initial [Alten R, al., Int J Rheum Dis 2014, 17 (1): 5-18]. The inventors analyzed the association of the inconsistent variant of human TLR10, 1473T, which is located in the domain

LRR18, with AR observing that the 1473T variant is not associated with propensity to AR. However, it was found that this variant correlates significantly with erosive disease in seropositive patients for antibodies against citrullinated proteins (ACPA) (p = O, 017 in total cohorle and p = O, 0049 in women) and with a lower treatment response with infliximab measured by the DAS28 index (p = O, 012) and by the EULAR criteria (p = O, 049), as shown in Figure 28.

The data shows that a genetic variant of TLR10 selects a group of patients with a more severe and resistant disease. Also, said genetic variant of TLR10 may be a good candidate marker of response to inHiximab or other anti-TNFa 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 ethnic response of a subject who has or is suspected of having an inflammatory disease to an anti-TNFa agent, wherein said method comprises:

to)

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.

s

In a preferred embodiment, 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-TNFa agent, wherein said method comprises:

10

to) determine, in an isolated biological sample of said subject, the genotype of the single nucleotide polymorphism (SNP) r511466657.

lS

In another aspect, the invention relates to an in vitro 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-TNFa agent, in which said method comprises stage a) according to the first aspect.

twenty

In a related aspect, the invention refers to an 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) defined above; and also includes:

25

b) select an anti-TNFa 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.

30

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-TNFa agent, the severity and / or prognosis of said subject. disease where said method comprises step a) defined above.

Also, the invention provides a method for treating an inflammatory disease, wherein said method comprises above: and further comprises:

subject that has a stage a) defined

5

b) administering to said subject an anti-TNFa agent when the allele (A) is present in at least one of the alleles in the SNP locus r511466657, preferably when the allele (A) is present in both alleles.

10

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

lS 20

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 polymomsms that give rise to a variant of TLR10 that has lost the ability to inhibit NFKB, where said kit it comprises: 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.

25

The invention also relates to the use of a kit, as described in the previous aspect, to predict the ethnic response of a subject who has an inflammatory disease, to identify a subject who has an inflammatory disease at risk of not responding. to a treatment with an anti-TNFa 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.

30

BRIEF DESCRIPTION OF THE FIGURES

35

Figure 1. Variants of the TLR10 gene. (A) Schematic representation of the TLR10 protrusion that illustrates the position of the contrasense variants (KmissenseB in English) identified by next generation sequencing (NGS). LRR, rich repetitions

in leucine; TM, transmembrane domain; TIR, Toll-inlerleucine receptor domain. (8) Genotype distribution of the 1473T variant in the independent cohort of 1493 healthy controls (He) and 1201 patients with rheumaloid arthritis (RA). (e) TLR10 genotype frequencies in HapMap populations (HapMap is a map of haplotypes in the human genome; International HapMap consortium el al. (2010), Integrating common and rare genetic variation in diverse human populations, Nature, 467, 52- 8).

Figure 2. Association of the 1473T variant with the severity of the disease in two cohorts of (A) 453 Y (B) 1201 patients with RA. The beta value (regression coefficient), used to evaluate the effect of the 1473T 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 positive for ACPA including the years of disease evolution; 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 infiiximab (EULAR response criteria: moderate to 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 variant 1473T. TO)

3D structure of TLR10 using the Jmol viewer showing the location of residue 1473. NAG, 2- (acetylamino} -2-deoxy-beta-O-glucopyranoside. (B) and (e) K562 and U937 cells were cotransfected (respectively) 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 TNFa 10 ng / ml for 24 h and then prepared and analyzed The cell extracts were used to determine luciferase activity, and K562 Y (O937) cells transcribed with the TLR10 variants indicated in the presence or absence of infliximab were treated with TNFn and the luciferase activity was analyzed. ± SD of three independent experiments. · P <O, 05.

Figure 4. Regulation of the levels of NFKB target genes by the 1473T variant. U937 (A, B, e) and K562 (O, E, F) cells were translected with TLR10 (wild type or mutated variant) and then treated or not treated with TNFa 20 ng / ml in the presence or absence of infiiximab ( IFX) for 24 h. The expression of NFKB target genes, CCL2 (A,

D, F) TRAIL (B, El and TNFa (C) by quantitative RT-PCR. P-actin was used for

standardization. e, cells transfected with empty vector as an additional control. The

Histograms show the mean ± SD of three independent experiments. · P <O, 05.

s

Detailed description of the invention

Definitions

The term · subject · as used herein refers to a subject

mammal. Preferably, it is selected from a human, companion animal, cattle not

10

Domestic 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 subject

Mammal is a human subject.

The term · subject who has a disease ~ as used herein is

fifteen

refers to those subjects who have been diagnosed with said disease. For example,

A ~ confirmed diagnosis of rheumatoid arthritis "or" definitive rheumatoid arthritis "may

be based on the confined presence of synovitis in at least one joint, absence of

an alternative diagnosis that better explains synovitis, and achieving a total score

of 6 or greater (from a maximum possible of 10) from the individual scores in

twenty

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 such

and as defined in the EULAR classification criteria (Ann Rheum Dis 2010; 69: 1580

1588).

2S

The term · subject suspected of having a disease ~ as used in the

This document refers to a subject that has one or more signs or symptoms.

indicative of said disease. A subject suspected of having a disease

It may also have one or more risk factors (i.e., a subject suspected of

30

will develop or present a risk of developing a disease). It also covers a

individual who has received a preliminary diagnosis but for which no

Confirmation test In addition, it includes those individuals in remission.

The term -examination-as used herein refers to identifying,

3S

determine or distinguish those subjects or individuals that present the characteristics or the

defined phenotype A uexamen test may be used when the presence of such characteristics or phenotype is suspected.

The term treatment · encompasses both prophylactic and therapeutic treatment. The term "therapeutic treatment" or "Mterapá" as used herein refers to bringing a body from a pathological state or disease back to its normal, healthy state. The term "prophylactic treatment · as used herein refers to preventing a pathological condition.

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 areata, amyloidosis, ulcerations, aphthous stomatitis, arteriosclerosis, arthritis, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, spondyloarthropathy, ankylosing spondylitis bronchial, Behcet's disease. Boeck's disease, inflammatory bowel disease, Crohn's disease, choroiditis, ulcerative colitis, celiac disease, cryoglobulinemia, macular degeneration, dermatitis, herpetiform dermatitis, dermatomyositis, insulin-dependent diabetes, juvenile diabetes, inflammatory demyelinating disease, Dupuytren's contracture, encephalomyelitis , allergic encephalomyelitis, endophthalmitis, allergic enteritis, auloimmune enteropathy syndrome, leprosy erythema, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, cystic fibrosis, gingivitis, glomerulonephritis, Goodpasture syndrome, Graves syndrome, Hashimoto's disease, chronic hepatitis, histiocytosis, regional ileilis, iritis, systemic lupus erythematosus, cutaneous lupus erythematosus, disseminated lupus erythomatous, lymphogranuloma, infectious mononucleosis, myasthenia gravis, transverse myelitis, primary idiopathic myxedema, nephrosis, obesity, great ophthalmia ulomatous orchitis, pancreatitis, panniculitis, pemphigus vulgaris, periodontitis, polyarteritis nodosa, chronic polyarthritis, polymyositis, acute polyradiculitis, psoriasis, chronic obstructive pulmonary disease, purpura, gangrenous pyoderma, Reiler syndrome, diabetic retinopathy, rosacea sarcoidosis, ataxic sclerosis, progressive systemic sclerosis, scleritis, scleroderma, multiple sclerosis, disseminated sclerosis, uveitis, vitiligo, Whipple's disease, diseases associated with AIDS, severe combined deficiency and the Epstein Barr virus: such as Sjogren's syndrome, osteoarticular tuberculosis and parasitic diseases: such as 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, ascites fluid, bone marrow aspirate, effusion pleural, 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 8 single nucleotide polymorphism ~ or ~ SNPM refers to a type of DNA polymodalism 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-coding gene regions or in intergenic regions between genes. The presence of a SNP within a gene or in a regulatory region near a gene may play a more direct role in the disease affecting the function of the gene.

The term "usonda" as used herein refers to synthetic or biologically produced nucleic acids, between 10 and 285 base pairs in length containing specific nucleotide sequences that allow specific and preferred hybridization under predetermined conditions to select as 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, "poly-deoxyadenosine tails).

The term "Mcebators" 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 polynucleotide 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 Rodriguez A al. (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 "Mhybridization" as used herein refers to a process whereby, under predetermined reaction conditions, two partially or completely complementary nucleic acid chains are allowed to join together in an antiparallel way to form a double stranded nucleic acid. with specific and stable hydrogen bridges, following explicit rules corresponding to which nucleic acid bases can be matched together.

The term "substantial 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. 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 0.9 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 nucleotide with nucleotide or amino acid with amino acid of two polypeptide or polynucleotide sequences or, respectively. Two or more sequences (of polynucleotides or amino acids) can be compared by determining their ~ percent identity. The percentage of 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.ncbi.nlm.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 in 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 cases of several units, the various analysis elements presented in a single unit, which should be considered as kits, may also be available.

Description

A method to predict the clinical response to an anti-TNFa agent

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-TNFa 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.

NF-kB (nuclear factor enhancing the kappa light chains of activated B cells) plays a key role in regulating 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 vitro method for predicting the clinical response of a subject who has or is suspected of having an inflammatory disease to an anti-TNFo agent, wherein said method comprises :

a) determine, 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 that has or is suspected

which has an inflammatory disease with a higher risk of not responding to treatment with an anti-TNFo agent .. in which 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-TNFa 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.ncbLnlm.nih.gov/projects/SNP/snp_ref.cgi?rs=11466657). 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 (SEO ID NO: 1). For this position (Iocus) the ancestral allele is T and an allelic change from AIT to A ~ T has been described which translates into a change in the protein sequence (NP_112218.2; SEO ID NO: 2) of isoleucine (lle) to threonine (Thr) at position 473 (1473T). When said polymorphism is

dete "" ina 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 1473T is referred to as allele (G).

According to the data presented in Example 2, patients with the 1473T variant show a significantly lower response to anti-TNFa agents, according to the DAS28 index (Prevoo ML, van 't Hof MA, Kuper HH, van Leeuwen MA , van de Putte LB, van Riel PL (1995) Modified disease activity scores that inelude twenty-eight-joint counts.

Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthrilis Rheum., 38, 44-48) And the EULAR response crilerios (Karonitsch T, Aletaha O, Boers M, Bombardieri S. Combe B, Dougados M, Emery P, Felson O, GomezReino J, Keystone E, Kvien TK, Martín-Mola E, Matucci-Cerinic M, Richards P, van Riel P, Siegel J, Smolen JS, Sokka T, van der Heijde D, van Vollenhoven R, Ward M, Wells G, Zink A, Landewe R. (2008) Melhods of deriving EULARlACR recommendalions on reporting disease activity in clinical tríals of patients with rheumatoid arthritis. Ann Rheurn Ois, 67 (10), 1365-73), which also indicates that this variant selects a group of patients with one more disease severe or severe

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 risk of not responding to the anti-inflammatory agent. In other words it is predictive of absence of clinical response or resistance to treatment.

The delermination of the presence or absence of said SNP can be delenminated 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 specific allele hybridization, primer extension ("first extension"), oligonucleotide ligation specific allele, sequencing, enzymatic cleavage, 5 'discrimination (flap ) endonuclease; and detection methods, such as fluorescence, chemiluminescence, and mass spectrometry. For example, the presence or absence of said pOlimorphisms can be detected in an AON sample, preferably after amplification. For example, the isolated AON 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; whereby the appearance of an amplification product is indicative of the presence of the polymorphism according to the invention. Alternatively, the AON 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 el al., 1991; Grampe. 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 pOlimorlism simply by direct PCR. Other strategies include, but are not limited to 8, direct sequencing, restriction fragment length polymorphisms (RFLP); hybridization with allele-specific oligonucleotides (ASO), allele-specific PCR; PCR using mutagenic primers; ligase-PeR, HOT creavage; 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 AON to be studied independently of the peR reaction, for example, rolling circle amplification (RCA), the Invader® assay, or the oligonucleotide ligand assay (OLA).

Another preferred quantification procedure is quantitative PCR (qPCR). The qPCR or real-time PCR is well known by an expert in the field. Different instruments for carrying out said reaction are marketed, such as ASI Prism 7700 SOS, GeneAmp 5700 SDS, ABI Prism 7900 HT SDS of Applied Biosyslems; iCycler iQ from BioRad; Cepheid Smart Cycler; 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, http://www.sigmaaldrich.com/technical-documents/protocols/biology/sybr-green-qpcr. htmlo

http://www.sigmaaldrich.com/1ife-science/molecular-biology/pcr/quantitative-pcr/qpcr

technical-guide.html. A review of the use of qPCR in the quantification of mRNA is

found for example in Wong ML and Medrana JF, Biotechniques 2005, 39 (1): 75-85.

5

Different biochemical detection are available for qPCR. You can use all

them with the qPCR instruments mentioned above. The term ~ biochemistry of

Detection - refers to a procedure to report the amplification of the product of

Specific PCR in real-time PCR. These biochemical detection are classified in

Two main groups. The first group comprises DNA interleaving molecules of

10

double chain: such as SYBR Green I and EvaGreen; and the second group includes

oligonucleotides typically labeled with a fluorophore. The latter, in turn, has

divided into three subgroups: (i) primers-probes (Scorpions, Amplifluor®, LUXTM, Cyclicons,

Angler®); (ii) hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization probes

(Hybprobe or FRET, Molecular Beacons, HyBeacon "', MGB-Pleiades, MGB-Eclipse,

fifteen

ResonSense®, Yin-Yang or displacing); and (iii) nucleic acid analogs (PNA, LNA®,

ZNA TM, unnatural bases: Plexor ™ primer, Tiny-Molecular Beacon), see E. Navarro eta l.

, Acim Chimica Clinic, Volume 439.15 January 2015, Pages 231-250.

For example, said probes are oligonucleotides can be double labeled, such as

twenty

hydrolysis probes or molecular beacons. The 5 'end of the oligonucleotide is typically

mark with a fluorescent reporter molecule such as FAM, TET or JOE

while the 3 'end is marked with a quencher molecule, such

such as TAM or BHQ1. The probe sequence is specific to a region of interest in the

amplified target molecule. In a preferred embodiment, said probe is a probe

2S

of hydrolysis that is designed so that the length of the sequence places the phorophore 5 '

and the deactivating molecule 3 'in close enough proximity to suppress the

fluorescence. Several indicator and extinguishing molecules for use in qPCR probes

They are well known in the art. These are available, for example, from

https: J / www.eurofinsgenomics.euJen / dna-rna-oligonucleotides / optimized-application

30

oligos / qpcr-probes.aspx.

Also, the present invention relates to oligonucleotide sequences (primers and / or

probes) that hybridize specifically with one of the alleles of the polymorphisms. He

Ténino M a primer and / or probe "specifically includes · primers and / or probes ~,

3S

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 between 18 and 22 nucleotides, and still much 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 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) defined above; and also includes:

b) select an anti-TNFa 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 in vivo. The antiTNFa and TNFa inhibitor are used interchangeably Here, in a preferred embodiment, said anti-TNFa agent is an antibody, preferably a specific anti-TNFa antibody Methods for obtaining antibodies against a known antigen are part of the state of the art and one skilled in the art You will know how to get anti-TNFa antibodies.

The term "antibody" is used in the broadest sense and includes fully assembled antibodies, chimeric antibodies, humanized antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), antigen-binding antibody fragments (eg. , Fab, Fab ', F (ab') "Fv, scFv, diabodies), single domain antibodies (sdAb) and recombinant peptides comprising the foregoing, as long as they show the desired biological activity. The term" antibody "is also refers to a fusion protein that includes a region equivalent to the Fc region of an immunoglobulin.

Anti-TNFa agents are well known in the state of the art. As an illustration. Anti-TNFa agents include but are not limited to anti-TNFa agents selected from the group consisting of etanercept, adalimimab, infliximab, golimumab, certolizumab, riluximab, abatacept, anakinra and tocizumab. In a preferred embodiment, said anti-TNFa agent is infiiximab. Singh et al., Arthritis & Rheumathology, 2016, 68 (1), 1-16 provides a manual for the treatment of RA from American College 01 Rheumatology. In particular, Table 1 provides a list of treatments for RA that includes anti-TNFa 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, systemic lupus erythematosus, Sjbgren syndrome, juvenile rheumatoid arthritis, and spondyloarthropathy.

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, Sjbgren's syndrome, juvenile rheumatoid arthritis spondyloarthropathy Uveltis 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 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 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

previously.

For each disease or group of inflammatory diseases criteria have been established by the ethnic 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 has been described above and the ACRlEULAR criteria are commonly used to determine the degree of RA severity. 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 prothena antigens (ACPA), presence of erosions and resistance (for example, DAS28 2: 3.2 or intolerance) to at least a disease-modifying antirheumatic drug (DMARDs) DMARDs agents are well known in the state of the art and include, for example, methotrexate, leflunomide, hydroxychloroquine 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 1473T variant is associated with an increase in erosion, especially in positive ACPA patients, since a lower response to anti-TNFa agents (Le., infliximab). So that. 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 rs11466657, preferably in both alleles, is indicative of an increase in severity of the inflammatory disease.

Also, the invention provides a method for treating a subject having an inflammatory disease, wherein said method comprises step a) defined above; and also includes:

b) administer to said subject an anti-TNFa 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-TNFa agent for use in a subject that has or is suspected of having an inflammatory disease, in which said subject has the allele (A) in at least one of the alleles in it. of SNP rs1 1466657, preferably wherein 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 biomarkers are selected from the group consisting of anti-cranial anti-proline proteins (ACPA), rheumatoid factor antibodies (FR), anti- antibodies · Manase binding lectin (MBL), erythrocyte sedimentation rate (ESR), e-reactive protein (CRP), platelet count, hemoglobin levels and hematocrit. More details on such biomarkers are provided for example in Gupta B aL J Autoimmun. 2006. 27: 125-133, Alzal 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.

The rheumaloid factor (RF) is the auloantibody that was first identified in rheumatoid arthritis. It is defined as an antibody against the Fc 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 joints of the hands, symmetric arthritis, rheumatoid nodules, and radiographic changes (see Table 1 of Rindflelsch 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 aspeclo of the invention relates to a method implemented by computer. wherein the method is any of the methods described herein or any combination thereof.

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, 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 for determining 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 SNP rs11466657, which comprises:

a reagent to determine the genotype of SNP rs11466657; - 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 SEa ID NO: 1 that amplifies a region comprising SNP rs11466657. Typically. said reagent includes a partially identical, preferably substantially identical, direct primer to a fragment of SEa ID NO: 1, and a probe and / or a reverse primer is partially complementary, preferably substantially complementary, to an SEO ID fragment NO: 1.

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

The oligonucleotide 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 nucleotides than the reference sequence.

In a particular embodiment, said kit comprises reagents for carrying out a real-time PCR reaction, which normally contains an AON polymerase, such as TaO DNA polymerase, a buffer, magnesium, dNTPs, and optionally 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 having an inflammatory disease, to identify a subject having an inflammatory disease at risk of not responding. to a treatment with an anti-TNFa agent, to select a treatment for a subject who has an inflammatory disease, or to determine the severity and / or prognosis of an inflammatory disease in a subject, in a method according to the above 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 refers. 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 Mun · or Muna · can mean Muna ·, but also matches the meaning of Muna or more ·, "at least one · and Muna or more than one ~. The use of the term Matra · can also refer to one or more. The use of the term "or · in the claims is used to mean · and / or" unless explicitly stated to refer only to alternatives

or that the alternatives are mutually exclusive.

As used herein and in the claim / claims, the words M comprising "(and any form of understanding, such as · comprising" and · comprising ")," having "(and any form of have, such as "have" and "have ·)," that includes "(and any way of including, such as" includes "and" include ") or" that 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 mentioned. The term "comprises" also expressly encompasses 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 novel (s) of the claimed invention. As used herein

document, 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.

5

The term MO combinations thereof · as used herein are

refers to all permutations and combinations of the listed items that

They precede the term. For example, MA, B. e, or combinations thereof · are intended

that includes at least one of: A, 8, e, AB, AC, Be or ABe, and if the order is important in a

10

particular context, also BA, CA, CS, CeA, seA, ACB, BAC or CAB. Continuing with

In this example, combinations containing repetitions of one or more are expressly included

more elements or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA,

CABABB, etc. 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 from

lS

Another way from context.

As used herein, approximation words such as, without

limitation, "approximately-," around "', ~ approximately ~ refer to a

condition that when modified in this way it is understood that it is not necessarily

twenty

absolute or perfect but could be considered close enough for experts

common 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 it can

institute a change and that a person skilled in the art can still recognize that

the modified distinctive feature still has the required characteristics and capabilities of the

2S

distinctive feature unmodified. In general, but subject to the preceding discussion, a value

numerical in this document that is modified by an approximation word such

as ~ approximately · may vary from the value set in ± 1, 2, 3, 4, 5,

6, 7, 8, 9, 10, 11, 12, 13, 14 or 15%. Preferably the term

"approximately" means exactly the indicated value (± 0%).

30

The following examples serve to illustrate the present invention and should not be interpreted

as limiting the scope of it.

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 5,453 patients not selected with follow-up at the Marqués University Hospital of

Valdecilla (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. et al., PLoS One 2015.

10 (4): e0122088]. Clinical information. including demographic data, characteristics of 10 disease and treatment, are summarized in table 1. All patients were diagnosed.

according to the classification criteria of the American COllege of Rheumatology [Arnett Fe, et al.,

Arthritis Rheum 1988, 31 (3): 315-324]. As a control population, they also genotyped

1702 healthy individuals of the same genetic background (i.e., a population of southern

Europe) [Julia A, et al., Gut 2013, 62 (10): 1440-1445]. All control individuals will

15 had examined to determine the presence of an autoimmune disease or family history of autoimmune disorders, and were excluded if they were positive.

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

Cohort I Cohort II (n = 453) (n = 1,201)

Women (%)

Mean age (years)

Duration of follow-up (months) Extra-articular manifestations (%) Presence of joint damage (%) Positive for FR (%)

Positive for ACPA (%)

Number of previous treatments with DMARDs Number of previous biological therapies

73.1 65.6 ± 14.5 '123.8 ± 91.5' 22.2

64.8 62.2 2.2 ± 1.5 '1.8 ± 1.2' 76.8 46.5 ± 14.5 '171, 6 ± 123.6'

100 74.6 74.1 1.7 ± 1.5 '0.6 ± 0.9'

FR, rheumatoid factor; DMARDs disease modifying antirheumatic drugs; ACPA, antibody antibodies citrullinated. 8 Half ± SD

20 1.2 Cell lines

K562 (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% felal bovine serum (FBS) (Lanza, Verviers, Belgium). The medium was replaced every 2-3 days.

1.3 Analysis of TLR10 gene sequencing The coding exons and the binding regions of the TLR10 gene were sequenced in 66 selected RA patients with severe disease. Patients with serious disease were considered those with posilivity for reurnatoid factor (FR) and / or antibodies against citrullinated protein antigens (ACPA), presence of erosions and resistance to at least one disease-modifying anti-rheumatic drug (OMAROs) and 30 healthy controls by next generation sequencing (NGS). The FR was measured by nephelometry (BN !!, Siemens Healthcare) and the cut-off value 22 IU I mI. ACPAs were measured by a commercial EUSA test (CCP2, Eurodiagnostica) and were considered positive above 50 U / ml. Those with 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 (Illumina, Madison, WI). For Sanger sequencing, whole blood DNA was extracted using the QIAamp blood DNA kit (Qiagen, Hamburg, Germany)

and amplified with primers for human TLR10 (SEO ID NO: 3): 5'-CATGGCCAGAAACTGTGGTC-3 'and (SEO ID NO: 4): 5'-ACCATCCAACCATCATGACC-3'.

Sequence analysis of the amplified fragment was carried out using an analyzer

Genetic (Applied Biosystems, Foster City, CA).

1.4 Single SNP analysis The 1473T variant of TLR10 (rs11466657;

htlp: llwww.ncbi.nlm.nih.gov/projectslSNP/snp_ref.cgi? rs = 11466657), in a cohort

independent of 1201 patients with RA and 1493 healthy controls using the platform of

TaqMan® genotyping (Assay Id C_25643390_30, Life Technologies, Carlsbad, CA). All

endpoint fluorescence and PCR reaction readings were performed using a

ABI PRISM 7900 HT (Applied Biosystems) sequence detection system. The

2.

PCR reaction conditions were as follows: 50 or e for 2 min and 95 or e

for 10 min, followed by 40 cycles of 92 or e for 15 s and 60 o e for 1 min.

estimated genotyping error by genotyping 20% of the samples in duplicate (error <1%)

[Lopez-Lasanta M, Julia A, Maymo J, Femandez-Gutierrez B, Urena-Gamica 1, Blanco FJ,

S

Canete JO, Alperi-Lopez M, Olive A, Corominas H el al: Variation at Interleukin-6 Receiver

Gene 15 Associated to Jalnt Damage in Rheumatoid Arthritis. Atthritis Res Ther 2015,

17: 242).

1.5 Transfections to perform the indicator gene assays

10

TLR10 cDNA (NM_030956; http://www.ncbLnlm.nih.gov/nuccore/NM_030956.3;

SEQ ID NO: 1) in pCMV6 (Origene, Rockville, MD). The 1473T variant was generated by

Site-directed mutagenesis using the Ouick Change mutagenesis kit (Agilent

Technologies, Santa Clara, CA) with the following primers: SEO ID NO: 5

5'-GGCCTTACGAGAACTAAATACTGCATTTAATTTTCTAACTGATC-3 'and SEQ ID NO: 6

fifteen

5'-ATCAGTTAGAAAATTAAATGCAGTATTTAGTTCTCGTAAGGCC-3 '. The sequence was sequenced

Modified graft to verify the mutation. K562 and U937 cells were co-transfected with

2) .1g of TLR10 constructs of natural or mutant type, 1) .1g of plasmid pBVI-Luc

indicator, which contained six tandem repetitions of the recognition sites of

NFKB within the promoter region linked to the luciferase gene [Inohara N, al., J Biol

twenty

ehem 2001, 276 (4): 2551-2554J and 0.2 ~ g of pRSV-p-gal using Lipofectamine 2000 (Sigma

Aldrich, St Louis, 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

25

tumor necrosis factor-alpha (TNFa) in the presence or absence of 200) lg / ml of

infliximab and after 24 h the cell extracts were prepared and analyzed to determine the

relative luciferase activity using an Oual-Light indicator gene test system

(Applied Biosystems). The results were normalized to determine the efficacy of

transfection with values obtained with pRSV-j3-gal.

30

1.7 NF1C8 target gene expression analysis

To evaluate the expression of individual genes, a complementary AON was generated and

amplified using primers for human TNFa SEO ID NO; 7

(5'-CAATGGCGTGGAGCTGAGAG-3 'and SEQ ID NO: 8

S'-GGCTGATGGTGTGGGTGAGG-3 '), CCL2 SEO ID No. 3 'and SEO ID NO: 12 5 S' -TGACGGAGTTGCCACTTGACT-3 '), and p-actin SEO ID NO: 13 (S'-GCTGCCTCAACACCTCAAC-3' and SEO ID NO: 14 S'-GATGGAGTTGAAGGTAGTTTCGTG-3 '), Quantitative real-time PCR was performed in

a 7000 sequence detection system (Applied Biosystems). The ratio of the abundance of differentiation markers to that of (3-actin transcripts) was calculated.

10 as 2n, where n is the threshold cycle value of p-actin minus the threshold cycle value of the corresponding mRNA and was normalized by the value of the sample with the lowest expression level of these genes. The specificity of the desired PCR products was determined with melting curve analysis.

15 Real-time PCR was carried out using the Applied Biosystems 7000 Real-Time PCR system (Bio-Rad), Each PCR reaction contains 12, S ~ I SYBR GREEN PCR Master Mix (Applied Byosystems), 400 nM of each primer, and 1 ~ I diluted cDNA (100 ng) in a total volume of 25 ~ I. The reactions were performed in a 96-well reaction plate under the following conditions: 2 min at 60 oC, 10 min at 95 oC, followed by 40 cycles

20 of IS s at 9S oC, 3 s and 1 min at 60 oC,

The delta Cl (.ó.Ct) method was used for the analysis of PCR matrix data. The value

(6Ct) normalized for each gene of interest (GOl) was calculated by subtracting the average Ct of the two

constitutive expression genes ("housekeeping genes N of the IC of each GOl. A

)

25 then, the double delta the (lillCt) for each GOl was calculated by subtracting the average .ó.Ct from

each GOl in the 6Ct control group of each GOL The change factor ("fold-change")

of each GOl compared to the control group was calculated as 2-MCt of the log, 02o6 Ct.

1.8 Statistical analysis

30 All statistical analyzes were performed using the SPSS 20 program (18M, Armonk, New York) and the 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 vitro analysis using the bilateral Student t test for

independent data. The level of significance was established at p <O, 05. They were made

genetic association analysis using linear and logistic regression models. As it

previously described, the covariates in these genetic analyzes included the years of

disease evolution and baseline disease activity score (OAS28) for

S

the association of the rs11466657 genotype with the level of joint damage [Lopez-Lasanta M, Julia

A, Maymo J, Fernandez-Gulierrez B, Urena-Gamica 1, Blanco FJ, Caneta JO, Alperi-Lopez

M, Oliva A, Corominas H et al: Variation at Interleukin-6 Receptor Gene 15 Associated to

Jaint Damage in Rheumatoid Arthritis. Arthritis Res Ther 2015, 17: 242], and the change in

DAS28 at 12 weeks [Acosta-Colman 1, Palau N, Tornero J, Fernandez-Nebro A, Blanco

10

F, Gonzalez-Alvaro 1, Canete JO, Maymo J, Ballina J, Fernandez-Gutierrez B el al: Gwas

Replication Study Confirms the Association of Pde3aSIco1c1 with Anti-Tnf Therapy

Response in Rheumatoid Arthritis. Pharmacogenomics 2013, 14 (7): 727-734],

respectively.

fifteen

Example 2.-Variant 1473T is associated with the severity of the disease and

response to treatment in patients with AA

The function of TLR10 is controversial and its association with RA has hardly been studied. With

in order to evaluate whether the TLR10 variants contribute to modify the development of the

twenty

disease in patients with RA, exons coding for the gene of

TLR10 in 66 patients with selected RA and 30 healthy controls. After filtering the bases

which had at least 30X sequence coverage, sixteen variants were identified (see

table 2 below).

25

Table 2. Sixteen allelic 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.

SNP ID

REF ALT MAF value of Change Class Prediction Prediction

reference

P From aa functional 6n through

Mediant

SNPs30

e SIFT

rs10776482 rs4129008

A c G T 0.29 0.01 0.2306 0.5652 0809 R799Q SILENT CONTRAST TIOO nr nr

31

SNP ID REF ALT MAF Exchange value Class Predicci Pn reference prediction of functional AA 6n by means of SNPs3D and SIFT

RS4129009 T e 0.271 0.1889 1775V CONTRASEN

TIDO r510776483 A G 0.302 0.3296 H724 SILENT nr nr rs11466658 G A 0.026 0.1599 R525W CONTRASEN

TIDO rs11466657 A G 0.089 0.04327 1473T CONTRASEN + + TIDO r511096955 T G 0.422 0.07296 1369L CONTRASEN

TIDO r511096956 e A 0.292 0.3918 P344 SILENT nr nr rs11466653 A G 0.026 0.5824 M326T CONTRASEN nr

TIDO rs11466652 T e 0.13 0.1403 K303 SILENT nr nr rs11466651 e T 0.026 0.5824 V2981 c ONTRASEN

TIDO rs11096957 T G 0.438 0.015 N241H CONTRASEN TIDO rs11466650 A G 0.0005 0.137 L167P CONTRASEN + + TIDO rs11466649 e A 0.026 0.5824 A163S CONTRASEN

TIDO rs10858837 e T 0.026 0.5824 T37 SILENT nr nr rs10856838 A T 0.146 0.5813 11 3 SILENT nr nr

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): 38125 4) and SNPs3D (Yue el al., BMC Bioinformalics. 2006, 7: 166). Most of the changes

found nonsense were located in the LRR domains (figure 1A). As a result of the selection procedure, variant 1473T (Table 2) was identified for additional association studies. First, a population of 453 patients was studied and genotype frequencies (86.6% AA, 11.9% AG, 1.5% GG) were found not to be significantly different from those of a population of 209 controls (84.5% of M, 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. They all indicated a

Trend correlation between severity and the GG genotype that was not observed with the genotype

5

AA (figure 2A). To reinforce this observation, a cohort greater than 1201 was studied

patients with RA and 1493 healthy controls. As previously noted. it was found

that the genotype distribution was 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’s interesting,

10

as shown in figure 28, that variant 1473T is highly associated with

erosive disease in RA positive for ACPA (p = O, 017 in the total cohort and p = O, 0049 in

female patients) and with a lower response to treatment with infliximab measured

by changing the OAS28 index (p = O, 012 in the total cohort) as well as by

EULAR response criteria (p = 0.025 in the total cohort), indicating that this

fifteen

variant selects a group of patients with a more serious disease.

Example 3.-Variant 1473T modifies the NFxB regulatory capacity of TLR10

Based on a 3D structure model using the Jmol viewer (Nepomnyachiy S1, et aL,

Structure 2015 May 5; 23 (5): 941-8; Figure 3A), position 473 is within a chain

twenty

bela in the LRR18 domain and is occupied by an isoleucine, a highly amino acid

hydrophobic, hidden inside the protein core. In the TLR10 variant, it is replaced

threonine isoleucine, a polar amino acid that can participate in hydrogen bonds and

It is usually located on the surface of the proterna. LRR domains provide

Featured framework to achieve various protein interactions. Therefore, this change

2S

structural decreases hydrophobic contacts and can alter protein interactions

functionally relevant protein. It is known that members of the TLR family are

NFKB activity regulators, a key route in inflammation. To prove

experimentally the functional consequences of the 1473T variant on activation

of NFKB, the codon alteration nuc1eotide was introduced into a construct containing

30

TLR10 cDNA by site-directed mutagenesis and the capacity of this was studied.

variant to modify the transcriptional activity of NFKB. As shown in the

Figures 3B and 3C, TLR10 of natural type regulates by decrease the transcriptional activity of

NFKB both under unstimulated conditions and under conditions stimulated by TNFa. in

the hematopoietic cell lines K562 and U937. However, the replacement of 1473T

35

blocks the inhibition capacity of TLR10. In order to translate the reduced response to

Infliximab in patients to the in vitro model, TNFa cells were stimulated in the presence or absence of infliximab. Consequently, cells expressing the 1473T variant had a higher level of NFKB activity that remained after treatment with infliximab compared to cells expressing the wild type variant (3D figures 5 and 3E). Then, this result was confirmed by analyzing the expression of NFKB target genes. The expression levels of CCL2, TRAIL and TNFo. they were regulated by decrease in the presence of wild-type TLR10 after stimulation of the NFKB route with TNFa. In line with the previous results, regulation by NF "B target gene reduction was canceled when cells were transfected with the construct containing the variant and

10 cultivated with TNFa. (figure 4AME). The expression of CCL2 also confirmed that the variant generates a lower response to infliximab (Figure 4F).

In conclusion, the 1473T variant leads to a reduced capacity of TLR10 to inhibit the activation of NFKB in response to inflammatory stimuli. This effect could be explained.

15 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, al., PLoS One 2010, 5 (9): e12713J.

Claims (21)

one,

In vitro method to predict the clinical response of a subject, who has or is

he suspects that he has an inflammatory disease, an anti-TNFa agent, in which

5

said method comprises:

a) determine in a biological sample isolated from said subject the genotype of the

single nucleotide polymorphism (SNP) rs11466657;

10

where the presence of the allele (G) in at least one of the alleles in the SNP locus

rs11466657 indicates an increased risk of not responding to the anti-TNFa agent .: and

where said inflammatory disease is selected from the group consisting of

inflammatory bowel disease, psoriasis. psoriasic arthritis, rheumatoid arthritis,

fifteen

systemic lupus erythematosus, Sjógren's syndrome, juvenile rheumatoid arthritis,

Spondyloarthropathy, uveftis and Behcet's disease.

2.

In vitro method to identify a subject, who has or is suspected of having a

inflammatory disease, which presents a higher risk of not responding to

twenty

treatment with an anti-TNFa agent, wherein 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 SNP's locus

rs11466657 indicates an increased risk of not responding to the anti-TNFa agent; Y

25

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 reurnatoid arthritis,

spondyloarthropathy, uvertis and Behcet's disease.

30

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 agent

TNFa.

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 it also includes: b) select an anti-TNFa agent when the allele (A) is present in both alleles in the SNP 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 Behcet's disease. 5. In vitro 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; Y where said inflammatory disease is selected from the group consisting of inflammatory bowel disease, psoriasis, psoriasis arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sj6gren's syndrome, juvenile rheumatoid arthritis, spondyloarthropathy, uveitis and Behcet's 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-TNFa agent, the severity and / or prognosis of said disease where said method comprises the stage 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, Sj6gren's syndrome, juvenile rheumatoid arthritis, spondyloarthropathy, uveitis and Behcet's disease.

8.

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

9.

The method according to any one 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.

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

12.

Method implemented by computer, wherein the method is as defined in any of claims 1 to 11.

13.

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

14.

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

fifteen.

Use of infliximab in the preparation of a medicament for the treatment of an inflammatory disease in a subject, which has or is suspected of having said inflammatory disease, in which said subject has the allele (A) in both alleles in it of the SNP rsl 1466657,

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, uvertis and Behcet's disease.

16.

The method according to any of claims 1 to 140 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 16.0 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 rsl1466657, comprising: - a reagent for determining the genotype of SNP rs11466657; - optionally, instructions for the use of said reagent in the genotype determination of SNP rs11466657 in an isolated biological sample.

19.

The kit according to claim 18, wherein said reagent is a specific SEO ID NO: 1 primer and / or probe that amplifies a region comprising SNP r511466657.

twenty.

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

twenty-one . 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 at risk of not responding to a treatment with an anti- agent TNFa, 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. 3.