WO2016097961A1

WO2016097961A1 - Genetic markers predictive of clinical response to biological drugs in psoriasis.

Info

Publication number: WO2016097961A1
Application number: PCT/IB2015/059573
Authority: WO
Inventors: Barbara MARINARI; Marina TALAMONTI; Antonio Costanzo; Elisabetta Botti
Original assignee: Università degli Studi di Roma “La Sapienza”
Priority date: 2014-12-17
Filing date: 2015-12-14
Publication date: 2016-06-23

Abstract

The present invention relates to a method predictive of response to treatment with ustekinumab in a patient affected from psoriasis comprising detecting in the genome of the subject the association of the HLA-Cw* 0602 allele and a single nucleotide polymorphism predictive of response to said biological drug. The method according to the invention is indicative of greater likelihood of clinical response to treatment, speed of clinical response to treatment and better maintenance to the therapeutic effects dell'ustekinumab and/or drugs blocking the IL12/23-IL17 axis.

Description

GENETIC MARKERS PREDICTIVE OF CLINICAL RESPONSE TO BIOLOGICAL DRUGS IN PSORIASIS

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Field of the invention

The present invention relates to the field of pharmacogenomics and, more precisely, relates to genetic markers associated with the response to a drug used in the treatment of psoriasis, and in particular of plaque psoriasis.

Background

Psoriasis is a multifactorial immune-mediated chronic skin disease [1] . The prevalence of psoriasis is variable, it is estimated that in the world about 125 million people suffer from psoriasis, among these about a third manifest moderate-severe and/or complicated forms [2] . The prevalence is higher in Northern Europe, lower in Southern America, Africa and Asia. Prevalence is around 3% of the population worldwide [ 3 ] .

The age of onset of psoriasis has a bimodal distribution: in 2/3 of the cases a peak incidence between 16 and 22 years is found, in the remaining third of cases between 57 and 60 years [4] .

Psoriasis is associated to a high incidence of other related conditions, namely psoriatic arthritis affecting about 30% of patients, metabolic syndrome, in turn characterized by atherogenic dyslipidemia, obesity, hypertension, insulin resistance and diabetes resulting in subsequent cardiovascular disease [5-6] . As psoriasis is a chronic condition, it often requires long-term therapy with immunosuppressants, which can affect other organs and contribute to the development of serious side effects and comorbidities. Psoriasis has a strong social impact with psychological and economic repercussions for patients, and often it is also associated to anxiety and/or depression [7] .

The pathogenic processes underlying psoriasis are complex and multifactorial, with initial local activation of the innate immune response and subsequent involvement of the acquired immune response; the predominant activation of Thl and Thl7 cells causes the production of cytokines, chemokines and specific growth factors that ultimately lead to epidermal hyperplasia and inflammation.

It is widely recognized a genetic predisposition to psoriasis. Whole genome association studies have identified several genomic regions of susceptibility to psoriasis including: HLA-C, IL12B, IL23A IL-23R IL23A, IL4/IL13, TNFAIP3, TNIP1, ZNF313 and LCE3B/3C1 and particular single nucleotide polymorphisms (SNPs) associated with psoriasis [8-12].

Literature has shown that HLA-Cw* 0602 is the most important susceptibility to psoriasis marker, 50-80% of Caucasian psoriasis patients carry thist allele, compared with 20% of the healthy population. Furthermore, HLA-Cw* 0602 is part of the haplotype associated to type I psoriasis forms, wherein the disease occurs before the age of 40 years [13, 14] . In patients HLA-Cw* 0602 positive an inverse relationship between the average age of onset and the severity of psoriasis has been observed: in fact, lower the age of onset, the greater the degree of severity [ 15] .

In the genome of a subject HLA-Cw* 0602 not only is a factor predisposing to the development of psoriasis, but it is also an indicator of response to the treatment with ustekinumab [16], as it has been found an association between the presence of this particular gene variant and a good clinical response to treatment with the biological drug. Patients carrying HLA-Cw* 0602 respond faster to the monoclonal antibody that specifically binds the p40 subunit of interleukin IL-12 and IL-23, inhibiting in such a way the activity thereof and causing lack of development of T helper 1 and 17, essential for the development of plaques psoriasis. About 90% of patients carrying HLA-Cw* 0602 reached PASI 50 4 weeks after the first injection of the drug, nearly all patients reached PASI 75 at 12 weeks and maintained the result over 40 weeks. On the other hand also minor drug response in patients without the allele but with early onset of psoriasis type 1 suggests that HLA-Cw* 0602 is a good predictor of response to ustekinumab.

The IL12B gene encodes the p40 subunit shared by IL12 and IL23, respectively the main cytokines involved in Thl and Thl7 response [17] . Therefore, inhibition of p40 also inhibiths production of IL17 family members that are blocked by specific drugs (namely, Secukinumab, ixekizumab and brodalumab) .

A recent publication has shown that two SNP of IL12B associated to psoriasis susceptibility (rs3212227 and rs6887695) [8] are able to regulate the expression of p40. Specifically, the haplotype A of rs3212227 and G of rs6887695 are associated to a p40 increase and to a lymphocyte polarization towards a Thl phenotype response, with increase of IL12, interferon-γ and CXCL10 expression, and simultaneous decrease of IL23 expression. This regulatory mechanism assumes that likely the psoriasis comprises several variants characterized by activation and/or modulation of various inflammatory pathways [18] .

Currently moderate to severe psoriasis with the involvement of more than 20% of the body surface area and not effectively responding to the topical therapies must be treated with systemic therapies. Systemic therapies for psoriasis involve immunosuppressants such as cyclosporine, retinoids, PUVA therapy and, more recently, in the case of ineffectiveness or contraindication to these drugs, use of biopharmaceuticals including anti-TNF alpha molecules (infliximab, etenercept and adalimumab) and an antibody against p40 (ustekinumab) [19] .

The European patent application EP2149612 relates to genetic markers associated to a particular clinical response toward treatment with an inhibitor of Lymphocyte Function-associated Antigen 1, also known as LFA-1, in individuals suffering from psoriasis relating to a method for genotyping individuals with psoriasis comprising detecting the presence or absence of at least a particular allele in the selected group of: SNP_A-2040233 (rs4043263), SNP_A- 1978609 (rsl7501861) , SNP_A- 1986846 (rs9294506), SNP_A-2223123 (rs6485472), SNP_A-2085239 (rsll251116) , SNP_A-4269573 ( rs 11037653 ) , SNP_A- 2010642 (rs4620711), SNP_A-4260850 ( rs 10838166 ) , SNP_A-4239787 (rs4573056), SNP_A-2213961 (rsl0755496) or SNP_A-2299781 (rs7742750) .

The international patent application WO 2008/124937 provides a method predictive of response to a biological agent in a patient with psoriasis involving detecting the allelle HLA-Cw* 0602 and the two polymorphisms TNFa 238 and TNFa 308.

The US patent application US2013/0216551 describes new SNPs, combinations of SNPs, haplotypes or diplotypes of SNPs associated with psoriasis, and in particular with an increased or decreased risk of developing the disease. The document proposes using such polymorphisms for designing diagnostic reagents and developing therapeutic agents in the treatment of psoriasis and related conditions. Object of the invention is thus the genotyping of SNPs rs3212227 and rs6887695 in the gene encoding the p40 subunit of interleukin IL-12 and IL-23 used to diagnose in a subject a state of altered, either decreased or increased, risk of developing psoriasis. Among the variants rs3212227 (A) and rs6887695 (G) are indicated as risk factors, while the variants rs3212227 (C) and rs6887695 (C) are referred to as protective factors against the disease. However, so far research and knowledge in the field are not able to establish a link between the particular arrangement of such genotypic markers and the response to a kind of treatment.

The development of biological drugs, that is those drugs mimicking in the organism the action of an active biological molecule, and therefore highly selective because they aim to precisely target a single structure (receptor, protein, DNA sequence) involved in a pathological process, thus reducing side effects and increasing the therapy effectiveness, has definitely revolutionized the treatment of psoriasis and other inflammatory and autoimmune diseases.

Data from a meta-analysis study of 22 clinical trials concerning the use of different biological drugs against psoriasis have shown that infliximab (INN), the recombinant chimeric monoclonal antibody against tumor necrosis factor a is the most clinically responsive drug. By PASI index enabling a measure of erythema, desquamation and thickness of plaques as well as the extent of involvement of each body region, the efficacy of the treatment is evaluated as a function of the score reduction compared to baseline. Therefore a 75% reduction is referred as PASI 75, a 90% reduction as PASI 90, and PASI 100 corresponds to a skin completely free from injuries. Following treatment with infliximab, PASI 75 and PASI 90 were obtained respectively in 80% and 54% of patients. By using ustekinumab, the fully human monoclonal antibody binding the p40 protein subunit of interleukin 12 and 23, PASI 75 and PASI 90, respectively, in 74% and 46% of patients is recorded. However, biological drugs, have proven not to always have clinical efficacy or the same response level in all patients; sometimes their use is associated to serious side events [20].

Furthermore, the biological therapies are very expensive. It is estimated that in Italy biological drugs account approximately for 13.7% of pharmaceutical expenditure of national health service. The average cost of a patient being treated with biological drug amounts about 15, 000 euros per year. Guidelines suggest using anti TNF amolecule as a first choice , if not contraindicated, as they are on the market for a long time and more clinical results and security data are available thereon.

Hence, in the common clinical practice anti TNFa is the most widely used biological drug. The efficacy is assessed after 12 weeks treatment and in case of failure, the next therapeutic strategy is usually based on implementing dosage or treating with another biologic drug, whose answer will be typically assessed at 24 weeks. The identification of the optimal therapeutic agent can expose the patient to 9 months of ineffective therapy and an increased likelihood of developing adverse effects and, for sure, to further costs, not only related to the drug, but also due to specialists, blood tests and declining productivity of the patient. It has been shown that during the first year of treatment with biological drugs, 36% of patients stop the treatment or change therapy. 18% of psoriasis patients treated with biological drugs stops the treatment, 12% shift to other drug, 7% increases the dosage.

Furthermore, several studies have shown that exposure to a biological drug may affect the response to a subsequent treatment with another biological agent. Hence, any initial ineffective therapeutic chioce, in addition to being a waste, can compromise the clinical response to other therapies, that instead, when used as a first approach, would be more effective. This clearly represents a disadvantage for those drugs that are currently prescribed at a later stage, as ustekinumab. It has been published that patients who have not received prior therapy with anti-TNFa have a better clinical response to ustekinumab; after 24 weeks of treatment, PASI 75 was achieved in 85% of treatment-naive patients compared with 50% of patients previously treated with anti- TNF-a (p = 0.0235) [21] .

It is hypothesized that the heterogeneous clinical response to a drug is due to a polymorphic genetic background, as gene variants act differently within specific molecular pathways [22] . Few data of pharmacogenetics in psoriasis are available. There are genetic prediction studies about some conventional treatments like methotrexate and acitretin [23-25] , but few data concerning biological drugs. A study showed the association between two polymorphisms in TNFAIP3 gene and response to anti- TNFa drug (etanercept, adalimumab and infliximab) . In particular, the presence of the G allele in SNP rs610604 or T in SNP rs2230926 are associated with a better clinical response [26] . Another study revealed the association of the CC genotype of SNP rsl799724 of TNFa and the TT genotype of SNP rsl061622 of TNFRSF1B with the response to etanercept [27] .

The Applicant has investigated the association of the HLA-Cw* 0602 and an improved clinical response to efalizumab, a monoclonal anti CDlla antibody, currently withdrawn from the market in Europe [28] . Since in clinical practice no genetic screening for deciding the therapeutic strategy is performed, the possibility of having available sure genetic markers would allow to customize therapy according to each individual patient suffering from psoriasis, in order to enable to choose, not empirically anymore, but on molecular basis, likely the most suitable drug in terms of clinical efficacy and safety, with significant cost savings in the long term. Having a validated technique to guide the choice of therapy would avoid waste money for not effective drugs for a genetically specific patient, and the patient would not be exposed to lost of time with ineffective therapy, and to risk of adverse events affecting not only the health, but also the ability to positively respond to subsequent therapies. In addition, a patient not responding to a given therapy, not only determined cost increase because multiple therapies or associations of therapies have to be tested, but, being dissatisfied about the health status, requires several specialized consulting and instrumental and laboratory investigations. In terms of benefit intervening with the right medication in the early stages of the disease potentially slows the progression of the disease and the establishment of co-morbidity .

Definitely, it is important to promptly diagnose psoriasis, but to identify the best therapy for a specific patient taking in account the hereditary genetic factors creating differences in the effectiveness of drugs in different individuals is important as well. The delayed identification of the correct therapy can dramatically alter the sometimes very disabling disease course, exposing the patient to the risk of side effects due to not optimal therapies .

Therefore, it is strongly felt the need to have available genetic markers predictive of the clinical response to biological drugs in psoriasis.

The present invention proposes to solve the problem of non-detected or delayed therapy for psoriasis more suitable for each patient, providing an indication of the response to treatment with the main therapeutic agents currently available.

Summary of the Invention

The invention provides a method for genetic screening indicative of the probability that the patient with psoriasis, in particular plaque psoriasis, respond to treatment with a biological agent, based on the presence of specific alleles or polymorphisms in two genetic loci.

The presence or absence of the allele and polymorphism is detected by analysis of a sample of genomic DNA obtained from the subject.

Brief description of the figures

Figure 1 graphically shows the trend of the clinical response to treatment with ustekinumab in a time interval between 0 and 52 weeks expressed as percentage of patients achieving PASI 75.

Figure 2 shows the clinical response after 4 weeks of treatment with ustekinumab in patients with various polymorphisms in the two loci considered in the method of the invention expressed as the percentage of patients achieving PASI 75.

Detailed description of the invention.

The invention is based on the presence of particular genetic markers in patients with psoriasis.

The method of the present invention is based on the discovery that particular alleles or polymorphisms in a subject suffering from psoriasis correlate with the response to certain biological drugs.

Surprisingly, it was identified that a particular allele or genotype of two specific genetic markers is associated with a positive clinical response to treatment of psoriasis with an inhibitor of interleukin 12/23.

Infact, it has been experimentally found that individuals with psoriasis who have said allele, or genotype, in said genetic markers are more likely to develop a very good clinical outcome, determined by the PASI (Psoriasis Area and Severity Index) value following treatment against psoriasis with biologic drug ustekinumab.

Therefore, aim of the present invention is to provide a method predictive of the response to biological agents acting on the IL12/23-IL17 pathogenetic axis in a subject suffering from psoriasis based on the analysis of certain genetic variants in two genetic loci of susceptibility to psoriasis.

The genotypes of the two genetic markers associated with the clinical response to treatment with ustekinumab of psoriasis can be detected in individuals with psoriasis. So, individuals can be identified and early addressed to treatment with the optimal drug based on genotype. Given the strict pathogenetic connection between IL23 and IL17, as a consequence, the same genetic markers will predict response to IL17 blockers ( secukinumab, ixekizumab and brodalumab) .

The term "subject" refers to humans and non-human primates and any other organism that may benefit from the use of the method according to the present disclosure. This subject can also be referred to as: patient, individual, host, user, organism. In some particularly preferred embodiments of the invention the subject is a human.

In the present disclosure of the invention with the term "psoriasis" it is intended to comprise a group of dermatological conditions characterized by red, scaly patches and plaques or lesions associated with the excessive production of epidermis and inflammation, including plaque psoriasis (psoriasis vulgaris) mild, moderate or severe and chronic plaque psoriasis .

The severity of psoriasis in a subject is typically assessed on the basis of affected body surface area, it is indicated as mild if there is involvement of less than 5% of the body surface, as moderate with affected surface between 5 and 10%, whereas psoriasis is deemed to be serious when symptoms interest cover more than 10% of the body.

The expression "response" to treatment of psoriasis with an inhibitor of IL12/23 or ustekinumab, or "clinical response" to treatment of psoriasis with an inhibitor of IL12/23 or ustekinumab, or the like used herein refers to the change of the symptoms in an individual suffering from psoriasis after treatment/drug administration, evaluated preferably by the classification PASI, or other rating scales known to the experts of the sector, such as the Physician Global Assessment (PGA) .

Therefore, specifically, the invention provides a method predictive of clinical response to ustekinumab in a patient with psoriasis wherein the method comprises detecting in the subject genome the presence, or absence, of HLA-Cw* 0602 and a single nucleotide polymorphism in the gene of p40 protein subunit of interleukin IL-12/23 (IL12B rs6887695) . Given the strict pathogenetic connection between IL23 and IL17, as a consequence, the same genetic markers will predict response to IL17 blockers ( secukinumab, ixekizumab and brodalumab) .

By the term "allele", as used herein, it is meant each of the two or more alternative forms of a gene occupying the same position (locus) on homologous chromosomes and controlling the expression of the same character.

By the term "polymorphism", as used herein, it is meant the set of two or more variants of the DNA sequence (genotypes) for a given character or locus. The possible sequence variants differ for only one or a few nucleotides. Thus, "single nucleotide polymorphism" SNP refers to one of the possible sequence variants at a particular genetic locus that differs by only a single nucleotide.

The term "allele HLA-Cw* 0602" refers to a particular allele (Cw6) present in the locus of the HLA-C major histocompatibility complex class I on the short arm of chromosome 6 in humans. For this locus at least 25 different alleles Cw were identified, identified by sequence analysis [29] .

The other genetic marker that in combination with the allele HLA-Cw* 0602 was found to be particularly useful to identify people with psoriasis who are likely to develop a positive response to treatment with ustekinumab is the single nucleotide polymorphism rs6887695.

The association between the risk of developing psoriasis and rs6887695 SNP was identified for the first time by M. Cargill [30] . This polymorphism is also associated with psoriatic arthritis and type 2 diabetes [ 31 ] .

As it will be more evident from the experimental section of this description, the method according to the invention provides that in association with the detection of the HLA-Cw* 0602 other polymorphisms predictive of response to treatment of a subject with ustekinumab psoriasis, including the IL12B polymorphism rs3212227 and rsl800795 IL6, can also be used as indicators.

In vitro studies suggest that different genotypes of rs3212227 functionally affect the production of IL12. The rs3212227 polymorphism was associated with the susceptibility to the development of certain autoimmune diseases [32], type 1 diabetes [33], multiple sclerosis [34-35] and Chagas disease [36]. Interleukin- 6 (IL6) gene promoter influencing the level of cytokine production, has a quite polymorphic site in Caucasians (SNP rsl800795) , while Asian and African populations are nearly monomorphic for the allelic form bearing the G.

The rsl800795 polymorphism was described for the first time in 1998, when it was shown that the allelic form C produces less IL6 than allele G. This observation supported the hypothesis that rsl800795 (C/C) could be a protective genotype against the juvenile rheumatoid arthritis onset. In fact, few patients with juvenile rheumatoid arthritis have this genotype .

More recent studies have identified potential associations of rsl800795 with heart disease, Kaposi's sarcoma, type 2 diabetes, stroke, obesity, Hodgkin's disease, sudden infant death syndrome, cancer (including breast cancer, gastric cancer, prostate cancer) , hypertension, periodontal disease and complications following organ transplants or grafts. However, some allele associations for rsl800795, do not concern the risk of developing a certain disease, but are referred to as a specific disorder can progress or respond to different treatments (reference for the main associations can be found in OMIM entry 147620) .

In the light of the above, in a general aspect the present invention aims to provide a method indicative of greater likelihood of clinical response to the treatment with the drug.

In another aspect, the invention intends to provide a method indicative of the speed of clinical response to the treatment with the drug.

In a further aspect, the invention intends to provide an indication of better maintenance of the therapeutic effects resulting from the use of the drug. These findings can be easily extended to other drugs belonging to the IL12/23-IL17 axis.

The results of a previous study carried out by the Applicant have shown that HLA-Cw6 positive patients have a best clinical response to treatment with ustekinumab compared to HLA-Cw6 negative patients [37] .

Surprisingly, it has been found that the association between the HLA-Cw* 0602 polymorphism and the G/G SNP rs6887695 in the IL12B gene increases the significance of the prediction of response to ustekinumab .

Specifically, the presence of positive HLA-Cw* 0602 aplotype and G/G SNP rs6887695 in IL12B gene in patients suffering from psoriasis is associated with: 1) a greater likelihood of clinical response to treatment with ustekinumab compared to patients having negative HLA-Cw* 0602 and C/G or C/C rs6887695 SNP;

2) a faster response to treatment with ustekinumab;

3) better maintenance of the benefit during continued treatment .

The presence or absence of a particular allele in the locus of a genetic marker in an individual can be determined by methods and techniques well known to the skilled person in the field, i.e. methods and techniques for genotyping.

Some examples of such techniques include, but are not limited to, direct DNA sequencing of the amplified region of interest, capillary electrophoresis, hybridization by allele specific probes, single- strand conformational polymorphism analysis, electrophoresis on denaturing gradient gel, electrophoresis in temperature gradient, detection of failure pairing between nucleotides, nucleic acid arrays, primer specific extension, protein detection, and other techniques well known in the art [38-39] . Typically, these methods involve the analysis of a sample of nucleic acid prepared from a biological sample obtained from the individual to determine the identity of a nucleotide, or nucleotide pair, present in the genetic marker, for example, without limitation, the SNP .

Samples of nucleic acids may be prepared from any biological sample. For example, suitable samples include whole blood, serum, semen, saliva, tears, feces, urine, sweat, material from the mouth, skin and hair.

In a preferred embodiment of the invention the DNA sample is obtained from a peripheral blood sample or saliva of the analyzed subject.

Nucleic acid samples can be prepared for analysis using any technique known to those skilled in the art.

Preferably, such techniques involve the production of genomic DNA sufficiently pure for determining the genotype of a desired set of genetic markers in the DNA molecule .

In a first embodiment of the invention the DNA analysis of the subject is carried out by direct DNA sequencing of the amplified region of interest.

Any DNA amplification technique known to the experts of the field can be used in carrying out the present invention including, without being limited to specific techniques of polymerase chain reaction (PCR) .

In a particularly preferred embodiment the amplification of the DNA region of interest is performed by means of DNA polymerase chain reaction (PCR) using materials and methods known to those skilled in the art.

According to the invention the DNA region of interest specifically amplified is then analyzed to determine the allele in the genetic loci of the marker.

The presence, or absence, of HLA-Cw* 0602 is tested through a PCR reaction with primers specific for the allele, as described in Talamonti et al . 2013. The rs6887695 locus was genotyped by Taqman assay using the specific probe TaqMan_SNP Genotyping Assay ID C 1994992_10 (Life Technologies) according to manufacturer's instructions.

In another aspect the invention relates to a kit for predicting the response to the treatment with a biological drug consisting in the antibody against p40 in a subject suffering from psoriasis according to the method of the invention. According to the invention the kit includes means for detecting the presence, or absence, of HLA-Cw* 0602 and the rs6887695 polymorphism predictive of response to treatment of psoriasis with the therapeutic agent ustekinumab and instructions for using the kit for predicting the response to treatment with a biologic therapy in a patient with psoriasis. For example, means for detecting the presence, or absence, of the polymorphism or allele predictive of response to treatment of psoriasis include:

- Preparations of nucleic acid in amount sufficient to detect by PCR the presence or absence of HLA Cw* 0602 and the rs6887695 polymorphism in a DNA sample from the subject, including primer pairs and probes specific for the genetic loci under genotypic investigation; DNA polymerase, reagents and reaction mixtures suitably balanced to conduct the amplification reaction of the DNA fragment of the predictive genomic allele and polymorphism.

- Preparations of nucleic acid to be used as DNA template in a control amplification reaction for the two polymorphisms.

- Sterile deionized water.

In a particularly preferred embodiment the kit of the invention provides all means to achieve the amplification of genomic DNA by "touchdown" PCR reaction, i.e. characterized by the progressive reduction of the annealing temperature. The initial annealing temperature is 65 °C at the first amplification cycle, and is progressively reduced by 0.5 °C at each cycle for 18 cycles, followed by 10 additional cycles of amplification at 57 °C. Preferably, the amplification reaction specific for HLA-Cw* 0602 is performed by using DNA polymerase KAPA2G robust (KAPA Biosystems) using the specific CG robust reaction buffer. However, the amplification reaction can be catalyzed by any DNA polymerase that has similar functional characteristics.

More particularly, a kit of the invention preferably include:

- Preparations of oligonucleotides specific for HLA- Cw* 0602: Fwd 5 ' -TACTACAACCAGAGCGAGGA-3 ' and Rev 5'- GGTCGCAGCCATACATCCA -3 ' ,

- Preparations of oligonucleotides and Taqman probe rs6887695 (TaqMan_SNP Genotyping Assay C1994992_10

ID) detecting C/G polymorphism located in position: Chr. 5: 158,822,645 (NCBI Build 37) in the frame of the nucleotide sequence:

GAGAGAAGCAGTGTAGTGTAGTGGT [C/G] AATAGTCTGGATTTACATCTTTG AT;

- Preparations of appropriate reagents and reaction mixtures balanced for the performance of amplification and genotyping reactions, such as non- restrictive example, lOmM each dNTP mix, 25mM MgCl₂, DNA polymerase 5υ/μ1, preparations of genomic DNA, plasmid DNA, cDNA, dye.

Further characteristics and advantages of the present invention will appear clearly from the following examples given for indicating and not limiting purposes .

Experimental part

Example 1 - Clinical study

The study aims to assess whether the presence of HLA- Cw* 0602 and the particular polymorphism SNP rs6887695 correlate with response to treatment of psoriasis patients treated with ustekinumab.

Patients

For the clinical study 64 patients were enrolled (18 females and 46 males) aged between 20 and 74 years who have been diagnosed with chronic plaque psoriasis stable for at least six months, with initial PASI ≥ 12 and according to PGA indexing moderate to severe. From the study were excluded: pregnant women, breast- feeding or intend to conceive a child during the study period, patients with positive screening for tuberculosis, HIV, hepatitis B and C, patients with a history of malignancy prior and/or concomitant, excluding non-melanoma skin cancer (NMSC) , patients with concomitant severe infections or severe infections within 2 months prior to screening, patients with present or past history of chronic or recurrent infections, patients requiring concomitant treatment with corticosteroids. All enrolled patients in the study have donated the sample of their DNA and given their informed consent to the economic exploitation through patenting of the results of the study and invention that ensued.

Patients were subjected to treatment with ustekinumab, after the therapeutic approach based on the use of at least two systemic drugs (methotrexate, cyclosporine, acitretin, UVB, PUVA therapy) has failed, or if its use was contraindicated .

Treatment and response

Forty five mg of ustekinumab were subcutaneously (sc) administered in patients weighing less than 100 kg, alternatively 90 mg sc in patients weighing more than 100 kg. A first administration was given at time 0, a second was made after four weeks and then every 12 weeks .

The clinical response of patients was assessed by the PASI 50, PASI 75 and PASI 90 indexes after 4, 12, 24 and 40 weeks of treatment.

After treatment patients showing PASI ≥ 75, or PASI 90 after 12 weeks of the first subcutaneous injection were considered responsive to therapy.

Extraction of DNA

From each patient participating in the study a sample of venous blood was withdrawnfrom which the DNA was isolated using standard techniques known to the experts of the sector.

Genotyping

HLA-Cw* 0602 was detected by standard PCR reaction where the two allele specific oligonucleotide:

Fwd 5 ' -TACTACAACCAGAGCGAGGA-3 ' and Rev 5'-

GGTCGCAGCCATACATCCA-3 ' were used as primers for the amplification reaction conducted by DNA polymerase under the same conditions described in Talamonti et al. 2013.

The locus rs6887695 was genotyped using Taqman assay using the specific probe TaqMan_SNP Genotyping Assay ID C1994992_10 (Life Technologies) enabling detection of the polymorphism C/G localized in position GAGAGAAGCAGTGTAGTGTAGTGGT [C/G] AATAGTCTGGATTTACATCTTTG AT .

The IL12B locus rs3212227 was genotyped using the allelic discrimination technique IL12B rs3212227 Life Technologies No cat. C_2084293_10 enabling detection of the polymorphism G/T located in the position ATTGTTTCAATGAGCATTTAGCATC [ G/ T ] AACTATACAAATACAGCAAAGAT AT according to the manufacturer indications.

The locus IL 6 rsl800795 was genotyped using the allelic discrimination technique Life Technologies (cat no. C_1839697_20) that permits detection of the polymorphism C/G localized in position

ACTTTTCCCCCTAGTTGTGTCTTGC [C/G] ATGCTAAAGGACGTCACATTGCA CA.

Results

The results of the study are reported in Table 1 and show that the association between the HLA-Cw* 0602 polymorphism and G/G SNP rs6887695 in the gene IL12B increases the significance of the prediction of response to the treatment with ustekinumab.

Table 1

% ratio

HLA-Cw* 0602 IL12B

rs6887695

positive G/G 91, 7 2,8 0, 136 negative C/G o C/C 66, 7

28 weeks treatment

Genotype PASI 75 odds P

% ratio

HLA-Cw* 0602 IL12B

rs6887695

positive G/G 91, 7 6, 56 0, 033 negative C/G o C/C 66, 7

40 weeks treatment

Genotype PASI 75 odds P

% ratio

HLA-Cw* 0602 IL12B

rs6887695

positive G/G 91, 7 7,27 0, 023 negative C/G o C/C 66, 7

52 weeks treatment

Genotype PASI 75 odds P

% ratio

HLA-Cw* 0602 IL12B

rs6887695

positive G/G 83, 3 5,2 0, 007 negative C/G o C/C 44, 4

91.7% of patients with positive HLA-Cw6 and G/G SNP rs6887695 compared to 66.7% of HLA-Cw* 0602 negative and C/G or C/C achieved PASI 75 after 12 weeks of treatment (odds ratio OR 2.81; p = 0.136) .

91.7% of patients with positive HLA-Cw* 0602 and G/G SNP rs6887695 compared to 66.7% of negative HLA-Cw* 0602 and C/G or C/C achieved PASI 75 after 28 weeks of treatment (OR 6.56; p = 0.033) (Table 1) .

The results also show that patients with positive HLA-Cw* 0602 and G/G SNP rs6887695 gene IL12B have a faster response to treatment with ustekinumab, as well as better maintenance of the benefit during continued therapy.

After 4 weeks of treatment (single dose) 50% of patients with positive HLA-Cw* 0602 and G/G SNP rs6887695 achieved PASI 75 (OR 10:49, p = 0.009), whereas no negative HLA-Cw* 0602 and C/G or C/C patients show symptoms remission (Table 1) .

After 52 weeks of treatment, 83.3% of patients with positive HLA-Cw* 0602 and G/G SNP rs6887695 achieved PASI 75 (OR 5.2, p = 0.007) compared to 44.4% of patients negative HLA-Cw* 0602 and C/G or C/C SNP rs6887695 polymorphism (Table 1) . The results are graphically shown in Figure 1.

The advantage from using the present invention is therefore clear: while the probability of obtaining a positive clinical response to ustekinumab, based on achieving PASI 75, using predictive methods of the prior art is within 69-74%, the association of HLA- Cw* 0602 and the presence of G/G SNP rs6887695 in the IL12B gene can increase this percentage up to 91.7%. In Table 2 the correlation between the percentage of patients who achieved PASI 75 as early as the fourth week of treatment with ustekinumab in various combinations of the two considered polymorphic loci is illustrated, wherein clearly appears that the combination of the positive CW6 haplotype and G/G rs6887695 polymorphism correlate to a remission index higher than other genomic variants expressed as a percentage of patients with a PASI 75.

Table 2

rs6887695 heterozigous o C/C

IL12B 25 rs6887695 G/G

C 6 positive - IL12B 50 rs6887695 G/G

C 6 positive - IL12B 16, 6 rs6887695 heterozigous o C/C

C 6 negative- IL12B 10 rs6887695 G/G

C 6 negative- IL12B 0

rs6887695 heterozigous o C/C

The data reported in the table are graphically presented in Figure 2.

The figure illustrate s that already at 4 weeks 50% of patients with positive HLA-Cw* 0602 haplotype and G/G SNP rs6887695 has an index PASI 75 (OR 10:49, p=0.009), whereas patients with different genotype for the two considered loci have a lower percentage of symptoms remission. The figure provides an explicit indication that the presence of positive HLA-Cw* 0602 and G/G SNP rs6887695 gene IL12B in psoriasis patients can be used as an indicator of speed of response to treatment with ustekinumab.

Example 2 - Comparative study

The effectiveness of the predictive method according to the invention was also evaluated with respect to the use of other genetic markers, either alone or in association with HLA-Cw* 0602, as predictors of the clinical efficacy of the psoriasis treatment with ustekinumab. Other polymorphisms considered were: IL12B rs3212227; IL6 rsl800795; IL23R rs7530511; IL23R rsll209026. This comparative study was conducted according to the procedure of Example 1 using pairs of primers and probes for the specific loci analyzed, showed that the rs7530511 and rsll209026 of IL23R are not statistically significant predictors of response to ustekinumab. Whereas, the predictability level of the polymorphisms of the gene IL12B rs3212227 and of the gene IL6 rsl800795, although statistically less significant than the association of HLA-Cw* 0602 with IL12B rs6887695 G/G, however, is significant, and can therefore be used as predictive indicators of clinical efficacy of the psoriasis treatment with ustekinumab.

Table 3

Genotype PASI 75 odds P

% ratio

CW6+/IL12B rs6887695 G/G 91,7 7,27 0, 023

CW6-/IL12B rs6887695 C/G C/C 66, 7

CW6+/IL12B rs3212227 C/A C/C 94, 1 6, 98 0, 02

CW6-/IL12B rs3212227 A/A 63,2

CW6+/IL6 rsl800795 C/G C/C 92, 3 8, 03 0, 013

CW6-/IL6 rsl800795 G/G 60, 0

52 weeks treatment

Genotype PASI 75 odds P

% ratio

CW6+/IL12B rs6887695 G/G 83,3 5,21 0, 007

CW6-/IL12B rs6887695 C/G C/C 44,4

CW6+/IL12B rs3212227 C/A C/C 82, 4 4,75 0,006

CW6-/IL12B rs3212227 A/A 42, 1

CW6+/IL6 rsl800795 C/G C/C 84,6 4,99 0, 005

CW6-/IL6 rsl800795 G/G 46, 7

Although even genotyping of rs3212227 and rsl800795 polymorphisms analysis, in association with the determination of the haplotype HLA-Cw* 0602 provides in a 12 weeks time interval predictive value comparable to that obtained by the analysis of the polymorphism rs6887695, the result at four weeks clearly shows the better efficacy of the combination of HLA-Cw* 0602 and the polymorphism G/G rs6887695 as early predictive agent of the outcome of the treatment of psoriasis with ustekinumab.

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Claims

1) A method for predicting response to treatment with a biological drug constituted by the anti-p40 antibody (ustekinumab) in a subject affected by psoriasis comprising detecting in the genome of the subject association of the HLA-Cw* 0602 allele and of a single-stranded polymorphism predictive of response to said anti-p40 antibody selected in the group of:

- IL-12B rs6887695;

- IL-12B rs3212227;

- IL-6 rsl800795.

2) The method according to Claim 1, wherein psoriasis is mild, moderate, serious plaque psoriasis, or chronic plaque psoriasis.

3) The method according to Claims 1 and 2, comprising detecting in the genome of a subject affected by psoriasis the HLA-Cw* 0602 allele and the single-stranded polymorphism present in the gene of the protein subunit p40 of the interleukins IL-12B (rs6887695) .

4) The method according to Claims 1 to 3, indicating the higher probability of clinical response to treatment with the anti-p40 antibody (ustekinumab) and/or drugs blocking the IL12/23-IL17 axis .

5) The method according to Claims 1 to 3, indicating the speed of clinical response to treatment with the anti-p40 antibody (ustekinumab) and/or drugs blocking the IL12/23-IL17 axis.

6) The method according to Claims 1 to 3, indicating better maintenance to the therapeutic effects deriving from use of the anti-p40 antibody and/or drugs blocking the IL12/23-IL17 axis. 7) The method according to Claims 1 to 6, wherein detection of the HLA-Cw* 0602 allele and of the single-stranded polymorphism predictive of response to the biological drug is obtained through genotyping techniques that enable determination of the genotype of the two genetic markers on the nucleic-acid molecule.

8) The method according to Claim 7, wherein the genotyping techniques involve analysis of a specimen of nucleic acid prepared from a biological specimen obtained from the patient for determining the identity of a nucleotide or pair of nucleotides present in the allele and in the single-stranded polymorphism.

9) The method according to Claim 8, wherein the biological specimen is chosen from among: whole blood, serum, sperm, saliva, tears, foecal material, urine, sweat, buccal material, skin, and hair.

10) The method according to Claims 1 to 9, wherein determination of the HLA-Cw* 0602 allele is obtained via a reaction of amplification of the genomic DNA of the patient of a "touchdown" type, wherein the initial annealing temperature of 65°C is progressively reduced by 0.5°C at each cycle for 18 cycles and is followed by 10 further cycles of amplification at 57°C.

11) The method according to Claim 10, wherein the "touchdown" reaction of amplification of the genomic DNA of the patient is conducted by KAPA2G Robust DNA Polymerase (KAPA Biosystems) with the specific CG robust reaction buffer.

12) A kit for predicting response to treatment with a biological drug constituted by an anti-p40 antibody in a subject affected by psoriasis, comprising :

preparations of nucleic acid in an amount sufficient to detect, by means of PCR, the presence or absence of the HLA Cw* 0602 allele and of the rs6887695 polymorphism in a specimen of DNA of the subject, amongst which pairs of primers and specific probes for the genetic loci undergoing genotype investigation;

- DNA polymerase,

- reagents and reaction mixtures appropriately balanced for conducting the reaction of amplification of the fragment of genomic DNA for the predictive allele and polymorphism,

- preparations of nucleic acid to be used as DNA template in a control amplification reaction for the two polymorphisms, and

- sterile deionized water,

for detecting the association of the HLA-Cw* 0602 allele and of the rs6887695 polymorphism predictive of response to treatment of psoriasis with the therapeutic agent and the instructions necessary for using said kit.

13) The kit according to Claim 12 comprising: preparations of the oligonucleotides specific for HLA-Cw* 0602: Fwd 5'-

TACTACAACCAGAGCGAGGA-3 ' and Rev 5'-

GGTCGCAGCCATACATCCA-3 ' ,

preparations of oligonucleotides and rs6887695 Taqman probe (TaqMan_SNP Genotyping Assay ID C1994992_10) that enable recognition of C/G polymorphism located in the position

GAGAGAAGCAGTGTAGTGTAGTGGT [C/G] AATAGTCTGGATTTACATCTTTG AT;

preparations of appropriate reagents and reaction mixtures balanced for development of the amplification and genotyping reactions;

deionized water; and

instructions for use of the kit.

14) Use of the association of the HLA-Cw* 0602 allele and of the single-stranded polymorphism of the gene of the subunit p40 of the interleukin IL-12B rs6887695 as marker predictive of the clinical response of a patient affected by psoriasis to treatment with ustekinumab and/or drugs blocking the IL12/23-IL17 axis.

15) Use of the association of the HLA-Cw* 0602 allele and of the single-stranded polymorphism of the gene of the subunit p40 of the interleukin IL-12B rs3212227 as marker predictive of the clinical response of a patient affected by psoriasis to treatment with ustekinumab and/or drugs blocking the IL12/23-IL17 axis.

16) Use of the association of the HLA-Cw* 0602 allele and of the single-stranded polymorphism of the gene of the interleukin IL-6 rsl800795 as marker predictive the clinical response of a patient affected by psoriasis to treatment with ustekinumab and/or drugs blocking the IL12/23-IL17 axis.