WO2022064069A1

WO2022064069A1 - Mmp-9 as a predictive marker of chronic allograft dysfunction

Info

Publication number: WO2022064069A1
Application number: PCT/EP2021/076696
Authority: WO
Inventors: Pierre-Joseph ROYER; Antoine MAGNAN; Sophie Brouard; Mallory PAIN; Adrien TISSOT
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Centre Hospitalier Universitaire De Nantes; Université de Nantes; Centre National De La Recherche Scientifique
Priority date: 2020-09-28
Filing date: 2021-09-28
Publication date: 2022-03-31

Abstract

The present invention relates to an in vitro method of predicting a risk of chronic solid organ allograft dysfunction (CAD) in a subject prior to the transplantation of said organ comprising: a) determining a control concentration of MMP-9 in a control sample; b) determining a test concentration of MMP-9 in the sample from the subject prior to the graft; c) comparing the control and test concentrations, wherein a test concentration higher than the control concentration predicts that the subject is at risk of developing a CAD.

Description

MMP-9 AS A PREDICTIVE MARKER OF CHRONIC ALLOGRAFT DYSFUNCTION

FIELD OF INVENTION

The present invention pertains to the field of allograft dysfunction. In particular the invention relates to a method of predicting chronic allograft dysfunction (CAD) and more specifically to a pre-graft method of predicting CAD.

BACKGROUND OF INVENTION

Chronic allograft dysfunction is the major cause for the failure of long-term graft survival. CAD is not reversible to date by any treatment when histologically detected, is not proven to be preventable by any immunosuppressive regimen and its pathogenesis is not fully understood but involving immunological as well as non-immunological factors.

Lung transplantation (LT) is the only option for patients with end-stage respiratory failure. Its efficacy has been demonstrated in selected patients in terms of life expectancy and post-transplant quality of life. LT is the fastest growing type of solid organ transplantation due to the increase in the incidence of severe respiratory diseases and in the rate of use of donor lungs. Unfortunately, long-term survival remains low with an overall 5-year survival of 57% and the main cause of late mortality is chronic lung graft dysfunction (CLAD) - CLAD being observed in almost 50% of patients at 5 years post-transplantation (TP).

The current classification identifies two different CLAD phenotypes: 1) bronchiolitis obliterative syndrome (BOS), the most common with 35% at 5 years, which histologically consists of obstruction of the small airways by tissue remodeling and extracellular matrix deposition with obstructive respiratory functional tests, and 2) restrictive allograft syndrome (RAS) characterized by a major fibrotic process, functional restrictive syndrome and a worse prognosis. Due to a poor understanding of the precise mechanisms of the disease and the lack of a robust clinical decision-making tool, CLAD is diagnosed late, by definition, when there is an irreversible loss of lung function. As a result, prognosis is poor with a median survival rates for BOS and RAS of 4 and 2 years respectively.

CLAD also represents an important economic weight in terms of public health. In the case of lung of the 4,000 transplants performed worldwide per year (50 000 to 100 000 € per procedure), 50% will result within 5 years post-transplantation in a CLAD with management costs multiplied by 2.5 due to re-hospitalizations, the use of expensive drugs and, when patient is eligible, re-transplantation (100 000 €).

The prediction of CLAD, and more generally of chronic solid organ allograft dysfunction (CAD) is therefore a major challenge. Early detection and earlier targeted intervention will reduce the risk of graft loss, improve quality of life, increase life expectancy, and improve the cost/benefit ratio of organ transplantation in recipients at risk of CLAD. This would allow a major advance in the field of transplantation.

There is an important need to have a reliable tool for prognosis of allograft dysfunction, the earliest as possible, i.e. before transplantation.

The present invention relates to a method of predicting chronic solid organ allograft dysfunction (CAD) in a subject prior to transplantation comparing the level of MMP-9 of the subject to be transplanted to a control value.

SUMMARY

This invention thus relates to an in vitro method of predicting a risk of chronic solid organ allograft dysfunction (CAD) in a subject prior to the transplantation of said organ comprising: a) determining a control concentration of MMP-9 in a control sample; b) determining a test concentration of MMP-9 in the sample from the subject prior to the graft; c) comparing the control and test concentrations, wherein a test concentration higher than the control concentration predicts that the subject is at risk of developing a CAD.

In a specific aspect of the invention, the organ is chosen among lung, heart, liver or kidney.

In another aspect of the invention wherein the organ is lung, and the Chronic Lung Allograft Dysfunction (CLAD). CLAD is either bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS).

In a more specific aspect of the invention, the concentration of MMP-9 is measured by ELISA or any method of protein dosage.

In a specific aspect of the invention, the tissue sample is a body fluid, preferably blood, plasma or serum even more preferably plasma.

In another aspect of the invention, the control concentration is determined in a population of transplanted recipients that did not develop a CAD after 36 months.

In an even more specific aspect of the invention, the control concentration of MMP-9 is comprised between 20- 250 ng/ml of MMP-9.

In a very specific aspect of the invention, the control concentration of MMP-9 is 246 ng/ml.

This invention also relates to an in vitro survival prognosis method comprising: a) determining a control concentration of MMP-9 in a control sample; b) determining a test concentration of MMP-9 in the sample from the subject prior to a graft; c) comparing the control and test concentrations, wherein a test concentration higher than the control concentration predicts a bad/low survival prognosis.

According to a specific aspect of the invention, the organ is chosen among lung, heart, liver or kidney. According to a further specific aspect of the invention, the concentration of MMP-9 is detected by ELISA or any method of protein dosage.

According to a still further specific aspect of the invention, the tissue sample is a body fluid, preferably blood, plasma or serum even more preferably plasma.

In a specific aspect of the invention, the control concentration of MMP-9 is comprised between 20 -250 ng/ml of MMP-9;

The present invention also relates to the use of MMP-9 as a biomarker for CAD in a subject prior to transplantation.

DEFINITIONS

In the present invention, the following terms have the following meanings:

“CAD” refers to Chronic solid organ Allograft Dysfunction.

“CLAD”: refers to Chronic Lung Allograft Dysfunction.

“FEV1”: Forced Expiratory Volume in 1 second

“BOS”: refers to bronchiolitis obliterative syndrome,

“RAS”: refers to restrictive allograft syndrome,

“MMP-9”: refers to Matrix -Metalloproteinase 9

DETAILED DESCRIPTION

The present invention deals with a method of predicting chronic solid organ allograft dysfunction (CAD) in a subject prior to transplantation comprising the step of a) determining a control concentration of MMP-9 in a control sample; b) determining a test concentration of MMP-9 in the sample from the subject prior to the graft; c) comparing the control and test concentrations and wherein a test concentration higher than the control concentration predicts that the subject is at risk of developing a CAD.

As used herein “chronic solid organ allograft dysfunction (CAD)” is defined as the definitive alteration of graft function.

In a specific embodiment of the invention, in the case where the solid organ to be graft is lung, CAD is CLAD: chronic lung allograft dysfunction. CLAD is defined as a substantial and persistent decline (>20%) in measured of the forced expiratory volume in one second (FEV1) value from the reference baseline (the baseline value is computed as the mean of the best 2 post-operative FEV1 measurements, taken >3 weeks apart.) The two described CLAD phenotypes are bronchiolitis obliterans syndrome (BOS), defined as a progressive and irreversible airway obstruction and restrictive allograft syndrome (RAS). The two phenotypes can be predicted using the methods of the invention. CLAD manifests as bronchiolitis obliterans syndrome (BOS) that involves mainly small airways and the more recently described restrictive allograft syndrome (RAS), with a parenchymal impairment (Verleden et al. 2014). In clinical practice, CLAD is a diagnosis of exclusion, once confounding factors related to allograft (persistent acute rejection, infection, anastomotic stricture, disease recurrence), or extra-allograft complications (pleural disease, diaphragm dysfunction or native lung hyperinflation) are ruled out (Meyer et al., 2014).

As used herein, the term "predicting" means that the subject to be analyzed by the method of the invention is allocated either into the group of subjects who will have a CAD, or into a group of subjects who will not have a CAD. Typically, said risk is elevated as compared to the average risk in a cohort of transplanted subjects. In the context of the invention, the risk of having a CAD in a subject shall be predicted. The term "predicting that the subject is at risk", as used herein, refers to assessing the probability according to which the subject (or patient) as referred to herein will develop a CAD if the transplantation is made. As will be understood by those skilled in the art, such an assessment is usually not intended to be correct for 100% of the subjects to be investigated. The term, however, requires that prediction can be made for a statistically significant portion of subjects in a proper and correct manner. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well-known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the probability envisaged by the invention allows that the prediction of an increased risk will be correct for at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort or population. The term, preferably, relates to predicting whether or not there is an increased risk of having CAD in a population of subjects rather than giving a precise probability for the said risk.

Within the meaning of the invention, the term "transplanted subject" also called as grafted subject, refers to a subject who has received an organ transplantation. The term "organ transplantation" or “organ graft” refers to the procedure of replacing diseased organs, or parts of organs, by healthy organs or tissues. The transplanted organ is obtained from another human donor (allograft). Transplanted organs may be artificial or natural, whole (such as lungs, kidney, heart and liver) or partial (such as heart valves). In a particular embodiment, the subject is a lung transplanted subject.

According to a specific aspect of the invention, the solid organs are chosen among lung, heart, liver or kidney.

Within the meaning of the invention, the measure of the concentration of MMP-9 is made in vitro in a sample obtained from a control subject (or a population of control subjects) or from a subject to be transplanted, in each case before transplantation.

The determination of the control concentration of MMP-9 is made in vitro by the measure of MMP-9 concentration in a control sample obtained before transplantation from a control subject (or a population of control subjects) whom were grafted but did not developed CAD within 3 years post-transplant and the measure of MMP-9 concentration in a control sample of a subject (or a population of control subjects) grafted and who developed CAD within 3 years post-transplant. As used herein, the term “sample” or "biological sample" refers to any sample obtained from a subject to be transplanted, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a tissue biopsy. In a particular embodiment, the biological sample is plasma. “Control sample” refers to the same type of sample (same origin) as the sample obtained from the subject to be tested. Those control sample are from a subject or a population of subject

According to the present invention the MMP-9 concentration is measured in a control sample. The “control sample” refers to the same type of sample (same origin) as the sample obtained from the subject to be tested, it is obtained from the subject (or a population of subjects) who is known to not having developed CAD.

As used herein, the term "control value" or “control concentration” refers to a threshold value or a cut-off value statistically significant, which was defined in a reference group of grafted subjects that did not develop a CAD.

According to the present invention a subj ect who is known for not having developed CAD is a patient with no signs of chronic dysfunction, e.g. no persistent decline in lung function in the 3 years (36 months) following the lung transplantation.

Matrix-Metalloproteinase 9 (MMP9) is a known molecule generally associated with remodeling and fibrosis processes observed in transplantation during chronic organ transplant dysfunction. MMP-9 is a predictive biomarker of CLAD in transplanted subjects. It allows the prediction of BOS and RAS 12 months before the functional diagnosis.

Within the meaning of the present invention the concentration of MMP-9 can be measured by any method known by the skilled man in the art and more preferably, by ELISA or any method of protein dosage.

According to a more specific aspect of the invention, the control concentration of MMP-9 is comprised between 20 - 250 ng/ml of MMP-9.

In a very specific aspect of the invention, the control concentration of MMP-9 is 246 ng/ml. This interval was statistically determined in a control population and has a value comprised between 20 and 250 ng/ml. According to the present invention, the control population includes without limitation, such subjects who underwent solid organ transplantation and who did not develop CAD within 3 years post transplantation, having similar age range, the same sex and the like.

According to a specific aspect of the invention, a concentration of MMP-9 measured in the sample of the subject that needs to be grafted that is above the concentration in the sample of the control predicts that the subject is at risk of developing CAD.

According to another aspect, the invention also relates to an in vitro survival prognosis method of a subject to be transplanted comprising: a) determining a control concentration of MMP-9 in a control sample; b) determining a test concentration of MMP-9 in the sample from the subject prior to a graft; c) comparing the control and test concentrations, and wherein a test concentration higher than the control concentration predicts a lower graft survival.

This in vitro survival prognosis method can be applied in the case of a solid organ transplantation such as lung, heart, liver or kidney.

Within said method, the concentration of MMP-9 can be established by ELISA or any method of protein dosage.

The sample is defined as above.

In a specific aspect of the method, the control concentration of MMP-9 is comprised between 20 - 250 ng/ml of MMP-9.

According to another aspect, the invention also relates to the use of MMP-9 as a biomarker for CAD in a subject prior to transplantation. According to another aspect, the invention also relates to a method for treating the transplanted/grafted subjects to prevent the occurrence of CAD comprising the steps of diagnosing of the subject before transplantation according to the method above described and administering to said subject as diagnosed a therapeutically effective amount of an agent that will delay and/or treat CAD.

In another embodiment, the method according to the invention is suitable to help the decision of treating the transplant recipient with a preventive care of subsequent CAD.

As used herein, the term "subject" refers to a grafted subject (also known as transplanted subject). Particularly, the subject who has received a solid organ transplantation.

According to a further aspect, the present invention refers to a method treatment of a chronic solid organ allograft dysfunction wherein the method comprises the steps of predicting a risk of chronic solid organ allograft dysfunction (CAD) in a subject prior to the transplantation of said organ comprising: a) determining a control concentration of MMP-9 in a control sample; b) determining a test concentration of MMP-9 in the sample from the subject prior to the graft; c) comparing the control and test concentrations, wherein a test concentration higher than the control concentration predicts that the subject is at risk of developing a CAD and treating the subject at risk of developing a CAD.

According to a further aspect, the present invention refers to a method treatment of subject predicted as having a bad/low survival prognosis comprising the steps of: a) determining a control concentration of MMP-9 in a control sample; b) determining a test concentration of MMP-9 in the sample from the subject prior to a graft; c) comparing the control and test concentrations, wherein a test concentration higher than the control concentration predicts a bad/low survival prognosis, and treating the subject having a bad/low survival prognosis.

As used herein, the terms "treating" or "treatment" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a ²²therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria).

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., immunosuppressive drug) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

A "therapeutically effective amount" is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

According to the present invention the treatment with azithromycin can be used to significantly reduce the prevalence of BOS.

According to the present invention "immunosuppressive drugs" also known as immunosuppressive agents or antirejection medications are drugs that inhibit or prevent the activity of immune system. Typically, the subject is treated with immunosuppressive drugs or other drugs that are currently known in the art or that will be identified in the future. In a particular embodiment, the subject is under immunosuppressive treatment, which means that the subject is administered with one or more immunosuppressive drugs. Immunosuppressive drugs that may be employed in transplantation procedures include corticosteroids, calcineurin inhibitors (cyclosporin, tacrolimus), azathioprine, mycophenolate mofetil and tyrosin kinase inhibitors (everolimus, sirolimus). These drugs may be used in monotherapy or in combination therapies. In a still further aspect, the present invention relates to a kit for predicting of CAD or survival prognosis in a subject prior to transplantation comprising at least one reagent for the determination of the concentration of MMP-9

As used herein, the term "a reagent for the determination of the concentration of MMP-9" is meant a reagent which specifically allows for the determination of said concentration, i.e. a reagent specifically intended for the specific determination of the concentration of MMP-9. This definition excludes generic reagents useful for the determination of the concentration of any protein, although such reagents may also be included in a kit according to the invention.

In some embodiments, the kit according to the invention may comprise instructions for determining whether a subject is at risk of developing a CAD, more particularly a CLAD. The instructions for determining whether a subject has CAD, CLAD, BOS or RAS may include at least one reference/control concentration.

The kit as described above is also used to predict the risk of having CAD.

The present invention also relates to a method of treating chronic solid organ allograft dysfunction (CAD) in a subject in need thereof.

In a more specific aspect, invention also relates to a method of treating Chronic Lung Allograft Dysfunction (CLAD) in a subject in need thereof, wherein CLAD is either bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS).

The term “treatment” and its declensions refer to the administration to a subject of a therapeutic regimen in order to abrogate, inhibit, slow or reverse the progression of a given disease or condition, and/or to ameliorate clinical symptoms of the given disease or condition, and/or to prevent the appearance of further clinical symptoms of the given disease or condition, e.g., CAD or to any measure taken to prevent or predict the appearance of further clinical symptoms of the given disease, e.g. placing said subject under active surveillance.

The terms “a subject in need thereof’ refers to a subject with a predict risk of CAD, said risk being identified by the method of the invention. In one embodiment, the method comprises a first step of diagnosing a risk of CAD in a subject, using the method detailed above.

In one embodiment, the method comprises a second step of treating the subject with an immunosuppressive therapy if or when said subject is diagnosed prior to the transplantation with a predicted a risk of CAD during the first step.

In one embodiment, treating the subject diagnosed with a predicted risk of CAD comprises resuming a previously-completed immunosuppressive therapy.

In one embodiment, treating the subject diagnosed with a risk of CAD comprises increasing the dosage regimen of a currently-administered immunosuppressive therapy.

In one embodiment, treating the subject diagnosed with a risk of CAD comprises changing the currently-administered immunosuppressive therapy for a more aggressive treatment.

Examples of suitable immunosuppressive therapies are well known to the skilled man in the art.

In one embodiment, the method comprises a first step of diagnosing a risk of CAD in a subject, using the method detailed above.

In one embodiment, the method comprises a second step of reducing and eventually suppressing an immunosuppressive therapy in the subject if or when said subject is not diagnosed with a risk of CAD during the first step.

In another embodiment, the method comprises a first step of diagnosing a risk of CAD in a subject, using the method detailed above.

In one embodiment, the method comprises a second step of placing said subject under active surveillance if the subject the subject is at risk of developing a CAD.

“Active surveillance” is considered in the field of medicine as a form of treatment (see in particular the Memorial Sloan Kettering Cancer center (MSKCC; https://www.mskcc.or.g/cancer-care/types/prostate/treatment/active-surveillance). In another embodiment, the method comprises a first step of diagnosing a risk of CAD in the subject, using the method detailed above.

In one embodiment, the method comprises a second step of treating the subject with an immunosuppressive therapy if or when said subject is diagnosed prior to the transplantation with a predicted a risk of CAD during the first step, comprises resuming a previously-completed immunosuppressive therapy, comprises increasing the dosage regimen of a currently-administered immunosuppressive therapy or comprises changing the currently-administered immunosuppressive therapy for a more aggressive treatment and

In one embodiment, the method comprises a third step concomitant or following the second step of placing said subject under active surveillance if or when said subject is diagnosed prior to the transplantation with a predicted a risk of CAD during the first step.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the plasmatic dosage of MMP9 realized before the graft. The patients are classified according to the pathology leading to transplantation. CF: cystic fibrosis, COPD: chronic obstructive pulmonary disease, IPD: interstitial pulmonary disease, PH: Pulmonary Arterial Hypertension

Figure 2 shows the predictive performance of the biomarker by ROC curve for patient of the groups "COPD" and "IPD" .

Figure 3 shows the survival without CLAD and overall survival of patients according to their plasma MMP9 concentration. Threshold value used: 246 ng/ml EXAMPLES

The present invention is further illustrated by the following examples.

Example 1 :

Materials and Methods

Material

Freezer -20°C and -80°C

→ Centrifuge for tube 10 mL at rotation to 2000g

→ Microplate reader (reading at 450 nm and 620 nm) and software

→ BD Vacutainer® Lithium Heparin Tube 9/10 mL

→ Complete™ Protease Inhibitor Cocktail (Roche #04693116001)

→ MMP9 Human ELISA kit (Thermo Fischer Scientific # BMS2016-2 or

# BMS2016-2TEN)

→ Micropipet P20, P200, Pl 000

→ Multipipet P400 (or P200)

→ Sterile tips

→ MiMrotube 1,5 mL or 2 mL

Methods

The blood samples collected in lithium heparin tubes are centrifuged at 2000 g during 20 minutes at 20°C. The plasma thus obtained is aliquoted by 500 pL to which are added 20 pL of diluted protease inhibitor (Roche #04693116001) before being frozen at -20°C.

The plasma is thawed at room temperature before dosage of MMP9 with the MMP9 Human ELISA kit (Thermo Fischer Scientific # BMS2016-2 or # BMS2016-2TEN). The protocol steps of the ELISA are explained in the manufacturer manual supplied in the kit. However, the samples are generally pre-diluted at 1 : 10 (25 pL of plasma) but it may be possible to dilute them more or less depending on the MMP9 rate in the sample. The absorbance obtained is read on the Spark® 10M multimode microplate reader of Tecan according the recommendations specified in the protocol (450 nm as the primary wave length and 620 nm as the reference wave length). The software SparkControl™ generates automatically the MMP9 concentrations in the samples taking into account the dilutions.

Results

Patients are grouped according to their initial pathology and our results show that for the COPD (chronic obstructive pulmonary disease) or fibrosis groups (IPD), pre-graft plasma MMP9 is higher for patients who will develop CLAD (Figure 1). For these two groups of patients, the performance of CLAD prediction was determined using a Receiver Operating Characteristic (ROC) curve. A plasma concentration threshold of 246 ng/ml predicts CLAD with a sensitivity of 73% and a specificity of 84%. The area under the curve is 0.82 (Figure 2). Analysis of the survival curves shows a higher CLAD-free survival for patients with a pre-graft MMP9 level below 246 ng/ml compared to patients with a level above 246 ng/ml. Patient survival, on the other hand, is not significantly different (Figure 3).

BIBLIOGRAPHIC REFERENCES Verleden GM, Raghu G, Meyer KC, Glanville AR, Corris P. A new classification system for chronic lung allograft dysfunction. J Heart Lung Transplant 2014;33: 127-33.

Meyer KC, Raghu G, Verleden GM, Corris PA, Aurora P, Wilson KC, et al. An international ISHLT/ATS/ERS clinical practice guideline: diagnosis and management of bronchiolitis obliterans syndrome. Eur Respir J 2014;44: 1479-503.

Claims

CLAIMS An in vitro method of predicting a risk of chronic solid organ allograft dysfunction (CAD) in a subject prior to the transplantation of said organ comprising: a) determining a control concentration of MMP-9 in a control sample; b) determining a test concentration of MMP-9 in the sample from the subject prior to the graft; c) comparing the control and test concentrations, wherein a test concentration higher than the control concentration predicts that the subject is at risk of developing a CAD. The method of claim 1, wherein the organ is chosen among lung, heart, liver or kidney. The method of claim 2 wherein the organ is lung, and the Chronic Lung Allograft Dysfunction (CLAD), CLAD is either bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). The method of any one of claims 1 to 3 wherein the concentration of MMP-9 is measured by ELISA or any method of protein dosage. The method according to any of claims 1 to 4, wherein the tissue sample is a body fluid, preferably blood, plasma or serum even more preferably plasma. The method of any one of the preceding claims wherein the control concentration is determined in a population of transplanted recipients that did not develop a CAD after 36 months. The method according to any one of the preceding claims wherein the control concentration of MMP-9 is comprised between 20-250 ng/ml of MMP-9. An in vitro survival prognosis method comprising: a) determining a control concentration of MMP-9 in a control sample; b) determining a test concentration of MMP-9 in the sample from the subject prior to a graft; c) comparing the control and test concentrations, wherein a test concentration higher than the control concentration predicts a bad/low survival prognosis.

9. The method of claim 8, wherein the organ is chosen among lung, heart, liver or kidney.

10. The method of any one of claims 8 or 9, wherein the concentration of MMP-9 is detected by ELISA or any method of protein dosage. 11. The method according to any of claims 8 to 10, wherein the tissue sample is a body fluid, preferably blood, plasma or serum even more preferably plasma.

12. The method according to any of claims 8 to 11 wherein the control concentration of MMP-9 is comprised between 20-250 ng/ml of MMP-9.

13. The method according to any of claims 8 to 12 wherein the control concentration of MMP-9 is 246 ng/ml of MMP-9

14. Use of MMP-9 as a biomarker for CAD in a subject prior to transplantation.